Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

PubMed

Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

2010-06-01

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

Alterations of type IV collagen alpha chains in patients with chronic acquired glomerulopathies: mRNA levels, protein expression and urinary loss.

PubMed

Sanna-Cherchi, Simone; Carnevali, Maria Luisa; Martorana, Davide; Cravedi, Paolo; Maggiore, Umberto; Alinovi, Rossella; Bovino, Achiropita; Mattei, Silvia; Orlandini, Guido; Gatti, Rita; Savi, Mario; Sado, Yoshikazu; Neri, Tauro M; Allegri, Landino

2007-01-01

Type IV collagen is a major structural component of the normal kidney glomerulus. However, its role in chronic acquired glomerulopathies has been only partially elucidated. Urinary levels of col(IV)alpha1, col(IV)alpha3 and col(IV)alpha5 collagen chains were analyzed in 107 patients with chronic acquired glomerulopathies. In a subgroup of 33 patients, tissue mRNA levels, protein expression and urinary excretion were evaluated for all col(IV)alpha chains, from col(IV)alpha1 to col(IV)alpha5. The renal specimens were examined to get a semiquantitative score of the acute and chronic activity of the histological lesions. Urines obtained from 13 healthy subjects and 10 normal renal tissue samples were used as controls. Urinary levels of col(IV)alpha1, col(IV)alpha3, col(IV)alpha5 chains were significantly higher in patients than in controls [p < 0.01 for all], while only col(IV)alpha1 and col(IV)alpha3 urinary excretion correlated with the degree of chronic histological damage [col(IV)alpha1 R = 0.44, p < 0.001; col(IV)alpha3: R = 0.47, p < 0.001]. Compared with controls, patients showed a renal expression of mRNA for col(IV)alpha5 chain significantly higher [p = 0.001], while having a significantly lower protein expression of col(IV)alpha3, col(IV)alpha4 and col(IV)alpha5 chains [p < 0.01 for all]. Patients with chronic acquired glomerulopathies show important alterations in the col(IV)alpha chain network mimicking some molecular features of the X-linked Alport's syndrome. Further studies are needed to show whether urinary levels of the col(IV)alpha chains may be used as markers for monitoring renal injury. Copyright 2007 S. Karger AG, Basel.

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

PubMed

Nagai, N; Nakano, K; Sado, Y; Naito, I; Gunduz, M; Tsujigiwa, H; Nagatsuka, H; Ninomiya, Y; Siar, C H

2001-10-01

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.

Distribution of Collagen I, III, and IV and Laminin in the Human Liver during Prenatal Development.

PubMed

Jović, Marko; Nikolić, Ivan; Todorović, Vera; Petrović, Aleksandar; Petrović, Vladimir; Mojsilović, Marijola; Denčić, Tijana

2018-06-27

In the absence of systematized data on the extracellular matrix components during prenatal liver development, the present study aimed to investigate the time of appearance and distribution of collagen types I, III, and IV and laminin. The study material included embryonic and fetal livers, aged 7-37 weeks, categorized into 3 trimesters. The material was stained using hematoxylin-eosin and immunohistochemistry methods for the identification of collagen I, III, and IV and laminin. Collagen I was detected near the end of the first trimester in the capsules and walls of interlobular veins. As the liver matures, collagen I is increasingly abundant in the capsules, portal area connective tissues, arterial walls, interlobular veins, sinusoids, and central veins. Collagen III and collagen IV appear in the middle of the first trimester in the capsules, portal areas, and walls of central veins, as well as the sinusoids particularly. In trimesters 2 and 3, these collagens are increasingly present in all the structures, but collagen IV is also present in nerve fibers. Laminin is sporadically present adjacent to the sinusoids in trimester 1, while in trimesters 2 and 3 this protein commonly appears in the walls of arteries and interlobular veins, in the basal membrane of bile ducts, and in nerve fibers. The contents of collagen I, III, and IV increase during prenatal development in the liver capsule, arterial and vein walls, sinusoids, and portal area. Laminin expression is consistent with that of the collagens with the exception that, within lobules, laminin disappears with liver maturation. © 2018 S. Karger AG, Basel.

Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues

PubMed Central

Toh, Wei Seong; Gomoll, Andreas H.; Olsen, Bjørn Reino; Spector, Myron

2014-01-01

Objective: The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Design: Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti–collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. Results: When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. Conclusions: We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional roles of these 2 extracellular matrix proteins, normally associated with BM. PMID:26069692

Circulating Vascular Basement Membrane Fragments are Associated with the Diameter of the Abdominal Aorta and Their Expression Pattern is Altered in AAA Tissue.

PubMed

Holsti, Mari; Wanhainen, Anders; Lundin, Christina; Björck, Martin; Tegler, Gustaf; Svensson, Johan; Sund, Malin

2018-04-12

Abdominal aortic aneurysm (AAA) is characterised by enhanced proteolytic activity, and extracellular matrix (ECM) remodelling in the vascular wall. Type IV and XVIII collagen/endostatin are structural proteins in vascular basement membrane (VBM), a specialised ECM structure. Here the association between plasma levels of these collagens with the aortic diameter and expansion rate is studied, and their expression in aortic tissue characterised. This was a retrospective population based cohort study. Type IV and XVIII collagen/endostatin were analysed in plasma by ELISA assay in 615 men, divided into three groups based on the aortic diameter: 1) normal aorta ≤ 25 mm, 2) sub-aneurysmal aorta (SAA) 26-29 mm, and 3) AAA ≥ 30 mm. Follow up data were available for 159 men. The association between collagen levels and aortic diameter at baseline, and with the expansion rate at follow up were analysed in ordinal logistic regression and linear regression models, controlling for common confounding factors. Tissue expression of the collagens was analysed in normal aorta (n = 6) and AAA (n = 6) by immunofluorescence. Plasma levels of type XVIII collagen/endostatin (136 ng/mL [SD 29] in individuals with a normal aorta diameter, 154 ng/ml [SD 45] in SAA, and 162 ng/ml [SD 46] in AAA; p = .001) and type IV collagen (105 ng/mL [SD 42] normal aorta, 124 ng/ml [SD 46] SAA, and 127 ng/ml [SD 47] AAA; p = .037) were associated with a larger aortic diameter. A significant association was found between the baseline levels of type XVIII/endostatin and the aortic expansion rate (p = .035), but in the multivariable model, only the initial aortic diameter remained significantly associated with expansion (p = .005). Altered expression patterns of both collagens were observed in AAA tissue. Plasma levels of circulating type IV and XVIII collagen/endostatin increase with AAA diameter. The expression pattern of VBM proteins is altered in the aneurysm wall. Copyright © 2018 European Society for Vascular Surgery. Published by Elsevier B.V. All rights reserved.

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Differential expression of basement membrane type IV collagen α2 and α6 chains as a prognostic factor in patients with extrahepatic bile duct carcinoma.

PubMed

Hirashima, Kotaro; Iyama, Ken-Ichi; Baba, Yoshifumi; Honda, Yumi; Sado, Yoshikazu; Ninomiya, Yoshifumi; Watanabe, Masayuki; Takamori, Hiroshi; Beppu, Toru; Baba, Hideo

2013-03-01

The destruction of the basement membrane (BM) is the first step in cancer invasion and metastasis. Type IV collagen is a major component of the BM, and is composed of six genetically distinct α(IV) chains; α1(IV) to α6(IV). The loss of α5(IV) and α6(IV) chains from the epithelial BM at the early stage of cancer invasion has been reported in several types of cancers. However, the expression of α5(IV) and α6(IV) chains in extrahepatic bile duct carcinoma (EBDC) remains unclear. We examined the expression of α(IV) chains by immunohistochemistry using 71 resected EBDC specimens. Prognostic significance of α(IV) chains was examined by Cox regression and Kaplan-Meier analyses. In the invasive cancer, the expression of α6(IV) chain in the BM was lost partially or completely preceded by the loss of α2(IV) chain. The loss of α6(IV) chain in the BM of the invasive cancer was related to the tumor classification, TNM stages, and the expression of α2(IV) chain. The patients with α2(IV)-negative and α6(IV)-negative chains had significantly poorer prognosis than those with α2(IV)-positive and α6(IV)-positive/negative chains (P = 0.04). The loss of α2(IV) and α6(IV) chains might be a useful prognostic factor in patients with EBDC. Copyright © 2012 Wiley Periodicals, Inc.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

A case of mild phenotype Alport syndrome caused by COL4A3 mutations.

PubMed

Kamijo, Masafumi; Kitamura, Mineaki; Muta, Kumiko; Uramatsu, Tadashi; Obata, Yoko; Nozu, Kandai; Kaito, Hiroshi; Iijima, Kazumoto; Mukae, Hiroshi; Nishino, Tomoya

2017-11-01

In a case of 41-year-old man with mild nephropathy, Alport syndrome (AS) was diagnosed from the renal biopsy. However, the α5 chain of type IV collagen expressed in the glomerular basement membrane, which was the atypical staining pattern of AS. Genetic testing suggested autosomal recessive AS from heterozygous mutations at two positions in the type IV collagen α3 chain. These two gene mutations represented a new pattern of mutation and was suggested the association with an atypical α5 chain expression and mild phenotype.

Synthesis of prolyl 4-hydroxylase alpha subunit and type IV collagen in hemocytic granular cells of silkworm, Bombyx mori: Involvement of type IV collagen in self-defense reaction and metamorphosis.

PubMed

Adachi, Takahiro; Tomita, Masahiro; Yoshizato, Katsutoshi

2005-04-01

The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.

Colorectal laterally spreading tumors show characteristic expression of cell polarity factors, including atypical protein kinase C λ/ι, E-cadherin, β-catenin and basement membrane component.

PubMed

Ichikawa, Yasushi; Nagashima, Yoji; Morioka, Kaori; Akimoto, Kazunori; Kojima, Yasuyuki; Ishikawa, Takashi; Goto, Ayumu; Kobayashi, Noritoshi; Watanabe, Kazuteru; Ota, Mitsuyoshi; Fujii, Shoichi; Kawamata, Mayumi; Takagawa, Ryo; Kunizaki, Chikara; Takahashi, Hirokazu; Nakajima, Atsushi; Maeda, Shin; Shimada, Hiroshi; Inayama, Yoshiaki; Ohno, Shigeo; Endo, Itaru

2014-09-01

Colorectal flat-type tumors include laterally spreading tumors (LSTs) and flat depressed-type tumors. The former of which shows a predominant lateral spreading growth rather than an invasive growth. The present study examined the morphological characteristics of LSTs, in comparison with polypoid- or flat depressed-type tumors, along with the expression of atypical protein kinase C (aPKC) λ/ι, a pivotal cell polarity regulator, and the hallmarks of cell polarity, as well as with type IV collagen, β-catenin and E-cadherin. In total, 37 flat-type (24 LSTs and 13 flat depressed-type tumors) and 20 polypoid-type colorectal tumors were examined. The LSTs were classified as 15 LST adenoma (LST-A) and nine LST cancer in adenoma (LST-CA). An immunohistochemical examination was performed on aPKC λ/ι, type IV collagen, β-catenin and E-cadherin. The LST-A and -CA showed a superficial replacing growth pattern, with expression of β-catenin and E-cadherin in the basolateral membrane and type IV collagen along the basement membrane. In addition, 86.6% of LST-A and 55.6% of LST-CA showed aPKC λ/ι expression of 1+ (weak to normal intensity staining in the cytoplasm compared with the normal epithelium). Furthermore, ~45% of the polypoid-type adenomas showed 2+ (moderate intensity staining in the cytoplasm and/or nucleus) and 66.7% of the polypoid-type cancer in adenoma were 3+ (strong intensity staining in the cytoplasm and nucleus). A statistically significant positive correlation was observed between the expression of aPKC λ/ι and β-catenin (r=0.842; P<0.001), or type IV collagen (r=0.823; P<0.001). The LSTs showed a unique growth pattern, different from the expanding growth pattern presented by a polypoid tumor and invasive cancer. The growth characteristics of LST appear to be caused by adequate coexpression of β-catenin, type IV collagen and aPKC λ/ι.

Early Diagnosis and Intervention Strategies for Post-Traumatic Heterotopic Ossification in Severely Injured Extremities

DTIC Science & Technology

2014-10-01

Adipoq, BMP4, Col4a3, IGF2, MyoD1, Smad3); 3-20 fold lower elevation of expression of Col1a1 ; and 3-10 fold higher expression of MMP9 and Spp1...XI, alpha 1 COL1A1 collagen, type I, alpha 1 COL2A1 collagen, type II, alpha 1 COL4A3 collagen, type IV, alpha 3 COMP cartilage oligomeric matrix

L-arginine mediated renaturation enhances yield of human, α6 type IV collagen non-collagenous domain from bacterial inclusion bodies

PubMed Central

Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Akul Sudhakar, Yakkanti

2012-01-01

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated. PMID:22512648

L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

PubMed

Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

2012-10-01

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

Myostatin regulates proliferation and extracellular matrix mRNA expression in NIH3T3 fibroblasts.

PubMed

Z Hosaka, Yoshinao; Ishibashi, Mika; Wakamatsu, Jun-Ichi; Uehara, Masato; Nishimura, Takanori

2012-12-01

The aim of this study was to clarify the effects of myostatin, which is a negative regulator of skeletal muscle mass, on the proliferation of NIH3T3 fibroblasts and the synthesis of extracellular matrix (ECM) by them. A proliferation assay revealed that myostatin attenuated cell growth at any of the doses used. High doses of myostatin strongly inhibited cell proliferation. Moreover, myostatin receptor, activin receptor type-2B (ActRIIB), was found to be distributed on cells and it was also clarified that myostatin increased the expression of cyclin-dependent kinase inhibitor p21 (p21). These results suggested that a high dose of myostatin inhibits fibroblast proliferation by the same mechanism as that for inhibition of myoblast proliferation. We then examined the effects of myostatin on the mRNA expression of ECM molecules (decorin, biglycan, type I collagen, type III collagen, type IV collagen and type V collagen) by real-time PCR. Real-time PCR showed that myostatin increased the mRNA of decorin, biglycan and collagen (types I, IV and V) in fibroblasts. The results suggest that myostatin regulates ECM synthesis in cultured fibroblasts.

Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Won Hee; Warrington, Junie P.; Sonntag, William E.

Purpose: Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. Methods and Materials: Animals received either whole-brain irradiation (a single dose of 10 Gy {gamma}-rays or a fractionated dose of 40 Gy {gamma}-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR).more » The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. Results: A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. Conclusions: The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.« less

Astragaloside IV controls collagen reduction in photoaging skin by improving transforming growth factor-β/Smad signaling suppression and inhibiting matrix metalloproteinase-1.

PubMed

Chen, Bin; Li, Ran; Yan, Ning; Chen, Gang; Qian, Wen; Jiang, Hui-Li; Ji, Chao; Bi, Zhi-Gang

2015-05-01

Exposure to ultraviolet (UV) light reduces levels of type I collagen in the dermis and results in human skin damage and premature skin aging (photoaging). This leads to a wrinkled appearance through the inhibition of transforming growth factor‑β (TGF‑β)/Smad signaling. UV irradiation increases type I collagen degradation through upregulating matrix metalloproteinase (MMP) expression. Astragaloside IV (AST) is one of the major active components extracted from Astragalus membranaceus. However, its multiple anti‑photoaging effects remain to be elucidated. In the present study, the effects of AST against collagen reduction in UV‑induced skin aging in human skin fibroblasts were investigated. The expression of type I procollagen (COL1), MMP‑1, TGF‑βRⅡ and Smad7 were determined using reverse transcription‑polymerase chain reaction, western blotting and ELISA, respectively. UV irradiation inhibits type I collagen production by suppressing the TGF‑β/Smad signaling pathway and increasing COL1 degradation by inducing MMP‑1 expression. Transforming growth factor‑β type II protein and COL1 mRNA decreased but MMP‑1 and Smad7 levels increased in the photoaging model group, which was reversed by topical application of AST. AST prevents collagen reduction from UV irradiation in photoaging skin by improving TGF‑β/Smad signaling suppression and inhibiting MMP‑1, thus AST may be a potential agent against skin photoaging.

Distribution of pericellular matrix molecules in the temporomandibular joint and their chondroprotective effects against inflammation

PubMed Central

Chu, Wern Cui; Zhang, Shipin; Sng, Timothy J; Ong, Yu Jie; Tan, Wen-Li; Ang, Vivien Y; Foldager, Casper B; Toh, Wei Seong

2017-01-01

The objectives of this study were to (1) determine the distribution and synthesis of pericellular matrix (PCM) molecules (collagen VI, collagen IV and laminin) in rat temporomandibular joint (TMJ) and (2) investigate the effects of PCM molecules on chondrocytes against inflammation in osteoarthritis. Four zones (fibrous, proliferating, mature and hypertrophic) of condylar cartilage and three bands (anterior, intermediate and posterior) of disc were analysed by immunohistochemistry for the presence of PCM molecules in rat TMJs. Isolated chondrocytes were pre-treated with PCM molecules before being subjected to interleukin (IL)-1β treatment to stimulate inflammation. The responses of the chondrocytes were analysed using gene expression, nitric oxide release and matrix metalloproteinase (MMP)-13 production measures. Histomorphometric analyses revealed that the highest areal deposition of collagen VI (67.4%), collagen IV (45.7%) and laminin (52.4%) was in the proliferating zone of TMJ condylar cartilage. No significant difference in the distribution of PCM molecules was noted among the three bands of the TMJ disc. All three PCM molecules were expressed intracellularly by chondrocytes cultured in the monolayer. Among the PCM molecules, pre-treatment with collagen VI enhanced cellular proliferation, ameliorated IL-1β-induced MMP-3, MMP-9, MMP-13 and inducible nitric oxide synthase gene expression, and attenuated the downregulation of cartilage matrix genes, including collagen I, aggrecan and cartilage oligomeric matrix protein (COMP). Concurrently, collagen VI pretreatment inhibited nitric oxide and MMP-13 production. Our study demonstrates for the first time the distribution and role of PCM molecules, particularly collagen VI, in the protection of chondrocytes against inflammation. PMID:28282029

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation.

PubMed

Bjørn, S F; Hastrup, N; Lund, L R; Danø, K; Larsen, J F; Pyke, C

1997-08-01

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Effects of extracellular matrix proteins on macrophage differentiation, growth, and function: comparison of liquid and agar culture systems

NASA Technical Reports Server (NTRS)

Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Both spaceflight and skeletal unloading suppress the haematopoietic differentiation of macrophages (Sonnenfeld et al., Aviat. Space Environ. Med., 61:648-653, 1990; Armstrong et al., J. Appl. Physiol., 75:2734-2739, 1993). The mechanism behind this reduction in haematopoiesis has yet to be elucidated. However, changes in bone marrow extracellular matrix (ECM) may be involved. To further understand the role of ECM products in macrophage differentiation, we have performed experiments evaluating the effects of fibronectin, laminin, collagen type I, and collagen type IV on macrophage development and function. Bone marrow-derived macrophages cultured on four different ECM substrates in liquid culture medium showed less growth than those cultured on plastic. Significant morphological differences were seen on each of the substrates used. Phenotypically and functionally, as measured by class II major histocompatibility molecule (MHCII) expression, MAC-2 expression, and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), these macrophages were similar. In contrast, bone marrow-derived macrophages cultured in suspension, using agar, showed no difference in growth when exposed to ECM proteins. However, IL-6 and TNF-alpha secretion was affected by fibronectin, laminin, collagen type I, and collagen type IV in a concentration-dependent manner. We conclude that the ECM products fibronectin, laminin, collagen type I, and collagen type IV have profound effects on macrophage development and function. Additionally, we suggest that an ECM-supplemented agar culture system provides an environment more analogous to in vivo bone marrow than does a traditional liquid culture system.

Sodium-dependent Vitamin C transporter 2 deficiency impairs myelination and remyelination after injury: Roles of collagen and demethylation.

PubMed

Röhr, Dominik; Halfter, Hartmut; Schulz, Jörg B; Young, Peter; Gess, Burkhard

2017-07-01

Peripheral nerve myelination involves rapid production of tightly bound lipid layers requiring cholesterol biosynthesis and myelin protein expression, but also a collagen-containing extracellular matrix providing mechanical stability. In previous studies, we showed a function of ascorbic acid in peripheral nerve myelination and extracellular matrix formation in adult mice. Here, we sought the mechanism of action of ascorbic acid in peripheral nerve myelination using different paradigms of myelination in vivo and in vitro. We found impaired myelination and reduced collagen expression in Sodium-dependent Vitamin C Transporter 2 heterozygous mice (SVCT2 +/- ) during peripheral nerve development and after peripheral nerve injury. In dorsal root ganglion (DRG) explant cultures, hypo-myelination could be rescued by precoating with different collagen types. The activity of the ascorbic acid-dependent demethylating Ten-eleven-translocation (Tet) enzymes was reduced in ascorbic acid deprived and SVCT2 +/- DRG cultures. Further, in ascorbic acid-deprived DRG cultures, methylation of a CpG island in the collagen alpha1 (IV) and alpha2 (IV) bidirectional promoter region was increased compared to wild-type and ascorbic acid treated controls. Taken together, these results provide further evidence for the function of ascorbic acid in myelination and extracellular matrix formation in peripheral nerves and suggest a putative molecular mechanism of ascorbic acid function in Tet-dependent demethylation of collagen promoters. © 2017 Wiley Periodicals, Inc.

Type XVII collagen (BP180) can function as a cell-matrix adhesion molecule via binding to laminin 332

PubMed Central

Van den Bergh, F.; Eliason, S.L.; Giudice, G.J.

2010-01-01

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knockdown of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix. PMID:21034821

Liposomal gene transfer of keratinocyte growth factor improves wound healing by altering growth factor and collagen expression.

PubMed

Pereira, Clifford T; Herndon, David N; Rocker, Roland; Jeschke, Marc G

2007-05-15

Growth factors affect the complex cascade of wound healing; however, interaction between different growth factors during dermal and epidermal regeneration are still not entirely defined. In the present study, we thought to determine the interaction between keratinocyte growth factor (KGF) administered as liposomal cDNA with other dermal and epidermal growth factors and collagen synthesis in an acute wound. Rats received an acute wound and were divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and Lac-Z gene (0.22 microg). Histological and immunohistochemical techniques were used to determine growth factor, collagen expression, and dermal and epidermal structure. KGF cDNA increased insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3), and fibroblast growth factor (FGF), decreased transforming growth factor-beta (TGF-beta), while it had no effect on platelet-derived growth factor (PDGF) levels in the wound. KGF cDNA significantly increased collagen Type IV at both the wound edge as well as the wound bed, while it had no effect on collagen Type I and III. KGF cDNA increased re-epithelialization, improved dermal regeneration, and increased neovascularization. Exogenous administered KGF cDNA causes increases in IGF-I, IGF-BP3, FGF, and collagen IV and decreases TGF-beta concentration. KGF gene transfer accelerates wound healing without causing an increase in collagen I or III.

Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

PubMed

Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

2015-01-01

Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

[Effects of Valeriana officinalis var. latifolia on expression of transforming growth factor beta 1 in hypercholesterolemic rats].

PubMed

Si, Xiao-yun; Jia, Ru-han; Huang, Cong-xin; Ding, Guo-hua; Liu, Hong-yan

2003-09-01

To evaluate the effect of Valeriana officinalis var latifolia(VOL) on expression of transforming growth factor beta 1 (TGF-beta 1) in hypercholesterolemic rats and study its possible mechanisms. Dietary-induced hypercholesterolemia was induced in male Wistar rats by given 4% cholesterol and 1% cholic acid diet for 16 weeks. Changes of serum lipid, urinary albumin, renal function and Mesangial matrix index were assessed. Moreover, immunohistochemical stain for TGF-beta 1 and type IV collagen were performed. VOL could reduce the serum levels of total cholesterol, low density lipoprotein, urinary albumin and serum creatinine. Light microscopy and immunohistochemical stain revealed that in the same time of lowing serum lipid, Mesangial matrix index was significantly reduced, accompanied by decreased expression of TGF-beta 1 and type IV collagen. VOL has the protective effect on lipid-induced nephropathy, and the inhibition of TGF-beta 1 expression might be the mechanism of VOL on renal protection.

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

PubMed Central

Steenhard, Brooke M.; Vanacore, Roberto; Friedman, David; Zelenchuk, Adrian; Stroganova, Larysa; Isom, Kathryn; St. John, Patricia L.; Hudson, Billy G.; Abrahamson, Dale R.

2012-01-01

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM. PMID:23236390

Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells.

PubMed

Li, Q S; Meng, F Y; Zhao, Y H; Jin, C L; Tian, J; Yi, X J

2017-08-01

This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing. Blood samples were obtained from patients with hand fracture or intra-articular calcaneal fracture and from healthy controls (HCs). Expression of miR-214-5p was monitored by qRT-PCR at day 7, 14 and 21 post-surgery. Mouse osteoblastic MC3T3-E1 cells were transfected with antisense oligonucleotides (ASO)-miR-214-5p, collagen type IV alpha 1 (COL4A1) vector or their controls; thereafter, cell viability, apoptotic rate, and the expression of collagen type I alpha 1 (COL1A1), type II collagen (COL-II), and type X collagen (COL-X) were determined. Luciferase reporter assay, qRT-PCR, and Western blot were performed to ascertain whether COL4A1 was a target of miR-214-5p. Plasma miR-214-5p was highly expressed in patients with bone fracture compared with HCs after fracture (p < 0.05 or p < 0.01). Inhibition of miR-214-5p increased the viability of MC3T3-E1 cells and the expressions of COL1A1 and COL-X, but decreased the apoptotic rate and COL-II expression (p < 0.05 or p < 0.01). COL4A1 was a target of miR-214-5p, and was negatively regulated by miR-214-5p (p < 0.05 or p < 0.01). Overexpression of COL4A1 showed a similar impact on cell viability, apoptotic rate, and COL1A1, COL-II, and COL-X expressions inhibiting miR-214-5p (p < 0.01). Inhibition of miR-214-5p promotes cell survival and extracellular matrix (ECM) formation of osteoblastic MC3T3-E1 cells by targeting COL4A1. Cite this article: Q. S. Li, F. Y. Meng, Y. H. Zhao, C. L. Jin, J. Tian, X. J. Yi. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells. Bone Joint Res 2017;6:464-471. DOI: 10.1302/2046-3758.68.BJR-2016-0208.R2. © 2017 Yi et al.

Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate.

PubMed Central

Knox, J. D.; Cress, A. E.; Clark, V.; Manriquez, L.; Affinito, K. S.; Dalkin, B. L.; Nagle, R. B.

1994-01-01

The epithelial basal lamina composition and integrin expression profile of normal and neoplastic human prostate was characterized using immunohistochemical analysis of frozen samples. The major components of the basal lamina surrounding normal acini were laminin, type IV collagen, entactin, and type VII collagen with variable amounts of tenascin. The basal lamina of neoplastic acini had a similar composition, except for the loss of type VII collagen, which was observed in all grades of carcinoma. The basal cells of the normal prostate express the alpha 6-, beta 1-, and beta 4-integrin subunits, suggesting that both the alpha 6 beta 1- and alpha 6 beta 4-integrin complexes are formed. In prostate carcinoma there is a complete loss of beta 4 expression and the alpha 6- and beta 1-integrin subunits, which are restricted to the basal and basal lateral surfaces of basal cells, are distributed diffusely throughout the cytoplasmic membrane. The differential expression of type VII collagen and beta 4 are discussed in relationship to their possible role in tumor progression. Images Figure 1 Figure 2 Figure 3 PMID:8030747

Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-β expression.

PubMed

Siddiqui, Shabeena; Ahsan, Haseeb; Khan, Mohammad Rashid; Siddiqui, Waseem A

2013-12-01

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175-200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. © 2013.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siddiqui, Shabeena; Ahsan, Haseeb; Khan, Mohammad Rashid

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175–200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage overmore » a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. - Highlights: • The nephroprotective effect of TRF in type-2 diabetic rats was investigated. • Treatment with TRF improved glycemic status, lipid profile and renal functions in rats. • TRF down-regulated the expression of TGF-β, fibronectin and collagen in rats' kidney. • TRF ameliorated nephropathy by hypoglycemic, hypolipidemic and antioxidant activity. • Tocotrienols modulate the expression of TGF-β in DN in type-2 diabetic rats.« less

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis.

PubMed

Carvalho, S N; Lira, D C; Oliveira, G P; Thole, A A; Stumbo, A C; Caetano, C E; Marques, R G; Carvalho, L

2010-11-01

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.

PubMed

Bates, Andreia L; Pickup, Michael W; Hallett, Miranda A; Dozier, E Ashley; Thomas, Stacy; Fingleton, Barbara

2015-04-01

Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel explanation of corneal clouding in a bone marrow transplant-treated patient with Hurler syndrome

PubMed Central

Yuan, Ching; Bothun, Erick D.; Hardten, David R.; Tolar, Jakub; McLoon, Linda K.

2016-01-01

One common complication of mucopolysaccharidosis I-Hurler (MPS1-H) is corneal clouding, which occurs despite current treatments, including bone marrow transplantation. Human corneas were obtained from a 14 year old subject with MPS1-H and visual disability from progressive corneal clouding despite a prior bone marrow transplant at age 2. This was compared to a cornea from a 17 year old donated to our eye bank after his accidental death. The corneas were analyzed microscopically after staining with Alcian blue, antibodies to collagen I, IV, VI, and α-smooth muscle actin. Differences in levels of expression of the indicated molecules were assessed. Corneas from Hurler and control mice were examined similarly to determine potential mechanistic overlap. The MPS1-H subject cornea showed elevations in mucopolysaccharide deposition. The MPS1-H and Hurler mice corneas showed increased and disorganized expression of collagen I and IV relative to the control corneas. The MPS1-H corneas also showed increased and disordered expression of collagen VI. Positive expression of α-smooth muscle actin indicated myofibroblast conversion within the MPS1-H cornea in both the patient and mutant mouse material compared to normal human and control mouse cornea. Increased deposition of collagens and smooth muscle actin correlate with corneal clouding, providing a potential mechanism for corneal clouding despite bone marrow transplantation in MPS1-H patients. It might be possible to prevent or slow the onset of corneal clouding by treating the cornea with drugs known to prevent myofibroblast conversion. PMID:27235795

Genetics Home Reference: familial porencephaly

MedlinePlus

... one component of a protein called type IV collagen. Type IV collagen molecules attach to each other to form complex ... and support cells in many tissues. Type IV collagen networks play an important role in the basement ...

Genetics Home Reference: multicentric osteolysis, nodulosis, and arthropathy

MedlinePlus

... to cut (cleave) a protein called type IV collagen. Type IV collagen is a major structural component of basement membranes, ... enzyme, preventing the normal cleavage of type IV collagen. It is unclear how a loss of enzyme ...

Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-GBM autoantibodies specific for α345NC1 hexamers

PubMed Central

Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J.; Wasiluk, Andrew; Heidet, Laurence; Kitching, A. Richard; Borza, Dorin-Bogdan

2012-01-01

Goodpasture disease is an autoimmune kidney disease mediated by autoAbs against NC1 monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis. We identified a novel type of human IgG4-restricted anti-GBM autoAbs associated with mild non-progressive glomerulonephritis, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoAbs were investigated in mouse models recapitulating this phenotype. Wild type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoAbs specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause glomerulonephritis. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG antibodies specific for α3α4α5NC1 hexamers, which were not subclass restricted. As heterologous antigen in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a and IgG2b autoAbs specific for α345NC1 hexamers and induced anti-GBM Ab glomerulonephritis. These findings indicate that tolerance toward autologous intact α3α4α5(IV) collagen is established in hosts expressing this antigen, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α3α4α5NC1 hexamers. This provides a mechanism eliciting autoAbs specific for α345NC1 hexamers, which are restricted to non-inflammatory IgG subclasses and non-nephritogenic. In Alport syndrome, lack of tolerance toward α3α4α5(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including pro-inflammatory IgG subclasses which mediate post-transplant anti-GBM nephritis. PMID:23303673

Effect of a nutrient mixture on the localization of extracellular matrix proteins in HeLa human cervical cancer xenografts in female nude mice.

PubMed

Roomi, M Waheed; Cha, John; Kalinovsky, Tatiana; Roomi, Nusrath; Niedzwiecki, Aleksandra; Rath, Matthias

2015-09-01

Cervical cancer is one of the most commonly diagnosed cancers and a significant cause of mortality in women worldwide. Although cervical cancer is fully treatable in the early stages, once it has metastasized, patient outcome is poor. The objective of the present study was to investigate the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on the expression of extracellular matrix (ECM) proteins in HeLa cell xenografts in nude female mice. After housing for 1 week, female athymic nude mice between 5 and 6 weeks of age (n=12) were inoculated subcutaneously with 3×10 6 HeLa cells in phosphate-buffered saline and Matrigel and randomly divided into two groups. These were the control group, in which the mice were fed with regular mouse chow, and the NM group, in which the mice were fed with the regular diet supplemented with 0.5% NM (w/w). After 4 weeks, the tumors were excised and processed for histology. Tumor growth was evaluated and the tumors were stained for the ECM proteins collagen I, collagen IV, fibronectin, laminin, periodic acid-Schiff (PAS) and elastin. NM strongly inhibited (by 59%, P=0.001) the growth of HeLa xenografts in nude mice. Tumors from control mice exhibited little to no collagen I expression either internally or in the fibrous capsule, while tumors from the NM group expressed collagen I in the fibrous capsule and within the tumor. Tumors from the control group showed diffuse cytoplasmic and capsular collagen IV with abundant nucleated cells. NM treatment substantially increased collagen IV production and induced a dense fibrous network of collagen IV with chambers that surrounded live nucleated cells and large amounts of necrotic cell debris. Tumors from the mice fed with the NM exhibited a well-defined border of fibronectin in the capsule and intense areas of staining internally whereas control group tumors showed less overall fibronectin with sporadic internal staining and little in the fibrous capsule. Although laminin appeared abundantly in control and NM-treated tumors, the NM group tumors exhibited a chamber-like network of laminin internally. Tumors from the control group exhibited internal areas of intense PAS staining, whereas tumors from the NM-treated group exhibited a more uniform diffuse pattern of PAS staining. In conclusion, NM supplementation of HeLa xenograft-bearing female nude mice demonstrated a potent inhibition of tumor growth and enhancement of ECM proteins, suggesting the therapeutic value of this specific nutrient complex in the treatment of cervical cancer.

Genetics Home Reference: hereditary angiopathy with nephropathy, aneurysms, and muscle cramps syndrome

MedlinePlus

... one component of a protein called type IV collagen . Type IV collagen molecules attach to each other to form complex ... and support cells in many tissues. Type IV collagen networks play an important role in the basement ...

LOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer.

PubMed

Choi, Sul Ki; Kim, Hoe Suk; Jin, Tiefeng; Moon, Woo Kyung

2017-02-14

Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.

PubMed

Aouacheria, Abdel; Geourjon, Christophe; Aghajari, Nushin; Navratil, Vincent; Deléage, Gilbert; Lethias, Claire; Exposito, Jean-Yves

2006-12-01

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

Noninvasive in situ evaluation of osteogenic differentiation by time-resolved laser-induced fluorescence spectroscopy.

PubMed

Ashjian, Peter; Elbarbary, Amir; Zuk, Patricia; DeUgarte, Daniel A; Benhaim, Prosper; Marcu, Laura; Hedrick, Marc H

2004-01-01

The clinical implantation of bioengineered tissues requires an in situ nondestructive evaluation of the quality of tissue constructs developed in vitro before transplantation. Time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) is demonstrated here to noninvasively monitor the formation of osteogenic extracellular matrix (ECM) produced by putative stem cells (PLA cells) derived from human adipose tissue. We show that this optical spectroscopy technique can assess the relative expression of collagens (types I, III, IV, and V) within newly forming osteogenic ECM. The results are consistent with those obtained by conventional histochemical techniques (immunofluorescence and Western blot) and demonstrate that TR-LIFS is a potential tool for monitoring the expression of distinct collagen types and the formation of collagen cross-links in intact tissue constructs.

Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis.

PubMed

Nagai, Toshihiro; Sato, Masato; Kobayashi, Miyuki; Yokoyama, Munetaka; Tani, Yoshiki; Mochida, Joji

2014-09-18

Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection. First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group). Histologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group. Considering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.

Extracellular chloride signals collagen IV network assembly during basement membrane formation

PubMed Central

Cummings, Christopher F.; Pedchenko, Vadim; Brown, Kyle L.; Colon, Selene; Rafi, Mohamed; Jones-Paris, Celestial; Pokydeshava, Elena; Liu, Min; Pastor-Pareja, Jose C.; Stothers, Cody; Ero-Tolliver, Isi A.; McCall, A. Scott; Vanacore, Roberto; Bhave, Gautam; Santoro, Samuel; Blackwell, Timothy S.; Zent, Roy; Pozzi, Ambra

2016-01-01

Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. We report that extracellular Cl− ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl− in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins. Phylogenetic evidence indicates this conformational switch is a fundamental mechanism of collagen IV network assembly throughout Metazoa. Using recombinant triple helical protomers, we prove that NC1 domains direct both protomer and network assembly and show in Drosophila that NC1 architecture is critical for incorporation into basement membranes. These discoveries provide an atomic-level understanding of the dynamic interactions between extracellular Cl− and collagen IV assembly outside cells, a critical step in the assembly and organization of basement membranes that enable tissue architecture and function. Moreover, this provides a mechanistic framework for understanding the molecular pathobiology of NC1 domains. PMID:27216258

Ultrastructural sinusoidal changes in extrahepatic cholestasis. Light and electron microscopic immunohistochemical localization of collagen type III and type IV.

PubMed

Gulubova, M V

1996-07-01

Extrahepatic cholestasis causes excessive extracellular matrix formation perisinusoidally. Ito cells, transitional and endothelial cells are considered to be a source of extracellular matrix proteins in experimental cholestasis. The localization of collagens type III and type IV in human liver in extrahepatic cholestasis was investigated immunohistochemically in the present study. Immersion fixation was used after modification to be applied to surgical biopsies with commercially available kits. Sinusoidal changes were observed that indicated excessive collagen and matrix formation. Light microscopically, increased immunostaining with the two collagen antibodies was found perisinusoidally and portally. Ultrastructurally, collagen type III positive fibres were found beneath basement membranes of vessels, in collagen bundles and as a fibrillar network in the space of Disse. Collagen type IV immunostaining was located in portal tracts and near hepatocyte microvilli. Intracellular staining with collagen type IV was detected in the rough endoplasmic reticulum of some transitional cells. Immunostaining was located around transitional cells, Ito cells or endothelial cells mainly. Our study indicates that Ito cells, transitional and endothelial cells are the main source of collagens type III and IV in the space of Disse in extrahepatic cholestasis in humans.

Inhibitory effect of collagen-derived tripeptides on dipeptidylpeptidase-IV activity.

PubMed

Hatanaka, Tadashi; Kawakami, Kayoko; Uraji, Misugi

2014-12-01

The collagen tripeptide fragments Gly-Ala-Hyp, Gly-Pro-Ala and Gly-Pro-Hyp were generated by hydrolyzing collagen from pig-skin, cattle-skin, fish-scales and chicken-feet, respectively, with Streptomyces collagenase. Collagenase treatment increased the concentration of tripeptides in the hydrolysates by 13-15% (w/w). Of the three peptides, Gly-Pro-Hyp was a true peptidic inhibitor of dipeptidylpeptidase-IV (DPP-IV), because DPP-IV could not hydrolyze the bond between Pro-Hyp. This tripeptide was a moderately competitive inhibitor (Ki=4.5 mM) of DPP-IV, and its level in the collagen hydrolysates could be greatly increased (4-9% [w/w]) using Streptomyces collagenase.

Tissue-associated self-antigens containing exosomes: Role in allograft rejection.

PubMed

Sharma, Monal; Ravichandran, Ranjithkumar; Bansal, Sandhya; Bremner, Ross M; Smith, Michael A; Mohanakumar, T

2018-06-15

Exosomes are extracellular vesicles that express self-antigens (SAgs) and donor human leukocyte antigens. Tissue-specific exosomes can be detected in the circulation following lung, heart, kidney and islet cell transplantations. We collected serum samples from patients who had undergone lung (n = 30), heart (n = 8), or kidney (n = 15) transplantations to isolate circulating exosomes. Exosome purity was analyzed by Western blot, using CD9 exosome-specific markers. Tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV), were identified using western blot. Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2-fold) and Kα1T (37.1-fold) than stable. Exosomes isolated from heart transplant recipients diagnosed with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Further, Kidney transplant recipients diagnosed with transplant glomerulopathy had circulating exosomes with a 2-fold increased expression of fibronectin and 2.5-fold increase in Col-IV compared with stable. We conclude that circulating exosomes with tissue associated SAgs have the potential to be a noninvasive biomarker for allograft rejection. Copyright © 2018. Published by Elsevier Inc.

Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.

PubMed

López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M

2017-10-13

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Goodpasture's autoimmune disease - A collagen IV disorder.

PubMed

Pedchenko, Vadim; Richard Kitching, A; Hudson, Billy G

2018-05-12

Goodpasture's (GP) disease is an autoimmune disorder characterized by the deposition of pathogenic autoantibodies in basement membranes of kidney and lung eliciting rapidly progressive glomerulonephritis and pulmonary hemorrhage. The principal autoantigen is the α345 network of collagen IV, which expression is restricted to target tissues. Recent discoveries include a key role of chloride and bromide for network assembly, a novel posttranslational modification of the antigen, a sulfilimine bond that crosslinks the antigen, and the mechanistic role of HLA in genetic susceptibility and resistance to GP disease. These advances provide further insights into molecular mechanisms of initiation and progression of GP disease and serve as a basis for developing of novel diagnostic tools and therapies for treatment of Goodpasture's disease. Copyright © 2017. Published by Elsevier B.V.

PPARgamma agonists inhibit TGF-beta-PKA signaling in glomerulosclerosis.

PubMed

Zou, Rong; Xu, Gang; Liu, Xiao-cheng; Han, Min; Jiang, Jing-jing; Huang, Qian; He, Yong; Yao, Ying

2010-01-01

To study the probable mechanisms of the anti-glomerulosclerosis effects induced by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists in rat intraglomerular mesangial cells (MCs). Cells were transfected with the pTAL-PPRE-tk-Luc(+) plasmid and then treated with different concentrations of PPARgamma agonist, either troglitazone or telmisartan, for the indicated times. Promega luciferase assays were subsequently used for the detection of PPARgamma activation. Protein expression levels were assessed by Western blot, and PepTag assays were used for the non-radioactive detection of protein kinase A (PKA) activity. The deposition of alpha-smooth muscle actin (alpha-SMA) and p-cyclic AMP responsive element binding protein (pCREB) were analyzed by confocal laser scanning. Both troglitazone and telmisartan remarkably inhibit the PKA activation and pCREB expression that is stimulated by TGF-beta. The PPARgamma agonists also inhibited alpha-SMA and collagen IV protein expression by blocking PKA activation. PPARgamma ligands effectively suppress the activation of MCs and the accumulation of collagen IV stimulated by TGF-beta in vitro. The renal protection provided by PPARgamma agonists is partly mediated via their blockade of TGF-beta/PKA signaling.

Effect of the Direct Renin Inhibitor Aliskiren on Urinary Albumin Excretion in Spontaneous Type 2 Diabetic KK-A y Mouse

PubMed Central

Furukawa, Masako; Horikoshi, Satoshi; Funabiki, Kazuhiko; Tomino, Yasuhiko

2013-01-01

Objective. Although angiotensin II-mediated inflammation and extracellular matrix accumulation are considered to be associated with the progression of diabetic nephropathy, these processes have not yet been sufficiently clarified. The objective of this study was to determine whether the correction of the abnormal renal expression of MMPs and its inhibitors (MMPs/TIMPs) and cytokines following the administration of aliskiren to KK-A y mice results in a renoprotective effect. Methods. KK-A y mice were divided into two groups, that is, untreated (saline) and treated (aliskiren) groups. Systolic BP, HbA1c levels, and the albumin-creatinine ratio (ACR) were measured. The renal expression of MMPs/TIMPs, fibronectin, type IV collagen, MCP-1, and (pro)renin receptor ((P)RR) was examined using real-time PCR and/or immunohistochemical staining. Renal MAPK and NF-κB activity were also examined by Western blot analyses and ELISA, respectively. Results. Significant decreases in systolic BP and ACR levels were observed in treated KK-A y mice compared with the findings in untreated KK-A y mice. Furthermore, increases in MMPs/TIMPs, fibronectin, type IV collagen, MCP-1, and (P)RR expression, in addition to MAPK and NF-κB activity, were significantly attenuated by aliskiren administration. Conclusions. It appears that aliskiren improves albuminuria and renal fibrosis by regulating inflammation and the alteration of collagen synthesis and degradation. PMID:23819050

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects.

PubMed

Guzmán-Uribe, Daniela; Alvarado-Estrada, Keila Neri; Pierdant-Pérez, Mauricio; Torres-Álvarez, Bertha; Sánchez-Aguilar, Jesus Martin; Rosales-Ibáñez, Raúl

2017-01-01

The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

Effects of repeated social defeat on adolescent mice on cocaine-induced CPP and self-administration in adulthood: integrity of the blood-brain barrier.

PubMed

Rodríguez-Arias, Marta; Montagud-Romero, Sandra; Rubio-Araiz, Ana; Aguilar, María A; Martín-García, Elena; Cabrera, Roberto; Maldonado, Rafael; Porcu, Francesca; Colado, María Isabel; Miñarro, José

2017-01-01

Social stress in adulthood enhances cocaine self-administration, an effect that has been related with an increase in extracellular signal-regulated kinase and p38α mitogen-activated protein kinase phosphorylation. A detrimental effect of cocaine on blood-brain barrier (BBB) integrity has also been reported. This study evaluates the effects of repeated social defeat (RSD) during adolescence on the reinforcing and motivational effects of cocaine in adult mice and the changes induced by RSD on BBB permeability. Cocaine self-administration, conditioned place preference and quantitative analysis of claudin-5, laminin, collagen-IV and IgG immunoreactivity took place 3 weeks after RSD. Mice socially defeated during adolescence developed conditioned place preference and exhibited reinstated preference with a non-effective dose of cocaine (1 mg/kg). RSD mice needed significantly more sessions than control animals for the preference induced by 25 mg/kg of cocaine to be extinguished. However, acquisition of cocaine self-administration (0.5 mg/kg per injection) was delayed in the RSD group. Mice exposed to RSD displayed significant changes in BBB structure in adulthood, with a marked reduction in expression of the tight junction protein claudin-5 and an increase in basal laminin degradation (reflected by a decrease in laminin and collagen-IV expression) in the nucleus accumbens and hippocampus. The detrimental effect induced by cocaine (25 mg/kg) on collagen-IV expression in the hippocampus was more pronounced in RSD mice. In summary, our findings suggest that stress and cocaine can increase the long-term vulnerability of the brain to subsequent environmental insults as a consequence of a sustained disruption of the BBB. © 2015 Society for the Study of Addiction.

Hepatocyte growth factor: a regulator of extracellular matrix genes in mouse mesangial cells.

PubMed

Laping, N J; Olson, B A; Ho, T; Ziyadeh, F N; Albrightson, C R

2000-04-01

The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.

The effects of PPARδ agonist and zinc on ovariectomized rats' vagina.

PubMed

Takacs, Peter; Jaramillo, Sindy; Zhang, Yanping; Datar, Ram; Williams, Anthony; Olczyk, Joseph; Candiotti, Keith; Medina, Carlos A

2013-01-01

This study aimed to measure the effects of peroxisome proliferator-activated receptor-δ (PPARδ) agonist GW501516 (GW) and zinc sulfate (ZS) on ovariectomized rats' vaginal histomorphology and collagen expression. Two weeks after ovariectomy, rats received daily treatment with vaginal suppositories containing placebo, ZS, GW, ZS with GW, or estradiol for 2 weeks. Macroscopic measurements were taken and the midsection of the vagina was used for histology. Immunofluorescence was performed with antibodies against collagen I, III, and anti-actin or collagen I and V and anti-actin. Gene expression analysis of 3 collagen genes was performed by qRT-PCR. Macroscopic measurements revealed that the genital hiatus was narrower in the ZS and ZS with GW groups, and the vagina was significantly longer in the animals treated with GW, ZS with GW, and estradiol compared to the placebo group. Microscopic measurements of the vaginal layers showed that the lamina propria and the vaginal muscularis were significantly thicker in the ZS and ZS with GW group compared to the placebo.The ratio of vaginal Col1a1/Col3a1 mRNA expression was significantly up-regulated by ZS with GW compared to placebo, whereas the ratio of vaginal Col1a1/Col5a1 expression was significantly up-regulated by ZS, GW, and ZS with GW. The ratio of vaginal collagen I/III protein expression was significantly up-regulated by ZS and ZS with GW, whereas the ratio of vaginal collagen I/V expression was significantly up-regulated by estradiol, ZS, and ZS with GW compared to control. Vaginal suppositories containing zinc and PPARδ agonist significantly altered the vagina of ovariectomized rats.

Urinary type IV collagen is related to left ventricular diastolic function and brain natriuretic peptide in hypertensive patients with prediabetes.

PubMed

Iida, Masato; Yamamoto, Mitsuru; Ishiguro, Yuko S; Yamazaki, Masatoshi; Ueda, Norihiro; Honjo, Haruo; Kamiya, Kaichirou

2014-01-01

Urinary type IV collagen is an early biomarker of diabetic nephropathy. Concomitant prediabetes (the early stage of diabetes) was associated with left ventricular (LV) diastolic dysfunction and increased brain natriuretic peptide (BNP) in hypertensive patients. We hypothesized that urinary type IV collagen may be related to these cardiac dysfunctions. We studied hypertensive patients with early prediabetes (HbA1c <5.7% and fasting glucose >110, n=18), those with prediabetes (HbA1c 5.7-6.4, n=98), and those with diabetes (HbA1c>6.5 or on diabetes medications, n=92). The participants underwent echocardiography to assess left atrial volume/body surface area (BSA) and the ratio of early mitral flow velocity to mitral annular velocity (E/e'). Left ventricular diastolic dysfunction (LVDD) was defined if patients had E/e'≥15, or E/e'=9-14 accompanied by left atrial volume/BSA≥32ml/mm(2). Urinary samples were collected for type IV collagen and albumin, and blood samples were taken for BNP and HbA1c. Urinary type IV collagen and albumin increased in parallel with the deterioration of glycemic status. In hypertensive patients with prediabetes, subjects with LVDD had higher levels of BNP and urinary type IV collagen than those without LVDD. In contrast, in hypertensive patients with diabetes, subjects with LVDD had higher urinary albumin and BNP than those without LVDD. Urinary type IV collagen correlated positively with BNP in hypertensive patients with prediabetes, whereas it correlated with HbA1c in those with diabetes. In hypertensive patients with prediabetes, urinary type IV collagen was associated with LV diastolic dysfunction and BNP. Copyright © 2014 Elsevier Inc. All rights reserved.

Dendritic cells: In vitro culture in two- and three-dimensional collagen systems and expression of collagen receptors in tumors and atherosclerotic microenvironments.

PubMed

Sprague, Leslee; Muccioli, Maria; Pate, Michelle; Singh, Manindra; Xiong, Chengkai; Ostermann, Alexander; Niese, Brandon; Li, Yihan; Li, Yandi; Courreges, Maria Cecilia; Benencia, Fabian

2014-04-15

Dendritic cells (DCs) are immune cells found in the peripheral tissues where they sample the organism for infections or malignancies. There they take up antigens and migrate towards immunological organs to contact and activate T lymphocytes that specifically recognize the antigen presented by these antigen presenting cells. In the steady state there are several types of resident DCs present in various different organs. For example, in the mouse, splenic DC populations characterized by the co-expression of CD11c and CD8 surface markers are specialized in cross-presentation to CD8 T cells, while CD11c/SIRP-1α DCs seem to be dedicated to activating CD4 T cells. On the other hand, DCs have also been associated with the development of various diseases such as cancer, atherosclerosis, or inflammatory conditions. In such disease, DCs can participate by inducing angiogenesis or immunosuppression (tumors), promoting autoimmune responses, or exacerbating inflammation (atherosclerosis). This change in DC biology can be prompted by signals in the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells. Building on those studies, herewith we analyzed the angiogenic profile of murine myeloid DCs upon interaction with 2D and 3D type-I collagen environments. As determined by PCR array technology and quantitative PCR analysis we observed that interaction with these collagen environments induced the expression of particular angiogenic molecules. In addition, DCs cultured on collagen environments specifically upregulated the expression of CXCL-1 and -2 chemokines. We were also able to establish DC cultures on type-IV collagen environments, a collagen type expressed in pathological conditions such as atherosclerosis. When we examined DC populations in atherosclerotic veins of Apolipoprotein E deficient mice we observed that they expressed adhesion molecules capable of interacting with collagen. Finally, to further investigate the interaction of DCs with collagen in other pathological conditions, we determined that both murine ovarian and breast cancer cells express several collagen molecules that can contribute to shape their particular tumor microenvironment. Consistently, tumor-associated DCs were shown to express adhesion molecules capable of interacting with collagen molecules as determined by flow cytometry analysis. Of particular relevance, tumor-associated DCs expressed high levels of CD305/LAIR-1, an immunosuppressive receptor. This suggests that signaling through this molecule upon interaction with collagen produced by tumor cells might help define the poorly immunogenic status of these cells in the tumor microenvironment. Overall, these studies demonstrate that through interaction with collagen proteins, DCs can be capable of modifying the microenvironments of inflammatory disease such as cancer or atherosclerosis. Copyright © 2014. Published by Elsevier Inc.

Lysyl oxidase interacts with AGE signalling to modulate collagen synthesis in polycystic ovarian tissue

PubMed Central

Papachroni, Katerina K; Piperi, Christina; Levidou, Georgia; Korkolopoulou, Penelope; Pawelczyk, Leszek; Diamanti-Kandarakis, Evanthia; Papavassiliou, Athanasios G

2010-01-01

Abstract Connective tissue components – collagen types I, III and IV – surrounding the ovarian follicles undergo drastic changes during ovulation. Abnormal collagen synthesis and increased volume and density of ovarian stroma characterize the polycystic ovary syndrome (PCOS). During the ovulatory process, collagen synthesis is regulated by prolyl hydroxylase and lysyl oxidase (LOX) activity in ovarian follicles. LOX catalyzes collagen and elastin cross-linking and plays essential role in coordinating the control of ovarian extracellular matrix (ECM) during follicular development. We have recently shown accumulation of advanced glycation end products (AGEs), molecules that stimulate ECM production and abnormal collagen cross-linking, in ovarian tissue. However, the possible link between LOX and AGEs-induced signalling in collagen production and stroma formation in ovarian tissue from PCOS remains elusive. The present study investigates the hypothesis of AGE signalling pathway interaction with LOX gene activity in polycystic ovarian (PCO) tissue. We show an increased distribution and co-localization of LOX, collagen type IV and AGE molecules in the PCO tissue compared to control, as well as augmented expression of AGE signalling mediators/effectors, phospho(p)-ERK, phospho(p)-c-Jun and nuclear factor κB (NF-κB) in pathological tissue. Moreover, we demonstrate binding of AGE-induced transcription factors, NF-κB and activator protein-1 (AP-1) on LOX promoter, indicating a possible involvement of AGEs in LOX gene regulation, which may account for the documented increase in LOX mRNA and protein levels compared to control. These findings suggest that deposition of excess collagen in PCO tissue that induces cystogenesis may, in part, be due to AGE-mediated stimulation of LOX activity. PMID:19583806

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

PubMed

Rubio-Araiz, Ana; Perez-Hernandez, Mercedes; Urrutia, Andrés; Porcu, Francesca; Borcel, Erika; Gutierrez-Lopez, Maria Dolores; O'Shea, Esther; Colado, Maria Isabel

2014-08-01

The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1β levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.

Halogens are key cofactors in building of collagen IV scaffolds outside the cell.

PubMed

Brown, Kyle L; Hudson, Billy G; Voziyan, Paul A

2018-05-01

The purpose of this review is to highlight recent advances in understanding the molecular assembly of basement membranes, as exemplified by the glomerular basement membrane (GBM) of the kidney filtration apparatus. In particular, an essential role of halogens in the basement membrane formation has been discovered. Extracellular chloride triggers a molecular switch within non collagenous domains of collagen IV that induces protomer oligomerization and scaffold assembly outside the cell. Moreover, bromide is an essential cofactor in enzymatic cross-linking that reinforces the stability of scaffolds. Halogenation and halogen-induced oxidation of the collagen IV scaffold in disease states damage scaffold function. Halogens play an essential role in the formation of collagen IV scaffolds of basement membranes. Pathogenic damage of these scaffolds by halogenation and halogen-induced oxidation is a potential target for therapeutic interventions.

Superoxide overproduction and kidney fibrosis: a new animal model

PubMed Central

Guimarães-Souza, Nadia Karina; Yamaleyeva, Liliya Marsovna; Lu, Baisong; Ramos, Ana Claudia Mallet de Souza; Bishop, Colin Edward; Andersson, Karl Erik

2015-01-01

Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. PMID:25993073

Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.

PubMed

Malone, Andrew F; Funk, Steven D; Alhamad, Tarek; Miner, Jeffrey H

2017-06-01

Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.

Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome

PubMed Central

Malone, Andrew F.; Funk, Steven D.; Alhamad, Tarek; Miner, Jeffrey H.

2016-01-01

Introduction Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. Methods We analyzed targeted next-generation sequencing results of an individual with Alport syndrome and confirmed results by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant’s effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. Results A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. We demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. Conclusions Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance. PMID:28013382

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

PubMed

Wang, Dongmao; Mohammad, Mardhiah; Wang, Yanyan; Tan, Rachel; Murray, Lydia S; Ricardo, Sharon; Dagher, Hayat; van Agtmael, Tom; Savige, Judy

2017-07-01

X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL 2 ) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 ( P  = 0.01 and P  = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels ( P  < 0.01), and reduced ER size ( P  < 0.01 by EM and P  < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P  < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only ( P  = 0.06). Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

Extracellular matrix components expression in human pluripotent stem cell-derived retinal organoids recapitulates retinogenesis in vivo and reveals an important role for IMPG1 and CD44 in the development of photoreceptors and interphotoreceptor matrix.

PubMed

Felemban, Majed; Dorgau, Birthe; Hunt, Nicola Claire; Hallam, Dean; Zerti, Darin; Bauer, Roman; Ding, Yuchun; Collin, Joseph; Steel, David; Krasnogor, Natalio; Al-Aama, Jumana; Lindsay, Susan; Mellough, Carla; Lako, Majlinda

2018-05-17

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis. The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1 & IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation. Copyright © 2018 Acta Materialia Inc. All rights reserved.

Vitronectin-Based, Biomimetic Encapsulating Hydrogel Scaffolds Support Adipogenesis of Adipose Stem Cells

PubMed Central

Clevenger, Tracy N.; Hinman, Cassidy R.; Ashley Rubin, Rebekah K.; Smither, Kate; Burke, Daniel J.; Hawker, Craig J.; Messina, Darin; Van Epps, Dennis

2016-01-01

Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as unpredictable volume loss and reabsorption. Human adipose-derived stem cells (ASCs), isolated from liposuction aspirate have great potential for use in soft tissue regeneration, especially when combined with a supportive scaffold. To design scaffolds that promote differentiation of these cells down an adipogenic lineage, we characterized changes in the surrounding extracellular environment during adipogenic differentiation. We found expression changes in both extracellular matrix proteins, including increases in expression of collagen-IV and vitronectin, as well as changes in the integrin expression profile, with an increase in expression of integrins such as αVβ5 and α1β1. These integrins are known to specifically interact with vitronectin and collagen-IV, respectively, through binding to an Arg-Gly-Asp (RGD) sequence. When three different short RGD-containing peptides were incorporated into three-dimensional (3D) hydrogel cultures, it was found that an RGD-containing peptide derived from vitronectin provided strong initial attachment, maintained the desired morphology, and created optimal conditions for in vitro 3D adipogenic differentiation of ASCs. These results describe a simple, nontoxic encapsulating scaffold, capable of supporting the survival and desired differentiation of ASCs for the treatment of soft tissue defects. PMID:26956095

Matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in paediatric burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors.

PubMed

Weremijewicz, Artur; Matuszczak, Ewa; Sankiewicz, Anna; Tylicka, Marzena; Komarowska, Marta; Tokarzewicz, Anna; Debek, Wojciech; Gorodkiewicz, Ewa; Hermanowicz, Adam

2018-06-01

The purpose of this study was the determination of matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in the blood plasma of burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors. 31 children scalded by hot water who were managed at the Department of Paediatric Surgery between 2014-2015, after primarily presenting with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2,5+1 years). There were 10 girls and 21 boys. Venous blood samples were drawn 2-6h, and 12-16h after the thermal injury, and on the subsequent days 3, 5 and 7. The matrix metalloproteinase-2, collagen type IV and laminin-5 concentrations were assessed using Surface Plasmon Resonance Imaging by the investigators blinded to the other data. The MMP-2, laminin-5 and collagen type IV concentrations in the blood plasma of patients with burns, were highest 12-16h after thermal injury, the difference was statistically significant. The MMP-2, laminin-5 and collagen type IV concentrations measured 3 days, 5 days and 7 days after the thermal injury, slowly decreased over time, and on the 7th day reached the normal range, when compared with the concentration measured in controls. Current work is the first follow-up study regarding MMP-2 in burns. MMP-2, laminin-5 and collagen type IV levels were elevated early after burn injury in the plasma of studied patients, and were highest 12-16h after the injury. MMP-2, laminin-5 and collagen type IV levels were not proportional to the severity of the burn. We believe in the possibility that the gradual decrease of MMP-2, collagen type IV and laminin-5 concentrations could be connected with the process of healing, but to prove it, more investigation is needed in this area. The SPR imaging biosensor is a good diagnostic tool for determination of MMP-2, laminin-5 and collagen type IV in blood plasma of patients with burns. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

Role of 17 beta-estradiol on type IV collagen fibers volumetric density in the basement membrane of bladder wall.

PubMed

de Fraga, Rogerio; Dambros, Miriam; Miyaoka, Ricardo; Riccetto, Cássio Luís Zanettini; Palma, Paulo César Rodrigues

2007-10-01

The authors quantified the type IV collagen fibers volumetric density in the basement membrane of bladder wall of ovariectomized rats with and without estradiol replacement. This study was conducted on 40 Wistar rats (3 months old) randomly divided in 4 groups: group 1, remained intact (control); group 2, submitted to bilateral oophorectomy and daily replacement 4 weeks later of 17 beta-estradiol for 12 weeks; group 3, sham operated and daily replacement 4 weeks later of sesame oil for 12 weeks; and group 4, submitted to bilateral oophorectomy and killed after 12 weeks. It was used in immunohistochemistry evaluation using type IV collagen polyclonal antibody to stain the fibers on paraffin rat bladder sections. The M-42 stereological grid system was used to analyze the fibers. Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rat bladder wall. Estradiol replacement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Surgical castration performed on rats induced an increasing volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall and the estradiol treatment had a significant effect in keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall.

Contribution of alpha3(IV)alpha4(IV)alpha5(IV) Collagen IV to the Mechanical Properties of the Glomerular Basement Membrane

NASA Astrophysics Data System (ADS)

Gyoneva, Lazarina

The glomerular basement membrane (GBM) is a vital part of the blood-urine filtration barrier in the kidneys. In healthy GBMs, the main tension-resisting component is alpha3(IV)alpha4(IV)alpha5(IV) type IV collagen, but in some diseases it is replaced by other collagen IV isoforms. As a result, the GBM becomes leaky and disorganized, ultimately resulting in kidney failure. Our goal is to understanding the biomechanical aspects of the alpha3(IV)alpha4(IV)alpha5(IV) chains and how their absence could be responsible for (1) the initial injury to the GBM and (2) progression to kidney failure. A combination of experiments and computational models were designed for that purpose. A model basement membrane was used to compare experimentally the distensibility of tissues with the alpha3(IV)alpha4(IV)alpha5(IV) chains present and missing. The experiments showed basement membranes containing alpha3(IV)alpha4(IV)alpha5(IV) chains were less distensible. It has been postulated that the higher level of lateral cross-linking (supercoiling) in the alpha3(IV)alpha4(IV)alpha5(IV) networks contributes additional strength/stability to basement membranes. In a computational model of supercoiled networks, we found that supercoiling greatly increased the stiffness of collagen IV networks but only minimally decreased the permeability, which is well suited for the needs of the GBM. It is also known that the alpha3(IV)alpha4(IV)alpha5(IV) networks are more protected from enzymatic degradation, and we explored their significance in GBM remodeling. Our simulations showed that the more protected network was needed to prevent the system from entering a dangerous feedback cycle due to autoregulation mechanisms in the kidneys. Overall, the work adds to the evidence of biomechanical differences between the alpha3(IV)alpha4(IV)alpha5(IV) networks and other collagen IV networks, points to supercoiling as the main source of biomechanical differences, discusses the suitability of alpha3(IV)alpha4(IV)alpha5(IV) networks to meet the mechanics and permeability needs of the GBM, and explores the role of biomechanics and enzymatic digestion in GBM remodeling.

Deferiprone attenuates inflammation and myocardial fibrosis in diabetic cardiomyopathy rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zou, Chunbo; Liu, Xiaogang; Xie, Rujuan

We attempted to investigate the therapeutic effects of deferiprone on DC rats and explore the underlying mechanism. Total 24 6-week-old male Wistar rats (weighing from 180 g to 220 g) were subjected to DC model construction and then randomly divided to three groups (8 rats per group): DC group, DC + 50 mg, and DC + 100 mg deferiprone treatment group. The 8 normal rats were considered as controls. After deferiprone treatment for 20 weeks, the blood samples were collected for the biochemical parameters test, including fasting glucose, HOMA-IR (homeostasis model assessment of the insulin resistance), serum iron, ferritin and transferrin saturation (TS). The oxidative stress was assessedmore » by detecting the level of malondialdehyde (MDA) and superoxide dismutase (SOD). Histopathologic changes were determined by Masson's trichrome staining and electron microscopy imaging. The expression levels of NF-κB (nuclear factor kappa B), COX2 (cytochrome c oxidase), tenascin C, collagen IV were measured by RT-PCR and western blotting. The expression of nitrotyrosine and MCP-1 (monocyte chemotactic protein 1) were determined by immunohistochemistry. Deferiprone treatment reduced iron deposition and IR in DC rats except for blood glucose. After deferiprone treatment, MDA level was significantly decreased and SOD level was increased significantly. The level of NF-κB, cyclooxygenase-2, tenascin C, collagen IV MCP-1 and nitrotyrosine were significantly reduced. There was no significant difference in the effect of deferiprone at 50 and 100 mg doses. Deferiprone showed therapeutic effects on DC by regulating the pro-inflammatory and pro-fibrotic factors. - Highlights: • The expression of serum iron, ferritin and TS were elevated in DC rats. • Oxidative stress related MDA and SOD were upregulated in DC rats. • NF-κB, COX2, tenascin C, collagen IV were accumulated in DC rats. • All the changes were reversed by deferiprone treatment.« less

Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

PubMed

Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

2016-01-01

The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that aberrant structure and function of the COL4A1 protein may also be a significant factor in modulating immunity.

Detection of altered extracellular matrix in surface layers of unstable carotid plaque: an optical spectroscopy, birefringence and microarray genetic analysis.

PubMed

Korol, Renee M; Canham, Peter B; Liu, Li; Viswanathan, Kasinath; Ferguson, Gary G; Hammond, Rob R; Finlay, Helen M; Baker, Henry V; Lopez, Cecilia; Lucas, Alexandra R

2011-01-01

Erosion and rupture of surface layers in atherosclerotic plaque can cause heart attack and stroke; however, changes in luminal surface composition are incompletely defined. Laser-induced fluorescence spectroscopy (LIFS), with limited tissue penetration, was used to investigate the surface of unstable carotid plaque and correlated with microscopy, birefringence and gene expression. Arterial matrix collagens I, III and elastin were assessed in unstable plaques (n = 25) and reference left internal mammary arteries (LIMA, n = 10). LIFS in addition to selective histological staining with picrosirius red, Movat pentachrome and immunostaining revealed decreased elastin and increased collagen I and III (P < 0.05) in carotid plaque when compared with LIMA. Within plaque, collagen I was elevated in the internal carotid region versus the common carotid region. Polarized light microscopy detected layers of aligned collagen and associated mechanical rigidity of the fibrous cap. Microarray analysis of three carotid and three LIMA specimens confirmed up-regulation of collagen I, III and IV, lysyl oxidase and MMP-12. In conclusion, LIFS analysis coupled with microscopy revealed marked regional differences in collagen I, III and elastin in surface layers of carotid plaque; indicative of plaque instability. Birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in ECM gene expression. © 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.

In vitro 3D full thickness skin equivalent tissue model using silk and collagen biomaterials

PubMed Central

Bellas, Evangelia; Seiberg, Miri; Garlick, Jonathan; Kaplan, David L.

2013-01-01

Current approaches to develop skin equivalents often only include the epidermal and dermal components. Yet, full thickness skin includes the hypodermis, a layer below the dermis of adipose tissue containing vasculature, nerves and fibroblasts, necessary to support the epidermis and dermis. In the present study, we developed a full thickness skin equivalent including an epidermis, dermis and hypodermis that could serve as an in vitro model for studying skin development, disease or as a platform for consumer product testing as a means to avoid animal testing. The full thickness skin equivalent was easy to handle and was maintained in culture for greater than 14 days while expressing physiologically relevant morphologies of both the epidermis and dermis, as seen by keratin 10, collagen I and collagen IV expression. The skin equivalent produced glycerol and leptin, markers of adipose tissue metabolism. This work serves as a foundation for our understanding of some of the necessary factors needed to develop a stable, functional model of full-thickness skin. PMID:23161763

Reduced immunohistochemical expression of adhesion molecules in vitiligo skin biopsies.

PubMed

Reichert Faria, Adriane; Jung, Juliana Elizabeth; Silva de Castro, Caio César; de Noronha, Lucia

2017-03-01

Because defects in adhesion impairment seem to be involved in the etiopathogenesis of vitiligo, this study aimed to compare the immunohistochemical expression of several adhesion molecules in the epidermis of vitiligo and non lesional vitiligo skin. Sixty-six specimens of lesional and non lesional skin from 33 volunteers with vitiligo were evaluated by immunohistochemistry using anti-beta-catenin, anti-E-cadherin, anti-laminin, anti-beta1 integrin, anti-collagen IV, anti-ICAM-1 and anti-VCAM-1 antibodies. Biopsies of vitiligo skin demonstrated a significant reduction in the expression of laminin and integrin. The average value of the immunohistochemically positive reaction area of the vitiligo specimens was 3053.2μm 2 , compared with the observed value of 3431.8μm 2 in non vitiligo skin (p=0.003) for laminin. The immuno-positive area was 7174.6μm 2 (vitiligo) and 8966.7μm 2 (non lesional skin) for integrin (p=0.042). A reduction in ICAM-1 and VCAM-1 expression in the basal layer of the epidermis in vitiligo samples was also observed (p=0.001 and p<0.001, respectively). However, no significant differences were observed with respect to the expression of beta-catenin, E-cadherin, and collagen IV between vitiligo and non lesional skin. Our results suggest that an impairment in adhesion exists in vitiligo skin, which is supported by the diminished immunohistochemical expression of laminin, beta1 integrin, ICAM-1 and VCAM-1. Copyright © 2017 Elsevier GmbH. All rights reserved.

Human soluble phospholipase A2 receptor is an inhibitor of the integrin-mediated cell migratory response to collagen-I.

PubMed

Watanabe, Kazunori; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-Ei; Kugiyama, Kiyotaka

2018-05-23

Murine membrane-bound phospholipase A 2 receptor 1 (PLA 2 R) is shed and released into plasma in a soluble form that retains all of the extracellular domains. Relatively little is known about human PLA 2 R. This study examined whether human soluble PLA 2 R may have biological functions and whether soluble PLA 2 R may exist in human plasma. Here, we showed that human recombinant soluble PLA 2 R (rsPLA 2 R) bound to collagen-I and inhibited interaction of collagen-I with the extracellular domain of integrin β1 on the cell surface of HEK293 cells. As a result, rsPLA 2 R suppressed integrin β1-mediated migratory responses of HEK293 cells to collagen-I in Boyden chamber experiments. Inhibition of phosphorylation of FAK Tyr397 was also observed. Similar results were obtained with experiments using soluble PLA 2 R released from HEK293 cells transfected with a construct encoding human soluble PLA 2 R. rsPLA 2 R lacking the fibronectin-like type II (FNII) domain had no inhibitory effects on cell responses to collagen-I, suggesting an important role of the FNII domain in the interaction of rsPLA 2 R with collagen-I. In addition, rsPLA 2 R suppressed the migratory response to collagen-IV and binding of collagen-IV to the cell surface of human podocytes that endogenously express membrane-bound full-length PLA 2 R. Immunoprecipitation and Western blotting showed the existence of immuno-reactive PLA 2 R in human plasma. In conclusion, human recombinant soluble PLA 2 R inhibits integrin β1-mediated cell responses to collagens. Further studies are warranted to elucidate whether immuno-reactive PLA 2 R in human plasma has the same properties as rsPLA 2 R.

Transient expression of collagen type II at epitheliomesenchymal interfaces during morphogenesis of the cartilaginous neurocranium.

PubMed

Thorogood, P; Bee, J; von der Mark, K

1986-08-01

In the avian embryo a matrix-mediated tissue interaction between retinal pigmented epithelium and neural crest-derived periocular mesenchyme leads to the differentiation of (scleral) cartilage. The composition of the extracellular matrix at the interface between these two tissues has been examined immunohistochemically, both during and after the interaction has taken place. Of the matrix components studied (fibronectin, laminin, and collagen types I, II, IV, and V) only collagen type II displayed a dramatic change in distribution between the two stages. During the interaction, at stage 15, type II was present in the extracellular compartment basal to the epithelium. After completion of the interaction, collagen type II was no longer detectable at the interface even though it was readily detectable in the vitreous humor, cornea, and perinotochordal sheath, and subsequently will be expressed by the chondrogenic tissue itself as overt differentiation commences. These results suggest that collagen type II might be causally involved in this particular epitheliomesenchymal interaction. Examination of the spatial and temporal patterns of collagen type II expression elsewhere in the developing craniofacial complex revealed a hitherto unreported pattern of distribution. In addition to its predictable locations (i.e., cornea, vitreous, and perinotochordal sheath) it was found to be present at certain other sites, for example, at the basal surfaces of some neuroepithelia. These additional locations are all known to be sites of chondrogenesis-promoting tissue interactions which result in the formation of the elements of the cartilaginous neurocranium (e.g., otic vesicle). Furthermore this spatial distribution exhibits a changing temporal pattern in that it is detectable at the time that the interactions are known to be taking place, but subsequently is no longer detectable by the immunohistochemical means employed. This definable pattern of transient collagen type II expression, occurring at very early stages of craniofacial development, is interpreted as reflecting one level of morphogenetic specification of chondrocranial/skull form in the developing vertebrate head.

Alport Syndrome Diagnosis

MedlinePlus

... the presence or absence of the type IV collagen alpha-3, alpha-4 and alpha-5 chains ( ... linked Alport syndrome) is suspected. The type IV collagen alpha-5 chain (COL4A5) is normally present in ...

Immunohistochemical assessment of collagen types I, III, IV and VI in biopsy samples of the bovine uterine wall collected during the oestrous cycle.

PubMed

Boos, A

2000-01-01

Uterine biopsies were collected at cycle days 1 (oestrous), 8, 15 and 19 in six cows. Unfixed cryostat sections were used to immunolocalise collagen types I, III, IV and VI by an indirect FITC method. Collagen I was sparsely found in the endometrium where it formed a fine meshwork of thin fibres directly below the surface epithelium, clearly visible only at cycle days 8 and 15. Collagen III formed the bulk of connective tissue fibres and was arranged in fine aggregates within the superficial endometrial stroma, while in the deeper areas it consisted of many thick fibre bundles. Collagen IV was found in basement membranes underlying all endometrial epithelia. Furthermore, it surrounded smooth muscle cells of blood vessels. A few single fibrils also stained positively within the endometrial stroma, more numerous at cycle days 1 and 19 as compared to days 8 and 15. Collagen VI formed a mesh of fine and pericellularly situated fibrils within the endometrial stroma. The contribution of the collagen types studied to the connective tissue of caruncles, blood vessels, lymph follicles, and myometrium is also reported. The results of the present study indicate that the connective tissue of the bovine uterine wall is composed of different collagen types, which exhibit a characteristic distribution pattern each. The day of cycle may influence amounts and organisation of collagen types I and IV as demonstrated here at the light-microscopical level. Copyright 2000 S. Karger AG, Basel

Biomimetic collagen I and IV double layer Langmuir-Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells.

PubMed

Sorkio, Anni E; Vuorimaa-Laukkanen, Elina P; Hakola, Hanna M; Liang, Huamin; Ujula, Tiina A; Valle-Delgado, Juan José; Österberg, Monika; Yliperttula, Marjo L; Skottman, Heli

2015-05-01

The environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaefer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruch's membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on air-subphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of small molecular weight substance. Maturation and functionality of hESC-RPE cells on double layer collagen LS films was further assessed by RPE-specific gene and protein expression, growth factor secretion, and phagocytic activity. Here, we demonstrated that the prepared collagen LS films have layered structure with oriented fibers corresponding to architecture of the uppermost layers of Bruch's membrane and result in increased barrier properties and functionality of hESC-RPE cells as compared to the commonly used dip-coated controls. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anti-atherogenic activity of wild grape (Vitis thunbergii) extract antagonizing smooth muscle cell proliferation and migration promoted by neighboring macrophages.

PubMed

Kang, Sang-Wook; Kim, Min Soo; Kim, Hyun-Sung; Lee, Yong-Jin; Kang, Young-Hee

2012-06-01

The proliferation and migration of vascular smooth muscle cells (SMCs) play critical roles in intimal thickening and neointimal hyperplasia in early-phase atherosclerosis. This study tested whether wild grape extract (WGE) suppressed the proliferation and migration of human aortic SMCs induced by neighboring macrophages. Cellular expression of fibrogenic connective tissue growth factor (CTGF) and secretion of collagen IV and matrix metalloproteinase (MMP)-2 were determined in SMCs exposed to THP-1-differentiated macrophage-conditioned media. Proliferation was enhanced in SMCs exposed to macrophage-conditioned media collected during the early stage of differentiation, which was attenuated by treatment with ≥ 10 µg/ml WGE. Increased secretion of CTGF and collagen IV macrophage-conditioned media was suppressed in WGE-supplemented SMCs. TGF-β1-promoted production of CTGF and collagen IV was suppressed by blocking TGF-β receptors of R1 and R2 in SMCs. WGE repressed macrophage-conditioned media-upregulated MMP-2 secretion, indicating that WGE had an ability to encumber plaque rupture within atherosclerotic lesions. In addition, ≥ 1 µg/ml WGE ameliorated the migration of SMCs promoted by neighboring macrophages. These results demonstrate that WGE retarded neointimal hyperplasia and thickening within atherosclerotic plaques largely comprising of macrophages and SMCs. Therefore, WGE may be developed as an anti-proliferative and anti-migratory agent targeting SMCs in the proximity of newly differentiated and resident macrophages.

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster.

PubMed

Shahab, Jaffer; Baratta, Cristina; Scuric, Bianca; Godt, Dorothea; Venken, Koen J T; Ringuette, Maurice J

2015-04-01

SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV. © 2014 Wiley Periodicals, Inc.

DR-nm23 expression affects neuroblastoma cell differentiation, integrin expression, and adhesion characteristics.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

2001-01-01

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Losartan improves resistance artery lesions and prevents CTGF and TGF-beta production in mild hypertensive patients.

PubMed

Gómez-Garre, D; Martín-Ventura, J L; Granados, R; Sancho, T; Torres, R; Ruano, M; García-Puig, J; Egido, J

2006-04-01

Although structural and functional changes of resistance arteries have been proposed to participate in arterial hypertension (HTA) outcome, not all therapies may correct these alterations, even if they normalize the blood pressure (BP). The aim of this study was to investigate the mechanisms of the protection afforded by the angiotensin receptor antagonist losartan in resistance arteries from patients with essential HTA. In all, 22 untreated hypertensive patients were randomized to receive losartan or amlodipine for 1 year and the morphological characteristics of resistance vessels from subcutaneous biopsies were evaluated. Protein expression of connective tissue growth factor (CTGF), transforming growth factor beta (TGF-beta), and collagens III and IV was detected by immunohistochemistry. In comparison with normotensive subjects, resistance arteries from hypertensive patients showed a significant media:lumen (M/L) ratio increment and a higher protein expression of CTGF, TGF-beta, and collagens. After 1 year of treatment, both losartan and amlodipine similarly controlled BP. However, M/L only decreased in patients under losartan treatment, whereas in the amlodipine-treated group this ratio continued to increase significantly. The administration of losartan prevented significant increments in CTGF, TGF-beta, and collagens in resistance arteries. By contrast, amlodipine-treated patients showed a higher vascular CTGF, TGF-beta, and collagen IV staining than before treatment. Our results show that the administration of losartan, but not amlodipine, to hypertensive patients improves structural abnormalities and prevents the production of CTGF and TGF-beta in small arteries, despite similar BP lowering. These data may explain the molecular mechanisms of the better vascular protection afforded by drugs interfering with the renin-angiotensin system.

The unique luminal staining pattern of cytokeratin 5/6 in adenoid cystic carcinoma of the breast may aid in differentiating it from its mimickers.

PubMed

Nakai, Tokiko; Ichihara, Shu; Kada, Akiko; Ito, Noriko; Moritani, Suzuko; Kawasaki, Tomonori; Uchiyama, Tomoko; Itami, Hiroe; Morita, Kouhei; Takano, Masato; Takeda, Maiko; Hatakeyama, Kinta; Ohbayashi, Chiho

2016-08-01

Adenoid cystic carcinoma (AdCC) of the breast is an uncommon but distinct neoplasm composed of a dual cell population polarized around true glandular (luminal) spaces and pseudolumina. The aim of this study was to clarify whether various immunohistochemical markers (CK7, EMA, CD117, p63, calponin, CD10, S100, CK5/6, CK14, vimentin, and type IV collagen) can distinguish between the two cell types in classical AdCC (n = 14) and in collagenous spherulosis (n = 5). The sensitivity and specificity of these 11 markers to distinguish luminal from abluminal cells were evaluated using a curve created by plotting the true-positive rate (sensitivity) against the false-positive rate (1 - specificity) at threshold settings of 0, 10, 50, and 70 %. The most sensitive and specific markers for luminal cells in AdCC were CK7 and EMA; those for abluminal cells were type IV collagen, p63, and vimentin. CD10 and S100 did not act as abluminal markers in AdCC. CK5/6, one of the basal/myoepithelial markers, was expressed more frequently in luminal than in abluminal cells of AdCC. Thus, CK5/6 immunostaining resulted in a reverse expression pattern, analogous to what we recently documented in clear cells in mammary adenomyoepithelioma. In conclusion, compared with myoepithelial/abluminal cells of normal breast or collagenous spherulosis, the neoplastic abluminal cells of classical AdCC are characterized by enhanced vimentin and attenuated CD10 and S100. Furthermore, the luminal cells of AdCC show a unique aberrant staining pattern for CK5/6 that may aid in the differential diagnosis.

Loss of Matrix Metalloproteinase-2 Amplifies Murine Toxin-Induced Liver Fibrosis by Upregulating Collagen I Expression

PubMed Central

Radbill, Brian D.; Gupta, Ritu; Ramirez, Maria Celeste M.; DiFeo, Analisa; Martignetti, John A.; Alvarez, Carlos E.; Friedman, Scott L.; Narla, Goutham; Vrabie, Raluca; Bowles, Robert; Saiman, Yedidya

2010-01-01

Background and Aims Matrix metalloproteinase-2 (MMP-2), a type IV collagenase secreted by activated hepatic stellate cells (HSCs), is upregulated in chronic liver disease and is considered a profibrotic mediator due to its proliferative effect on cultured HSCs and ability to degrade normal liver matrix. Although associative studies and cell culture findings suggest that MMP-2 promotes hepatic fibrogenesis, no in vivo model has definitively established a pathologic role for MMP-2 in the development and progression of liver fibrosis. We therefore examined the impact of MMP-2 deficiency on liver fibrosis development during chronic CCl4 liver injury and explored the effect of MMP-2 deficiency and overexpression on collagen I expression. Methods Following chronic CCl4 administration, liver fibrosis was analyzed using Sirius Red staining with quantitative morphometry and real-time polymerase chain reaction (PCR) in MMP-2−/− mice and age-matched MMP-2+/+ controls. These studies were complemented by analyses of cultured human stellate cells. Results MMP-2−/− mice demonstrated an almost twofold increase in fibrosis which was not secondary to significant differences in hepatocellular injury, HSC activation or type I collagenase activity; however, type I collagen messenger RNA (mRNA) expression was increased threefold in the MMP-2−/− group by real-time PCR. Furthermore, targeted reduction of MMP-2 in cultured HSCs using RNA interference significantly increased collagen I mRNA and protein, while overexpression of MMP-2 resulted in decreased collagen I mRNA. Conclusions These findings suggest that increased MMP-2 during the progression of liver fibrosis may be an important mechanism for inhibiting type I collagen synthesis by activated HSCs, thereby providing a protective rather than pathologic role. PMID:20563750

Prostaglandin E1 inhibits collagen expression in anti-thymocyte antibody-induced glomerulonephritis: possible role of TGF beta.

PubMed

Schneider, A; Thaiss, F; Rau, H P; Wolf, G; Zahner, G; Jocks, T; Helmchen, U; Stahl, R A

1996-07-01

To test whether or not prostaglandins mediate extracellular matrix formation in immune-mediated glomerular disease, rats with anti-thymocyte antibody-induced glomerulonephritis were treated with prostaglandin E1 (PGE1) (250 micrograms/twice daily/s.c.). Glomerular expression of collagen types III and IV was assessed by Northern blotting, immunohistology and Western blotting. Proliferation of glomerular cells was evaluated by staining for the proliferating cell nuclear antigen (PCNA) and consecutive cell counting. At day five after induction of the disease, glomerular mRNA levels of collagen types III and IV were three- to fivefold higher compared with non-nephritic controls. Similarly glomerular deposition of these collagens was markedly increased when assessed by immunohistology. The treatment of nephritic rats with PGE1 reduced the increased glomerular mRNA levels as well as the protein concentration and the deposition of extracellular collagens. The number of PCNA positive cells which was significantly higher in nephritic rats when compared with control animals (24 hr, nephritis 2.53 +/- 0.33 and Control 0.26 +/- 0.06, P = 0.011; 5 days, nephritis 5.10 +/- 1.13 and Control 0.75 +/- 0.08, cells per glomerular cross section, P = 0.03) was reduced by PGE1 (24 hr, nephritis+PGE1 0.44 +/- 0.30, P = 0.0001; 5 days, nephritis +/- PGE1 1.91 +/- 1.84 cells per glomerular cross section, P = 0.001). Prostaglandin E1 also ameliorated the glomerular infiltration of monocytes at 24 hours (nephritis 4.36 +/- 2.82, nephritis + PGE1 2.20 +/- 1.82, cells per glomerular cross section) and five days (nephritis 1.51 +/- 0.58, nephritis+PGE1 1.12 +/- 0.61, cells per glomerular cross section). To further characterize possible mechanisms by which PGE1 reduces extracellular matrix deposition, the glomerular expression of transforming growth factor (TGF-beta), and interleukin 1 beta (IL-1 beta) was assessed by Northern blotting. Nephritic glomeruli showed increased mRNA levels of TGF-beta at day 5 and IL-1 beta at 24 hours when compared with control kidneys. Treatment of the animals with PGE1 inhibited the mRNA expression of TGF-beta and IL-1 beta. These data demonstrate that PGE1 reduces the glomerular expression of extracellular matrix proteins in anti-thymocyte antibody-induced glomerulonephritis, suggesting a beneficial role of prostaglandins in this proliferative glomerular immune injury. The effects of PGE1 might be mediated by inhibition of TGF-beta and IL-1 beta production.

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

PubMed

Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette; Stensballe, Allan; Moos, Torben

2017-03-01

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)] 2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789. © 2016 International Society for Neurochemistry.

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Interleukin-34 as a fibroblast-derived marker of liver fibrosis in patients with non-alcoholic fatty liver disease

PubMed Central

Shoji, Hirotaka; Yoshio, Sachiyo; Mano, Yohei; Kumagai, Erina; Sugiyama, Masaya; Korenaga, Masaaki; Arai, Taeang; Itokawa, Norio; Atsukawa, Masanori; Aikata, Hiroshi; Hyogo, Hideyuki; Chayama, Kazuaki; Ohashi, Tomohiko; Ito, Kiyoaki; Yoneda, Masashi; Nozaki, Yuichi; Kawaguchi, Takumi; Torimura, Takuji; Abe, Masanori; Hiasa, Yoichi; Fukai, Moto; Kamiyama, Toshiya; Taketomi, Akinobu; Mizokami, Masashi; Kanto, Tatsuya

2016-01-01

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic non-viral liver disease. Activation of macrophages and hepatic stellate cells is a critical step that promotes liver fibrosis. We aimed to explore the feasibility of interleukin-34 (IL-34), a key regulator of macrophages, as a fibrosis marker in patients with NAFLD. We enrolled 197 liver biopsy-proven NAFLD patients. We evaluated the serum levels of IL-34, macrophage-colony stimulating factor (M-CSF), soluble CD163 (sCD163), 40 cytokines/chemokines, hyaluronic acid, type IV collagen 7s, and clinically-approved fibrosis scores. IL-34 increased with the progression of fibrosis and was an independent marker for liver fibrosis. Immunostaining experiments, using resected liver specimens from NAFLD patients, revealed that IL-34 was mainly expressed on liver fibroblasts. IL-34 based fibrosis score (0.0387*IL-34 (pg/ml) + 0.3623*type IV collagen 7s (ng/ml) + 0.0184*age (year)–1.1850) was a practical predictive model of liver fibrosis. Using receiver-operating characteristic analyses, the area under the curve, sensitivity, and specificity of IL-34 based fibrosis score were superior or comparable to the other fibrosis biomarkers and scores. In conclusion, the IL-34 based fibrosis score, including serum IL-34, type IV collagen 7s and age, is a feasible diagnostic marker of liver fibrosis in NAFLD patients. PMID:27363523

Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture

PubMed Central

McCall, A. Scott; Cummings, Christopher F.; Bhave, Gautam; Vanacore, Roberto; Page-McCaw, Andrea; Hudson, Billy G.

2014-01-01

Summary Bromine is ubiquitously present in animals as ionic bromide (Br−) yet has no known essential function. Herein, we demonstrate that Br− is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a post-translational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br-deficiency is lethal in Drosophila while Br-replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br−, collagen IV, and peroxidasin. We establish that Br− is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals and its deficiency may be relevant to BM alterations observed in nutritional and smoking related disease. PMID:24906154

Feasibility of Repairing Glomerular Basement Membrane Defects in Alport Syndrome

PubMed Central

Lin, Xiaobo; Suh, Jung Hee; Go, Gloriosa

2014-01-01

Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen α3α4α5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the α3α4α5(IV) network, the α1α1α2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen α3α4α5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of α3α4α5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function. PMID:24262794

Feasibility of repairing glomerular basement membrane defects in Alport syndrome.

PubMed

Lin, Xiaobo; Suh, Jung Hee; Go, Gloriosa; Miner, Jeffrey H

2014-04-01

Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen α3α4α5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the α3α4α5(IV) network, the α1α1α2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen α3α4α5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of α3α4α5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function.

Endothelin-1 Mediated Induction of Extracellular Matrix Genes in Strial Marginal Cells Underlies Strial Pathology in Alport Mice

PubMed Central

Meehan, Daniel T.; Delimont, Duane; Dufek, Brianna; Zallocchi, Marisa; Phillips, Grady; Gratton, Michael Anne; Cosgrove, Dominic

2016-01-01

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ETARs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ETAR antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ETAR blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ETARs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology. PMID:27553900

A Novel Functional Role of Collagen Glycosylation

PubMed Central

Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

Alport syndrome: a unified classification of genetic disorders of collagen IV α345: a position paper of the Alport Syndrome Classification Working Group.

PubMed

Kashtan, Clifford E; Ding, Jie; Garosi, Guido; Heidet, Laurence; Massella, Laura; Nakanishi, Koichi; Nozu, Kandai; Renieri, Alessandra; Rheault, Michelle; Wang, Fang; Gross, Oliver

2018-05-01

Mutations in the genes COL4A3, COL4A4, and COL4A5 affect the synthesis, assembly, deposition, or function of the collagen IV α345 molecule, the major collagenous constituent of the mature mammalian glomerular basement membrane. These mutations are associated with a spectrum of nephropathy, from microscopic hematuria to progressive renal disease leading to ESRD, and with extrarenal manifestations such as sensorineural deafness and ocular anomalies. The existing nomenclature for these conditions is confusing and can delay institution of appropriate nephroprotective therapy. Herein we propose a new classification of genetic disorders of the collagen IV α345 molecule with the goal of improving renal outcomes through regular monitoring and early treatment. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

A unique evolution of the kidney phenotype in a patient with autosomal recessive Alport syndrome.

PubMed

Vischini, Gisella; Kapp, Meghan E; Wheeler, Ferrin C; Hopp, Laszlo; Fogo, Agnes B

2018-03-09

Alport syndrome is due to mutations in one of the genes encoding (α3,4,5) type IV collagen resulting in defective type IV collagen, a key component of the glomerular basement membrane (GBM). The GBM is initially thin, and with ongoing remodeling, develops a thickened basket-woven appearance. We report a unique case of a 9-year-old boy who was biopsied for hematuria and proteinuria, diagnosed as IgA nephropathy, with normal GBM appearance and thickness. Due to a family history of hematuria and chronic kidney disease, he subsequently underwent genetic evaluation and a mutation of α3 type IV collagen (COL4A3) was detected. Additional studies of the initial biopsy demonstrated abnormal type IV collagen immunostaining. A repeat biopsy 4years later showed characteristic glomerular basement membrane morphology of Alport syndrome, and scarring consistent with sequelae of IgA nephropathy. This is the first description of this unusual transition from an initial normal appearance of the glomerular basement membrane to the classic Alport phenotype. Copyright © 2018. Published by Elsevier Inc.

Identification of type IV collagen exposure as a molecular imaging target for early detection of thoracic aortic dissection

PubMed Central

Xu, Ke; Xu, Chen; Zhang, Yanzhenzi; Qi, Feiran; Yu, Bingran; Li, Ping; Jia, Lixin; Li, Yulin; Xu, Fu-jian; Du, Jie

2018-01-01

Thoracic aortic dissection (TAD) is an aggressive and life-threatening vascular disease and there is no effective means of early diagnosis of dissection. Type IV collagen (Col-IV) is a major component of the sub-endothelial basement membrane, which is initially exposed followed by endothelial injury as early-stage event of TAD. So, we want to build a noninvasive diagnostic method to detect early dissection by identifying the exposed Col-IV via MRI. Methods: Col-IV-targeted magnetic resonance/ fluorescence dual probe (Col-IV-DOTA-Gd-rhodamine B; CDR) was synthesized by amide reaction and coordination reaction. Flow cytometry analysis was used to evaluate the cell viability of SMC treated with CDR and fluorescence assays were used to assess the Col-IV targeting ability of CDR in vitro. We then examined the sensitivity and specificity of CDR at different stages of TAD via MRI and bioluminescence imaging in vivo. Results: The localization of Col-IV (under the intima) was observed by histology images. CDR bound specifically to Col-IV-expressing vascular smooth muscle cells and BAPN-induced dissected aorta. The CDR signal was co-detected by magnetic resonance imaging (MRI) and bioluminescence imaging as early as 2 weeks after BAPN administration (pre-dissection stage). The ability to detect rupture of dissected aorta was indicated by a strong normalized signal enhancement (NSE) in vivo. Moreover, NSE was negatively correlated with the time of dissection rupture after BAPN administration (r2 = 0.8482). Conclusion: As confirmed by in vivo studies, the CDR can identify the exposed Col-IV in degenerated aorta to monitor the progress of aortic dissection from the early stage to the rupture via MRI. Thus, CDR-enhanced MRI proposes a potential method for dissection screening, and for monitoring disease progression and therapeutic response. PMID:29290819

Analysis of morphological characteristics and expression levels of extracellular matrix proteins in skin wounds to determine wound age in living subjects in forensic medicine.

PubMed

Fronczek, Judith; Lulf, Ronald; Korkmaz, H Ibrahim; Witte, Birgit I; van de Goot, Franklin R W; Begieneman, Mark P V; Krijnen, Paul A J; Rozendaal, Lawrence; Niessen, Hans W M; Reijnders, Udo J L

2015-01-01

Wound age determination in living subjects is important in routine diagnostics in forensic medicine. Macroscopical description of a wound to determine wound age however is inadequate. The aim of this study was to assess whether it would be feasible to determine wound age via analysis of morphological characteristics and extracellular matrix proteins in skin biopsies of living subjects referred to a forensic outpatient clinic. Skin biopsies (n=101), representing the border area of the wound, were taken from skin injuries of known wound age (range: 4.5h-25 days) in living subjects. All biopsies were analyzed for 3 morphological features (ulceration, parakeratosis and hemorrhage) and 3 extracellular matrix markers (collagen III, collagen IV and α-SMA). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. For hemorrhage, collagen III, collagen IV and α-SMA expression no relation with wound age was found. Ulceration was only found in wounds of 0.2-2, 2-4 and 4-10 days old, implying that the probability that a wound was more than 10 days old in case of ulceration is equal to 0%. Also parakeratosis was almost exclusively found in wounds of 0.2-2, 2-4 and 4-10 days old, except for one case with a wound age of 15 days old. The probability scoring system of all analyzed markers, as depicted above, however can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. We have developed a probability scoring system, analysing morphological characteristics and extracellular matrix proteins in superficial skin biopsies of wounds in living subjects that can be applied in forensic medicine for wound age determination. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

PubMed Central

Huang, Ke jian; Wu, Wei dong; Jiang, Tao; Cao, Jun; Feng, Zhen zhong; Qiu, Zheng jun

2011-01-01

Aims Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression. Methods A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression. Results STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered. Conclusion These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells. PMID:21991388

A combination of low-dose bevacizumab and imatinib enhances vascular normalisation without inducing extracellular matrix deposition.

PubMed

Schiffmann, L M; Brunold, M; Liwschitz, M; Goede, V; Loges, S; Wroblewski, M; Quaas, A; Alakus, H; Stippel, D; Bruns, C J; Hallek, M; Kashkar, H; Hacker, U T; Coutelle, O

2017-02-28

Vascular endothelial growth factor (VEGF)-targeting drugs normalise the tumour vasculature and improve access for chemotherapy. However, excessive VEGF inhibition fails to improve clinical outcome, and successive treatment cycles lead to incremental extracellular matrix (ECM) deposition, which limits perfusion and drug delivery. We show here, that low-dose VEGF inhibition augmented with PDGF-R inhibition leads to superior vascular normalisation without incremental ECM deposition thus maintaining access for therapy. Collagen IV expression was analysed in response to VEGF inhibition in liver metastasis of colorectal cancer (CRC) patients, in syngeneic (Panc02) and xenograft tumours of human colorectal cancer cells (LS174T). The xenograft tumours were treated with low (0.5 mg kg -1 body weight) or high (5 mg kg -1 body weight) doses of the anti-VEGF antibody bevacizumab with or without the tyrosine kinase inhibitor imatinib. Changes in tumour growth, and vascular parameters, including microvessel density, pericyte coverage, leakiness, hypoxia, perfusion, fraction of vessels with an open lumen, and type IV collagen deposition were compared. ECM deposition was increased after standard VEGF inhibition in patients and tumour models. In contrast, treatment with low-dose bevacizumab and imatinib produced similar growth inhibition without inducing detrimental collagen IV deposition, leading to superior vascular normalisation, reduced leakiness, improved oxygenation, more open vessels that permit perfusion and access for therapy. Low-dose bevacizumab augmented by imatinib selects a mature, highly normalised and well perfused tumour vasculature without inducing incremental ECM deposition that normally limits the effectiveness of VEGF targeting drugs.

Kidney diseases caused by glomerular basement membrane type IV collagen defects in dogs.

PubMed

Lees, George E

2013-01-01

To review the pathogenesis, as well as the clinical and pathologic features of canine glomerular diseases caused by genetic type IV collagen defects. Original studies and review articles from human and veterinary medical fields. Presence in glomerular basement membranes (GBM) of a network composed of α3.α4.α5 heterotrimers of type IV collagen is required to maintain structure and function of glomerular capillary walls. Hereditary nephropathy (HN) is the most commonly used name for kidney diseases that occur in dogs due to genetic type IV collagen abnormalities. To date, 4 different collagen IV gene mutations have been identified in dogs with HN; 2 are COL4A5 mutations that cause X-linked HN (XL-HN), and 2 are COL4A4 mutations that cause autosomal recessive HN (AR-HN). Affected males with XL-HN and affected males and females with AR-HN develop juvenile-onset kidney disease manifested by proteinuria typically starting at 3-6 months of age and followed by progressive kidney disease leading to terminal failure usually at 6-24 months of age. Carrier female dogs with XL-HN also develop proteinuria starting at 3-6 months of age, but progressive disease causing kidney failure is uncommon until they are >5 years old. The distinctive pathologic lesions of HN are extensive multilaminar splitting and thickening of the GBM, as demonstrated by electron microscopy, and abnormal type IV collagen α-chain content of basement membranes, as demonstrated by immunolabeling. Identification of the underlying gene mutations has permitted genetic testing and selective breeding practices that currently are minimizing HN in breeds known to be at risk. Canine HN is a rare disease that should be considered whenever a dog exhibits a juvenile-onset kidney disease characterized partly by proteinuria, but highly specialized methods are required to pursue a definitive diagnosis. © Veterinary Emergency and Critical Care Society 2013.

Immunohistochemical distribution of collagens type I, III, IV and VI, of undulin and of tenascin in oral fibrous hyperplasia.

PubMed

Becker, J; Schuppan, D; Müller, S

1993-11-01

The distribution of collagens type I, IV and VI, of procollagen type III, of undulin and of tenascin was studied in 10 lesions which were clinically and histologically diagnosed as localized oral fibrous hyperplasias. The immunohistochemical distribution of these proteins was similar to that observed for normal oral mucosa. Undulin showed a pattern of parallel fibers throughout. Collagen type VI was pronounced in the subepithelial connective tissue, whereas the collagen fiber bundles were equally reactive for collagens type I and III. Tenascin was observed close to the subepithelial basement membrane and in proximity to collagen fiber bundles in the upper connective tissue. The present findings indicate that oral fibrous hyperplasias that are probably caused by inflammation or chronic irritation show the differentiated and ordered pattern of extracellular matrix proteins characteristic of normal oral mucosa.

Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

PubMed

Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

2009-10-01

The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions.

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis

PubMed Central

Seehusen, Frauke; Al-Azreg, Seham A.; Raddatz, Barbara B.; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

2016-01-01

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease. PMID:27441688

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

PubMed

Seehusen, Frauke; Al-Azreg, Seham A; Raddatz, Barbara B; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

2016-01-01

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

WY14,643, a PPARalpha ligand, attenuates expression of anti-glomerular basement membrane disease.

PubMed

Archer, D C; Frkanec, J T; Cromwell, J; Clopton, P; Cunard, R

2007-11-01

Peroxisome proliferator-activated receptor alpha (PPARalpha) ligands are medications used to treat hyperlipidaemia and atherosclerosis. Increasing evidence suggests that these agents are immunosuppressive. In the following studies we demonstrate that WY14,643, a PPARalpha ligand, attenuates expression of anti-glomerular basement membrane disease (AGBMD). C57BL/6 mice were fed 0.05% WY14,643 or control food and immunized with the non-collagenous domain of the alpha3 chain of Type IV collagen [alpha3(IV) NC1] in complete Freund's adjuvant (CFA). WY14,643 reduced proteinuria and greatly improved glomerular and tubulo-interstitial lesions. However, the PPARalpha ligand did not alter the extent of IgG-binding to the GBM. Immunohistochemical studies revealed that the prominent tubulo-interstitial infiltrates in the control-fed mice consisted predominately of F4/80(+) macrophages and WY14,643-feeding decreased significantly the number of renal macrophages. The synthetic PPARalpha ligand also reduced significantly expression of the chemokine, monocyte chemoattractant protein (MCP)-1/CCL2. Sera from mice immunized with AGBMD were also evaluated for antigen-specific IgGs. There was a significant increase in the IgG1 : IgG2c ratio and a decline in the intrarenal and splenocyte interferon (IFN)-gamma mRNA expression in the WY14,643-fed mice, suggesting that the PPARalpha ligand could skew the immune response to a less inflammatory T helper 2-type of response. These studies suggest that PPARalpha ligands may be a novel treatment for inflammatory renal disease.

WY14,643, a PPARα ligand, attenuates expression of anti-glomerular basement membrane disease

PubMed Central

Archer, D C; Frkanec, J T; Cromwell, J; Clopton, P; Cunard, R

2007-01-01

Peroxisome proliferator-activated receptor alpha (PPARα) ligands are medications used to treat hyperlipidaemia and atherosclerosis. Increasing evidence suggests that these agents are immunosuppressive. In the following studies we demonstrate that WY14,643, a PPARα ligand, attenuates expression of anti-glomerular basement membrane disease (AGBMD). C57BL/6 mice were fed 0·05% WY14,643 or control food and immunized with the non-collagenous domain of the α3 chain of Type IV collagen [α3(IV) NC1] in complete Freund's adjuvant (CFA). WY14,643 reduced proteinuria and greatly improved glomerular and tubulo-interstitial lesions. However, the PPARα ligand did not alter the extent of IgG-binding to the GBM. Immunohistochemical studies revealed that the prominent tubulo-interstitial infiltrates in the control-fed mice consisted predominately of F4/80+ macrophages and WY14,643-feeding decreased significantly the number of renal macrophages. The synthetic PPARα ligand also reduced significantly expression of the chemokine, monocyte chemoattractant protein (MCP)-1/CCL2. Sera from mice immunized with AGBMD were also evaluated for antigen-specific IgGs. There was a significant increase in the IgG1 : IgG2c ratio and a decline in the intrarenal and splenocyte interferon (IFN)-γ mRNA expression in the WY14,643-fed mice, suggesting that the PPARα ligand could skew the immune response to a less inflammatory T helper 2-type of response. These studies suggest that PPARα ligands may be a novel treatment for inflammatory renal disease. PMID:17888025

[Study on the tectology change of rectum wall above the hemorrhoids].

PubMed

Zhang, Li; Yang, Bin; Zhang, Yu-Chao; Fu, Yu-Ru; Chen, Shuang

2009-06-15

To investigate the histomorphological characteristics and its significance of rectum wall above hemorrhoids. Tissues of rectum wall above hemorrhoids were obtained after stapled hemorrhoidopexy from 21 patients with grade III-IV internal hemorrhoids. Seven macroscopically normal rectal tissues collected from upper rectal cancer patients without a history of hemorrhoids served as control. Masson trichrome staining was performed for detecting smooth muscles and collagen in the tissues. The expression of type III collagen was detected by using immunohistochemical staining in the two groups. Morphological abnormalities, such as fragment, rupture, disorganization were found in smooth muscle of proximal rectal tissues above the piles, and it was statistically different from the distal rectal tissues above the piles and control tissues (all P < 0.05). Moreover, hyperplasia of type III collagen in both muscularis mucosa and rectum wall in tissues above hemorrhoids were observed, no such changes was found in the control tissues. The range of pathological changes in hemorrhoids is beyond the anal cushions. The pathological changes of the smooth muscle and the type III collagen in the tissues above the piles are the pathological basis of hemorrhoids.

Endothelin-1 mediated induction of extracellular matrix genes in strial marginal cells underlies strial pathology in Alport mice.

PubMed

Meehan, Daniel T; Delimont, Duane; Dufek, Brianna; Zallocchi, Marisa; Phillips, Grady; Gratton, Michael Anne; Cosgrove, Dominic

2016-11-01

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ET A Rs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ET A R antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ET A R blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ET A Rs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology. Copyright © 2016 Elsevier B.V. All rights reserved.

Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age- and sex-dependent remodeling of the extracellular matrix.

PubMed

Kehlet, Stephanie N; Willumsen, Nicholas; Armbrecht, Gabriele; Dietzel, Roswitha; Brix, Susanne; Henriksen, Kim; Karsdal, Morten A

2018-01-01

The extracellular matrix (ECM) plays a vital role in maintaining normal tissue function. Collagens are major components of the ECM and there is a tight equilibrium between degradation and formation of these proteins ensuring tissue health and homeostasis. As a consequence of tissue turnover, small collagen fragments are released into the circulation, which act as important biomarkers in the study of certain tissue-related remodeling factors in health and disease. The aim of this study was to establish an age-related collagen turnover profile of the main collagens of the interstitial matrix (type I and III collagen) and basement membrane (type IV collagen) in healthy men and women. By using well-characterized competitive ELISA-assays, we assessed specific fragments of degraded (C1M, C3M, C4M) and formed (PINP, Pro-C3, P4NP7S) type I, III and IV collagen in serum from 617 healthy men and women ranging in ages from 22 to 86. Subjects were divided into 5-year age groups according to their sex and age. Groups were compared using Kruskal-Wallis adjusted for Dunn's multiple comparisons test and Mann-Whitney t-test. Age-specific changes in collagen turnover was most profound for type I collagen. PINP levels decreased in men with advancing age, whereas in women, the level decreased in early adulthood followed by an increase around the age of menopause (age 40-60). Sex-specific changes in type I, III and IV collagen turnover was present at the age around menopause (age 40-60) with women having an increased turnover. In summary, collagen turnover is affected by age and sex with the interstitial matrix and the basement membrane being differently regulated. The observed changes needs to be accounted for when measuring ECM related biomarkers in clinical studies.

DD-RT-PCR identifies 7-dehydrocholesterol reductase as a key marker of early Leydig cell steroidogenesis.

PubMed

Anbalagan, M; Yashwanth, R; Jagannadha Rao, A

2004-04-30

Postnatal Leydig cell development in rat involves an initial phase of proliferation of progenitor Leydig cells (PLCs) and subsequent differentiation of these cells into immature Leydig cells (ILCs) and adult Leydig cells (ALCs). With an objective to identify the molecular changes associated with Leydig cell differentiation, the mRNA population in PLCs and ILCs were analyzed by the technique of differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed differential expression of several transcripts in PLCs and ILCs. Of the several differentially expressed transcripts, the expression of transcripts corresponding to collagen IV alpha6 (Col IV alpha6) and ribosomal protein L 41 (RpL41) decreased during the differentiation of PLC to ILC. Also there was an increase in the expression of transcripts encoding enzymes such as microsomal glutathione-S-transferase (mGST 1) and 7-dehydrocholesterol reductase (7-DHCR) during this process. While Col IV alpha6 and RpL41 are known to be involved in cellular proliferation, mGST 1 and 7-DHCR are essential for normal Leydig cell steroidogenesis. A detailed study on 7-DHCR expression in Leydig cells revealed that this enzyme plays a crucial role in steroidogenesis. Interestingly expression of this enzyme is not under acute regulation by Luteinizing hormone (LH). Copyright 2004 Elsevier Ireland Ltd.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

PubMed Central

GUZMÁN-URIBE, Daniela; ALVARADO-ESTRADA, Keila Neri; PIERDANT-PÉREZ, Mauricio; TORRES-ÁLVAREZ, Bertha; SÁNCHEZ-AGUILAR, Jesus Martin; ROSALES-IBÁÑEZ, Raúl

2017-01-01

Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues. PMID:28403359

Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries From Patients With Type 2 Diabetes Mellitus and Lower Levels Among Metformin Users.

PubMed

Preil, Simone A R; Kristensen, Lars P; Beck, Hans C; Jensen, Pia S; Nielsen, Patricia S; Steiniche, Torben; Bjørling-Poulsen, Marina; Larsen, Martin R; Hansen, Maria L; Rasmussen, Lars M

2015-10-01

The increased risk of cardiovascular diseases in type 2 diabetes mellitus has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes mellitus and moreover hypothesized that metformin intake influences the protein composition. We analyzed nonatherosclerotic repair arteries gathered at coronary bypass operations from 30 patients with type 2 diabetes mellitus and from 30 age- and sex-matched nondiabetic individuals. Quantitative proteome analysis was performed by isobaric tag for relative and absolute quantitation-labeling and liquid chromatography-mass spectrometry, tandem mass spectrometry analysis on individual arterial samples. The amounts of the basement membrane components, α1-type IV collagen and α2-type IV collagen, γ1-laminin and β2-laminin, were significantly increased in patients with diabetes mellitus. Moreover, the expressions of basement membrane components and other vascular proteins were significantly lower among metformin users when compared with nonusers. Patients treated with or without metformin had similar levels of hemoglobin A1c, cholesterol, and blood pressure. In addition, quantitative histomorphometry showed increased area fractions of collagen-stainable material in tunica intima and media among patients with diabetes mellitus. The distinct accumulation of arterial basement membrane proteins in type 2 diabetes mellitus discloses a similarity between the diabetic macroangiopathy and microangiopathy and suggests a molecular explanation behind the alterations in vascular remodeling, biomechanical properties, and aneurysm formation described in diabetes mellitus. The lower amounts of basement membrane components in metformin-treated individuals are compatible with the hypothesis of direct beneficial drug effects on the matrix composition in the vasculature. © 2015 American Heart Association, Inc.

[Alport syndrome: Hereditary nephropathy associated with mutations in genes coding for type IV collagen chains].

PubMed

Heidet, Laurence; Gubler, Marie-Claire

2016-12-01

Alport syndrome is an inherited disorder characterized by the association of a progressive haematuric nephropathy with ultrastructural abnormalities of the glomerular basement membranes, a progressive sensorineural hearing loss and sometimes ocular involvement. Its incidence is less than 1 per 5000 individuals and the disease is the cause of about 2% of end stage renal disease in Europe and the United States. Alport syndrome is clinically and genetically heterogeneous. It is related to mutations in the genes encoding one of three chains, α3, α4 α5 of type IV collagen, the main component of basement membranes, expressed in the glomerular basement membrane. COL4A5 mutations are associated with X-linked Alport syndrome, which represents 80 to 85% of cases and is more severe in boys than in girls. Mutations in COL4A3 or COL4A4 are associated with autosomal Alport syndrome. The expression of collagen chains in skin and kidney basement membranes allows for the diagnosis and characterization of the mode of transmission in most patients. It is necessary to diagnose this syndrome because its family involvement, its severity, and the importance of genetic counseling. Angiotensin blockers are increasingly prescribed in proteinuric patients. Prospective studies are needed to assess the effectiveness of these treatments on proteinuria and progression of kidney failure, and to specify indications. Animal studies have shown the potential value of different molecules (protease inhibitors, chemokine receptor blockers, transforming growth factor-β1 inhibitors, hydroxy-methyl-coenzyme A reductase inhibitors, bone morphogenetic protein-7 inhibitors), hematopoietic stem cells, and of a anti-micro-RNA. Copyright © 2016. Published by Elsevier SAS.

Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV.

PubMed

Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J

2002-12-06

Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

Polarity and transport properties of rabbit kidney proximal tubule cells on collagen IV-coated porous membranes.

PubMed

Genestie, I; Morin, J P; Vannier, B; Lorenzon, G

1995-07-01

A high degree of functional polarity has been obtained in primary cultures of rabbit kidney proximal tubule cells grown on collagen IV-coated porous membranes. Tight confluency was attained 6 days after seeding and maintained for at least 6 more days, as shown by analysis of paracellular inulin diffusion. From day 6 onward, L-lactate, ammonia, and D-glucose concentration gradient and a pH difference of approximately 1 unit developed between the two nutrient medium compartments. Confluent monolayers expressed organic ion transport properties higher than those formerly reported for other cell models. Transcellular transport of 20 microM tetraethylammonium was directed from basal to apical compartment and was specifically inhibited by mepiperphenidol (1 mM). Unidirectional transport of 2.4 microM p-aminohippurate also occurred from basal to apical compartment, was saturable, and specifically inhibited by probenecid (1 mM). These results suggest that rabbit kidney proximal tubule cells, cultured under the experimental conditions described here, may be a useful model for the in vitro study of highly polarized renal transport processes.

Matrix Gelatinases in Atherosclerosis and Diabetic Nephropathy: Progress and Challenges.

PubMed

Dimas, Grigorios G; Didangelos, Triantafyllos P; Grekas, Dimitrios M

2017-01-01

Matrix metalloproteinases (MMPs) are zinc-dependent proteases that degrade components of the extracellular matrix (ECM). In glomerular disease, MMPs are major regulators of ECM degradation as well as structural and functional integrity in the glomerulus. In altered matrix composition diseases, glomerular damage is due to increased degradation of kidney and vessel basement membranes (BMs) by MMPs. MMP -2 and -9 are both considered as the main enzymes that degrade collagen type-IV (coll-IV), which represents the key collagenous component of ECM and constitutes the architectural structure of vessels and glomerular BM. There is growing evidence implicating MMPs in atherosclerosis as well as in cardiovascular disease (CVD) and chronic kidney disease (CKD). Specific endogenous tissue inhibitors of MMPs (TIMPs) are also implicated in CKD, CVD and diabetic nephropathy (DN). The present review discusses the role of MMPs -2 and -9 in DN, as a leading cause of endstage renal disease and as a model of the link between progressive glomerulosclerosis and MMP expression. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The adherent abilities of Clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis.

PubMed

Wade, Ben; Keyburn, Anthony L; Haring, Volker; Ford, Mark; Rood, Julian I; Moore, Robert J

2016-12-25

Necrotic enteritis of poultry is an emerging disease of substantial economic importance, but aspects of the pathogenesis of this multi-factorial disease are still unclear. We recently demonstrated that the ability of avian strains of the causative bacterium, Clostridium perfringens, to bind to specific collagen types correlated strongly with their virulence and we postulated that binding of the pathogen to collagen types IV and V and gelatin may involve the putative adhesin-encoding gene cnaA, which is found in the VR-10B locus. In this study we have used site-directed mutagenesis to demonstrate that disruption of the cnaA gene leads to a reduction in the expression of the three genes immediately downstream of cnaA and reduced adherence to collagen types IV and V and gelatin. In addition, a cnaA mutant of strain EHE-NE18 was no longer capable of causing necrotic enteritis in a chicken disease induction model and had a significantly reduced ability to colonise the chicken intestinal mucosa. These results were confirmed by generating and analysing a similar mutant in an independent necrotic enteritis causing C. perfringens strain. This study expands our understanding of the mechanisms involved in necrotic enteritis pathogenesis by demonstrating the importance of C. perfringens adherence to extracellular matrix proteins. Copyright © 2016. Published by Elsevier B.V.

Interstitial inflammation in Alport syndrome.

PubMed

Jedlicka, Jan; Soleiman, Afschin; Draganovici, Dan; Mandelbaum, Jana; Ziegler, Urs; Regele, Heinz; Wüthrich, Rudolf P; Gross, Oliver; Anders, Hans-Joachim; Segerer, Stephan

2010-04-01

The Alport syndrome is a hereditary glomerular disease linked to structural abnormalities of collagen IV. In a mouse model of Alport syndrome, the interstitial lymphocyte influx was important for disease progression. CXCR3 is a chemokine receptor involved in lymphocyte recruitment to the kidney. We hypothesized that CXCR3-positive T cells might be involved in human Alport syndrome. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsies from 17 patients with Alport syndrome, 10 with immunoglobulin A (IgA) nephropathy, and 11 healthy donor kidneys. We investigated the expression of the alpha5 chain of collagen IV to confirm the morphologic diagnosis, the chemokine receptor CXCR3 and CD3-positive T cells. Alport syndrome biopsies demonstrated a complete loss of the alpha5 chain of collagen IV from the glomerular basement membrane and the morphologic features consistent with Alport syndrome on electron microscopy. A prominent number of CXCR3-positive cells were found in the tubulointerstitium. Most of the CXCR3-positive cells were CD3-positive T cells, demonstrated by double-labeling in selected biopsies. The number of CXCR3-positive cells in kidneys with Alport syndrome correlated with serum creatinine (P < .05) and with morphologic features of a progressive disease (eg, interstitial fibrosis, glomerulosclerosis, and tubular atrophy). The severity of interstitial CXCR3-positive cell influx was similar in Alport syndrome as compared to immunoglobulin A nephropathy. The noninflammatory glomerular lesion of Alport syndrome is associated with prominent interstitial accumulation of CD3- and CXCR3-positive lymphocytes. The degree of infiltration correlated with renal function. We speculate that targeting T lymphocytes, for example, by CXCR3 blocking agents, might be a novel approach to inhibit disease progression in patients with Alport syndrome. Copyright 2010 Elsevier Inc.

HANAC Syndrome Col4a1 Mutation Causes Neonate Glomerular Hyperpermeability and Adult Glomerulocystic Kidney Disease

PubMed Central

Chen, Zhiyong; Migeon, Tiffany; Verpont, Marie-Christine; Zaidan, Mohamad; Sado, Yoshikazu; Kerjaschki, Dontscho; Ronco, Pierre

2016-01-01

Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman’s capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44, α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman’s capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders. PMID:26260163

HANAC Syndrome Col4a1 Mutation Causes Neonate Glomerular Hyperpermeability and Adult Glomerulocystic Kidney Disease.

PubMed

Chen, Zhiyong; Migeon, Tiffany; Verpont, Marie-Christine; Zaidan, Mohamad; Sado, Yoshikazu; Kerjaschki, Dontscho; Ronco, Pierre; Plaisier, Emmanuelle

2016-04-01

Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman's capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44,α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman's capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders. Copyright © 2016 by the American Society of Nephrology.

The invasion mode of GH(3) cells is conditioned by collagen subtype, and its efficiency depends on cell-cell adhesion.

PubMed

Azorín, Erika; Solano-Agama, Carmen; Mendoza-Garrido, M Eugenia

2012-12-15

The adaptation of GH(3) cells to different microenvironments is a consequence of a partial compromise with the tumor phenotype. A collagen type IV enriched microenvironment favors an invasive phenotype and increases the substrate adhesion capacity, whereas it decreases the phosphorylation of the regulatory myosin light chain and the aggregation capacity. In contrast, the higher internal tension and increased aggregation capacity induced by collagen type I/III are factors that reduce the invasion rate. Our results show, for the first time, the importance of collagen subtypes in determining the migratory strategy: collagen I/III favors mesenchymal-like motility, whereas collagen type IV induces an ameboid-type displacement. The reciprocal modulation of the myosin light chain kinase and the Rho-kinase determines the invasive capacity through changes in tissue cohesion, extracellular matrix affinity, regulatory myosin light chain phosphorylation and spatial distribution. The collagen subtype determines which of the mechano-transduction signaling pathways will regulate the tensional homeostasis and affect the invasion ability as well as the preferred migration strategy of the cells. Copyright © 2012 Elsevier Inc. All rights reserved.

The effect of montelukast and antiadhesion barrier solution on the capsule formation after insertion of silicone implants in a white rat model.

PubMed

Yang, J-D; Kwon, O-H; Lee, J-W; Chung, H-Y; Cho, B-C; Park, H-Y; Kim, T-G

2013-01-01

Capsular contracture is one of the most severe complications that can occur in breast surgery following silicone implant insertion. The purpose of this study was to investigate the effect of montelukast and antiadhesion barrier solution (AABS) on reducing capsular formation and their possible synergism. This study was approved by the Animal Ethics Committee (Reference No. KNU 2012-33) and was conducted in accordance with the Kyungpook National University - Institutional Animal Care and Use Committee, Animal Ethics Committee. The experiments in this study were conducted in vivo in 4 groups of 24 rats. Following silicone implant insertion, the pocket was injected with different agents. Group I (control group) was given normal saline injections into the pocket and fed with pure water. Group II was given injections of AABS and fed with pure water. Group III was given injections of normal saline and the medication montelukast during the experimental period. Group IV was given injections of AABS and montelukast as postoperative medication. Peri-implant capsules were excised after 8 weeks and were evaluated for transparency, inflammatory cell content, capsule thickness, collagen pattern and TGF-β expression. The capsules in the experimental groups (i.e., groups II-IV) were significantly more transparent than those in group I (controls; p < 0.05, Student's t test). The mean capsule thickness of the experimental groups II (296 ± 14.76 μm), III (280 ± 14.77 μm) and IV (276 ± 39.28 μm) was smaller than that of the control group I (361 ± 35.43 μm). Compared to the control group, the histologic findings in the experimental groups suggested a decreased inflammatory response occurring in the peri-implant capsules as they exhibited minor vascularization and a reduced number of mast cells and macrophages. The collagen patterns in the experimental groups were of a lower density than in the control group with the former showing a loose, tidy collagen pattern. The amounts of TGF-β and collagen I were higher in the control group than in the experimental groups. Group IV (the synergic effect group) had a more pronounced effect on all the parameters examined than that in groups II and III with separate drug administration. Montelukast and AABS reduced the thickness, the inflammatory cell infiltrate and the myofibroblast content of the peri-implant capsules around silicone implants in this white rat model. They lowered the expression of the fibrotic mediator, TGF-β, and inhibited the peri-implant capsular fibrosis. Therefore, montelukast and AABS are effective in the reduction of silicone-induced peri-implant capsular formation.

Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics.

PubMed

Valtink, Monika; Gruschwitz, Rita; Funk, Richard H W; Engelmann, Katrin

2008-01-01

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. Copyright 2008 S. Karger AG, Basel.

Urinary type IV collagen excretion predicts subsequent declining renal function in type 2 diabetic patients with proteinuria.

PubMed

Katavetin, Pisut; Katavetin, Paravee; Susantitaphong, Paweena; Townamchai, Natavudh; Tiranathanagul, Khajohn; Tungsanga, Kriang; Eiam-Ong, Somchai

2010-08-01

Baseline urinary type IV collagen excretion was negatively correlated with the subsequent GFR change (r(s)=-0.39, p=0.04) in our cohort of 30 type 2 diabetic patients with proteinuria. Therefore, it could be used to predict subsequent declining renal function in type 2 diabetic patients with proteinuria. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Cell stiffness, contractile stress and the role of extracellular matrix

DOE Office of Scientific and Technical Information (OSTI.GOV)

An, Steven S., E-mail: san@jhsph.edu; Kim, Jina; Ahn, Kwangmi

Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genesmore » in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.« less

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

PubMed

Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Collagen Type IV and Laminin Expressions during Cartilage Repair and in Late Clinically Failed Repair Tissues from Human Subjects

PubMed Central

Foldager, Casper Bindzus; Toh, Wei Seong; Christensen, Bjørn Borsøe; Lind, Martin; Gomoll, Andreas H.; Spector, Myron

2016-01-01

Objective To identify the collagen type IV (Col4) isoform in articular cartilage and to evaluate the expressions of Col4 and laminin in the pericellular matrix (PCM) in damaged cartilage and during cartilage repair. Design The Col4 isoform was determined in chondrocytes isolated from 6 patients cultured up to 6 days and in 21% O2 or 1% O2, and the gene expression of Col4 α-chains was investigated. The distribution of Col4 and laminin in traumatically damaged cartilage (n = 7) and clinically failed cartilage repair (microfracture, TruFit, autologous chondrocyte implantation; n = 11) were investigated using immunohistochemistry. Normal human cartilage was used as control (n = 8). The distribution during clinical cartilage repair procedures was investigated in a minipig model with 6-month follow-up (untreated chondral, untreated osteochondral, microfracture, autologous chondrocyte implantation; n = 10). Results The Col4 isoform in articular cartilage was characterized as α1α1α2, which is an isoform containing antiangiogenic domains in the NC1-terminals (arresten and canstatin). In normal cartilage, laminin and Col4 was exclusively found in the PCM. High amounts (>50%) of Col4 in the PCM significantly decreased in damaged cartilage (P = 0.004) and clinically failed repair tissue (P < 0.001). Laminin was only found with high expression (>50%) in 4/8 of the normal samples, which was not statistically significantly different from damaged cartilage (P = 0.15) or failed cartilage repair (P = 0.054). Conclusions Col4 in cartilage contain antiangiogenic domains and may play a role in the hypoxic environment in articular cartilage. Col4 and laminin was not found in the PCM of damaged and clinically failed repair. PMID:26958317

Beneficial effects of previous exercise training on renal changes in streptozotocin-induced diabetic female rats

PubMed Central

Amaral, Liliany S de Brito; Silva, Fernanda A; Correia, Vicente B; Andrade, Clara EF; Dutra, Bárbara A; Oliveira, Márcio V; de Magalhães, Amélia CM; Volpini, Rildo A; Seguro, Antonio C; Coimbra, Terezila M

2016-01-01

This study evaluated the effects of aerobic exercise performed both previously and after the induction of diabetes mellitus on changes of renal function and structure in streptozotocin-induced diabetic rats. Female wistar rats were divided into five groups: sedentary control (C + Se); trained control (C + Ex); sedentary diabetic (D + Se); trained diabetic (D + Ex) and previously trained diabetic (D + PEx). The previous exercise consisted of treadmill running for four weeks before the induction of diabetes mellitus. After induction of diabetes mellitus with streptozotocin, the D + PEx, D + Ex and C + Ex groups were submitted to eight weeks of aerobic exercise. At the end of the training protocol, we evaluate the serum glucose, insulin and 17β-estradiol levels, renal function and structure, proteinuria, and fibronectin, collagen IV and transforming growth factor beta 1 (TGF-β1) renal expressions. Induction of diabetes mellitus reduced the insulin and did not alter 17β-estradiol levels, and exercise did not affect any of these parameters. Previous exercise training attenuated the loss of body weight, the blood glucose, the increase of glomerular filtration rate and prevented the proteinuria in the D + PEx group compared to D + Se group. Previous exercise also reduced glomerular hypertrophy, tubular and glomerular injury, as well as the expressions of fibronectin and collagen IV. These expressions were associated with reduced expression of TGF-β1. In conclusion, our study shows that regular aerobic exercise especially performed previously to induction of diabetes mellitus improved metabolic control and has renoprotective action on the diabetic kidney. PMID:26490345

Beneficial effects of previous exercise training on renal changes in streptozotocin-induced diabetic female rats.

PubMed

Amaral, Liliany S de Brito; Silva, Fernanda A; Correia, Vicente B; Andrade, Clara E F; Dutra, Bárbara A; Oliveira, Márcio V; de Magalhães, Amélia C M; Volpini, Rildo A; Seguro, Antonio C; Coimbra, Terezila M; Soares, Telma de J

2016-02-01

This study evaluated the effects of aerobic exercise performed both previously and after the induction of diabetes mellitus on changes of renal function and structure in streptozotocin-induced diabetic rats. Female wistar rats were divided into five groups: sedentary control (C + Se); trained control (C + Ex); sedentary diabetic (D + Se); trained diabetic (D + Ex) and previously trained diabetic (D + PEx). The previous exercise consisted of treadmill running for four weeks before the induction of diabetes mellitus. After induction of diabetes mellitus with streptozotocin, the D + PEx, D + Ex and C + Ex groups were submitted to eight weeks of aerobic exercise. At the end of the training protocol, we evaluate the serum glucose, insulin and 17β-estradiol levels, renal function and structure, proteinuria, and fibronectin, collagen IV and transforming growth factor beta 1 (TGF-β1) renal expressions. Induction of diabetes mellitus reduced the insulin and did not alter 17β-estradiol levels, and exercise did not affect any of these parameters. Previous exercise training attenuated the loss of body weight, the blood glucose, the increase of glomerular filtration rate and prevented the proteinuria in the D + PEx group compared to D + Se group. Previous exercise also reduced glomerular hypertrophy, tubular and glomerular injury, as well as the expressions of fibronectin and collagen IV. These expressions were associated with reduced expression of TGF-β1. In conclusion, our study shows that regular aerobic exercise especially performed previously to induction of diabetes mellitus improved metabolic control and has renoprotective action on the diabetic kidney. © 2016 by the Society for Experimental Biology and Medicine.

Chondroitin sulphates A, B and C, collagen types I-IV and fibronectin in venous sinus of the red pulp in human spleen.

PubMed

Rovenská, E; Michalka, P; Papincák, J; Durdík, S; Jakubovský, J

2005-01-01

The morphological relationship of chondroitin sulphates A, B, and C, collagen types I-IV and fibronectin in the wall of venous sinuses of the red pulp in human spleen has not been a focus of interest among morphologists. Regarding the hypothesis that the structure of the spleen lends it the function of a blood filter the substances described in our study might play a significant role in the functional morphology. Of 146 human spleen surgical specimens, groups of 12 specimens each were examined under a light microscope using the method of antibodies against fibronectin, against collagen types I-IV and against chondroitin sulphates A, B, and C. The sections of the red pulp of human spleen stained with hematoxylin and eosin provided limited information about the wall of the sinuses. Chondroitin sulphates A and B were observed on the surface of sinus-lining cells (SLC), and fibronectin was detected on the surface of the annular fibers. Collagen type 11 was observed almost in the same places as chondroitin sulphates A and B. Collagen type IV was present in annular fibers of the wall of the sinus and in the basement membrane, like fibronectin. Chondroitin sulphate was not present in the walls of sinuses. Binding of antibodies against chondroitin sulphate A and against chondroitin sulphate B indicates the presence of chondroitin sulfates on the surface of SLC, where they probably play a role in helping the human organism to recognize alien and self substances. The presence of chondroitin,sulphates A and B probably affects inhibition of binding of cells with collagen type I, but not with fibronectin.

Incidence and specificity of antibodies to types I, II, III, IV, and V collagen in rheumatoid arthritis and other rheumatic diseases as measured by 125I-radioimmunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stuart, J.M.; Huffstutter, E.H.; Townes, A.S.

1983-07-01

Antibodies to human native and denatured types I, II, III, IV, and V collagens were measured using 125I-radioimmunoassay. Mean levels of binding by sera from 30 rheumatoid arthritis patients were significantly higher than those from 20 normal subjects against all of the collagens tested. The relative antibody concentration was higher in synovial fluid than in simultaneously obtained serum. Many patients with gout or various other rheumatic diseases also had detectable anticollagen antibodies. With a few notable exceptions, the majority of the reactivity detected in all patient groups was directed against covalent structural determinants present on all of the denatured collagens,more » suggesting a secondary reaction to tissue injury.« less

Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome.

PubMed

Daga, Sergio; Baldassarri, Margherita; Lo Rizzo, Caterina; Fallerini, Chiara; Imperatore, Valentina; Longo, Ilaria; Frullanti, Elisa; Landucci, Elisa; Massella, Laura; Pecoraro, Carmine; Garosi, Guido; Ariani, Francesca; Mencarelli, Maria Antonietta; Mari, Francesca; Renieri, Alessandra; Pinto, Anna Maria

2018-02-01

Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end-stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes-targeted therapeutic strategies have been hampered by the difficulty of non-invasively isolating them and transcripts-based diagnostic approaches are complicated by the inaccessibility of other COL4 chains-expressing cells. We firstly isolated podocyte-lineage cells from ATS patients' urine samples, in a non-invasive way. RT-PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patient's urine derived podocyte-lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease-relevant cells. © 2017 Wiley Periodicals, Inc.

Hyperglycemia-Induced Modulation of the Physiognomy and Angiogenic Potential of Fibroblasts Mediated by Matrix Metalloproteinase-2: Implications for Venous Stenosis Formation Associated with Hemodialysis Vascular Access in Diabetic Milieu.

PubMed

Janardhanan, Rajiv; Kilari, Sreenivasulu; Leof, Edward B; Misra, Sanjay

2015-01-01

It is hypothesized that venous stenosis formation associated with hemodialysis vascular-access failure is caused by hypoxia-mediated fibroblast-to-myofibroblast differentiation accompanied by proliferation and migration, and that diabetic patients have worse clinical outcomes. The aim of this study was to determine the functional and gene expression outcomes of matrix metalloproteinase-2 (Mmp-2) silencing in fibroblasts cultured under hyperglycemia and euglycemia with hypoxic and normoxic stimuli. AKR-2B fibroblasts were stably transduced using lentivirus-mediated shRNA-Mmp-2 or scrambled controls and subjected to hypoxia or normoxia under hyperglycemic or euglycemic conditions for 24 and 72 h. Gene expression of vascular endothelial growth factor-A (Vegf-A), Vegfr-1, Mmp-2, Mmp-9 and tissue inhibitors of matrix metalloproteinases (Timps) were determined by RT-PCR. Collagen I and IV secretion and cellular proliferation and migration were determined. Under hyperglycemic conditions, there is a significant reduction in the average gene expression of Vegf-A and Mmp-9, with an increase in Timp-1 at 24 h of hypoxia (p < 0.05) in Mmp-2-silenced fibroblasts when compared to controls. In addition, there is a decrease in collagen I and IV secretion and cellular migration. The euglycemic cells were able to reverse these findings. These findings demonstrate the rationale for using anti-Mmp-2 therapy in dialysis patients with hemodialysis vascular access in helping to reduce stenosis formation. © 2016 The Author(s) Published by S. Karger AG, Basel.

Tissue distribution of very late activation antigens-1/6 and very late activation antigen ligands in the normal thymus and in thymoma.

PubMed Central

Ruco, L. P.; Paradiso, P.; Pittiglio, M.; Diodoro, M. G.; Gearing, A. J.; Mainiero, F.; Gismondi, A.; Santoni, A.; Baroni, C. D.

1993-01-01

The expression of very late activation antigens (VLAs)-1/6 was correlated with that of the VLA ligands fibronectin, laminin, collagen, and vascular cell adhesion molecule-1 in sections of normal thymus, in thymocyte suspensions, and in 10 cases of thymoma. Capsular epithelial cells are VLA-2+, VLA-3+, and VLA-6+ and face the thymic basement membrane, which is rich in fibronectin, laminin, and collagen type IV. Cortical epithelial cells are VLA-2+ and are embedded in a reticular meshwork of nonorganized extracellular matrix (ECM) that is rich in fibronectin. Cortical thymocytes, identified as CD3dim cells by using immunofluorescence in suspension, are highly positive for VLA-4, a fibronectin ligand. Most cortical macrophages are positive for vascular cell adhesion molecule-1, a molecule recognized by VLA-4. Medullary epithelial cells are VLA-2+/VLA-3+ and are codistributed with fibrous strands of organized ECM that are positive for fibronectin, collagen, and laminin. Medullary thymocytes, identified as CD3bright cells, are positive for VLA-4 and VLA-6, a ligand for laminin. Our findings suggest that intrathymic thymocyte maturation is associated with changes in expression of VLA molecules, which are apparently correlated with the presence of VLA ligands in the tissue microenvironment. Thymomas were classified as cortical (three), common (five), or medullary (two) type. Expression of VLA molecules and distribution of ECM in the three histological subtypes were reminiscent of those observed in the respective regions of the normal thymus. All cases of thymoma were characterized by overexpression of VLA molecules on neoplastic cells, which was associated with increased deposition of organized ECM rich in fibronectin, laminin, and collagen. Images Figure 1 Figure 3 PMID:8456937

Bovine Respiratory Syncytial Virus and Histophilus somni Interaction at the Alveolar Barrier

PubMed Central

Agnes, J. T.; Zekarias, B.; Shao, M.; Anderson, M. L.; Gershwin, L. J.

2013-01-01

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection. PMID:23649093

Bovine respiratory syncytial virus and Histophilus somni interaction at the alveolar barrier.

PubMed

Agnes, J T; Zekarias, B; Shao, M; Anderson, M L; Gershwin, L J; Corbeil, L B

2013-07-01

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection.

Thrombosis Is Reduced by Inhibition of COX-1, but Unaffected by Inhibition of COX-2, in an Acute Model of Platelet Activation in the Mouse

PubMed Central

Armstrong, Paul C.; Kirkby, Nicholas S.; Zain, Zetty N.; Emerson, Michael; Mitchell, Jane A.; Warner, Timothy D.

2011-01-01

Background Clinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI2, formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice. Methodology/Principal Findings A non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg−1, i.v.) and parecoxib (0.5 mg.kg−1, i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A2-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist. Conclusions/Significance Inhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA2, but not that induced by U46619, which is independent of platelet TXA2. These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis. PMID:21629780

The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

PubMed Central

Fink, Doran L.; Green, Bruce A.; St. Geme III, Joseph W.

2002-01-01

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Haps, from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Haps. Purified Haps binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Haps to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract. PMID:12183535

Effect of [6]-gingerol on myofibroblast differentiation in transforming growth factor beta 1-induced nasal polyp-derived fibroblasts.

PubMed

Park, Sook A; Park, Il-Ho; Cho, Jung-Sun; Moon, You-Mi; Lee, Seung Hoon; Kim, Tae Hoon; Lee, Sang Hag; Lee, Heung-Man

2012-01-01

[6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp-derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2″,7″-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1-induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.

Glomerular basement membrane injuries in IgA nephropathy evaluated by double immunostaining for α5(IV) and α2(IV) chains of type IV collagen and low-vacuum scanning electron microscopy.

PubMed

Masuda, Yukinari; Yamanaka, Nobuaki; Ishikawa, Arimi; Kataoka, Mitue; Arai, Takashi; Wakamatsu, Kyoko; Kuwahara, Naomi; Nagahama, Kiyotaka; Ichikawa, Kaori; Shimizu, Akira

2015-06-01

The glomerulus contains well-developed capillaries, which are at risk of injury due to high hydrostatic pressure, hyperfiltration, hypertension and inflammation. However, the pathological alterations of the injured glomerular basement membrane (GBM), the main component of the glomerular filtration barrier, are still uncertain in cases of glomerulonephritis. We examined the alterations of the GBM in 50 renal biopsy cases with IgA nephropathy (31.8 ± 17.6 years old) using double immunostaining for the α2(IV) and α5(IV) chains of type IV collagen, and examining the ultrastructural alterations by transmission electron microscopy (TEM) and low-vacuum scanning electron microscopy (LV-SEM). The GBM of IgA nephropathy cases showed various morphological and qualitative alterations. In the TEM findings, thinning, gaps, rupture, thickening with a lamellar and reticular structure and double contours were detected in the GBM. Double immunostaining for α5(IV) and α2(IV) showed thickening of the GBM with reduced α5(IV) and increased α2(IV), or mosaic images of α5(IV) and α2(IV), and holes, fractures, spiny projections and rupture of α5(IV) in the GBM. In addition, LV-SEM showed an etched image and multiple holes in a widening and wavy GBM. These findings might be associated with the development of a brittle GBM in IgA nephropathy. Glomerular basement membrane alterations were frequently noted in IgA nephropathy, and were easily evaluated by double immunostaining for α2(IV) and α5(IV) of type IV collagen and LV-SEM. The application of these analyses to human renal biopsy specimens may enhance our understanding of the alterations of the GBM that occur in human glomerular diseases.

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts.

PubMed

Bratka-Robia, Christine B; Mitteregger, Gerda; Aichinger, Amanda; Egerbacher, Monika; Helmreich, Magdalena; Bamberg, Elmar

2002-02-01

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.

Type V Collagen is Persistently Altered after Inguinal Hernia Repair.

PubMed

Lorentzen, L; Henriksen, N A; Juhl, P; Mortensen, J H; Ågren, M S; Karsdal, M A; Jorgensen, L N

2018-04-01

Hernia formation is associated with alterations of collagen metabolism. Collagen synthesis and degradation cause a systemic release of products, which are measurable in serum. Recently, we reported changes in type V and IV collagen metabolisms in patients with inguinal and incisional hernia. The aim of this study was to determine if the altered collagen metabolism was persistent after hernia repair. Patients who had undergone repairs for inguinal hernia (n = 11) or for incisional hernia (n = 17) were included in this study. Patients who had undergone elective cholecystectomy served as controls (n = 10). Whole venous blood was collected 35-55 months after operation. Biomarkers for type V collagen synthesis (Pro-C5) and degradation (C5M) and those for type IV collagen synthesis (P4NP) and degradation (C4M2) were measured by a solid-phase competitive assay. The turnover of type V collagen (Pro-C5/C5M) was slightly higher postoperatively when compared to preoperatively in the inguinal hernia group (P = 0.034). In addition, the results revealed a postoperatively lower type V collagen turnover level in the inguinal hernia group compared to controls (P = 0.012). In the incisional hernia group, the type V collagen turnover was higher after hernia repair (P = 0.004) and the postoperative turnover level was not different from the control group (P = 0.973). Patients with an inguinal hernia demonstrated a systemic and persistent type V collagen turnover alteration. This imbalance of the collagen metabolism may be involved in the development of inguinal hernias.

Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens.

PubMed Central

Gilmour, D T; Lyon, G J; Carlton, M B; Sanes, J R; Cunningham, J M; Anderson, J R; Hogan, B L; Evans, M J; Colledge, W H

1998-01-01

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin/BM40) is a secreted Ca2+-binding glycoprotein that interacts with a range of extracellular matrix molecules, including collagen IV. It is widely expressed during embryogenesis, and in vitro studies have suggested roles in the regulation of cell adhesion and proliferation, and in the modulation of cytokine activity. In order to analyse the function of this protein in vivo, the endogenous Sparc locus was disrupted by homologous recombination in murine embryonic stem cells. SPARC-deficient mice (Sparctm1Cam) appear normal and fertile until around 6 months of age, when they develop severe eye pathology characterized by cataract formation and rupture of the lens capsule. The first sign of lens pathology occurs in the equatorial bow region where vacuoles gradually form within differentiating epithelial cells and fibre cells. The lens capsule, however, shows no qualitative changes in the major basal lamina proteins laminin, collagen IV, perlecan or entactin. These mice are an excellent resource for further studies on how SPARC affects cell behaviour in vivo. PMID:9524110

Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

PubMed

Kawaguchi, Kohei; Endo, Akinori; Fukushima, Toshiaki; Madoka, Yuka; Tanaka, Toshiaki; Komada, Masayuki

2018-05-15

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion. Copyright © 2018 Elsevier Inc. All rights reserved.

Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

PubMed Central

2014-01-01

Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases. PMID:24935095

Advanced Glycation End-Products Induce Connective Tissue Growth Factor-Mediated Renal Fibrosis Predominantly through Transforming Growth Factor β-Independent Pathway

PubMed Central

Zhou, Guihua; Li, Cai; Cai, Lu

2004-01-01

Advanced glycation end-products (AGEs) play a critical role in diabetic nephropathy by stimulating extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) is a potent inducer of ECM synthesis and increases in the diabetic kidneys. To determine the critical role of CTGF in AGE-induced ECM accumulation leading to diabetic nephropathy, rats were given AGEs by intravenous injection for 6 weeks. AGE treatment induced a significant renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) accumulation in glomeruli, and a mild renal dysfunction, as shown by increases in urinary volume and protein content. AGE treatment also caused significant increases in renal CTGF and transforming growth factor (TGF)-β1 mRNA and protein expression. Direct exposure of rat mesangial cells to AGEs in vitro significantly induced increases in fibronectin and Col IV production, which could be completely prevented by pretreatment with anti-CTGF antibody. AGE treatment also significantly increased both TGF-β1 and CTGF mRNA expression; however, inhibition of TGF-β1 mRNA expression by shRNA or neutralization of TGF-β1 protein by anti-TGF-β1 antibody did not significantly prevent AGE-increased expression of CTGF mRNA and protein. These results suggest that AGE-induced CTGF expression, predominantly through a TGF-β1-independent pathway, plays a critical role in renal ECM accumulation leading to diabetic nephropathy. PMID:15579446

Radio frequency glow discharge-induced acidification of fluoropolymers.

PubMed

Krawczyk, Benjamin M; Baltrusaitis, Jonas; Yoder, Colin M; Vargo, Terrence G; Bowden, Ned B; Kader, Khalid N

2011-12-01

Fluoropolymer surfaces are unique in view of the fact that they are quite inert, have low surface energies, and possess high thermal stabilities. Attempts to modify fluoropolymer surfaces have met with difficulties in that it is difficult to control the modification to maintain bulk characteristics of the polymer. In a previously described method, the replacement of a small fraction of surface fluorine by acid groups through radio frequency glow discharge created a surface with unexpected reactivity allowing for attachment of proteins in their active states. The present study demonstrates that 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) reacts with the acid groups on fluoropolymer surfaces in a novel reaction not previously described. This reaction yields an excellent leaving group in which a primary amine on proteins can substitute to form a covalent bond between a protein and these surfaces. In an earlier study, we demonstrated that collagen IV could be deposited on a modified PTFE surface using EDC as a linker. Once collagen IV is attached to the surface, it assembles to form a functional stratum resembling collagen IV in native basement membrane. In this study, we show data suggesting that the fluorine to carbon ratio determines the acidity of the fluoropolymer surfaces and how well collagen IV attaches to and assembles on four different fluoropolymer surfaces. Copyright © 2011 Wiley Periodicals, Inc.

The Collagen Binding Protein Cnm Contributes to Oral Colonization and Cariogenicity of Streptococcus mutans OMZ175

PubMed Central

Miller, James H.; Avilés-Reyes, Alejandro; Scott-Anne, Kathy; Gregoire, Stacy; Watson, Gene E.; Sampson, Edith; Progulske-Fox, Ann; Koo, Hyun; Bowen, William H.; Lemos, José A.

2015-01-01

Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm+ native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm+ isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity. PMID:25733523

TRAF3IP2 Mediates Aldosterone/Salt-Induced Cardiac Hypertrophy and Fibrosis

PubMed Central

Sakamuri, Siva S.V.P; Valente, Anthony J.; Siddesha, Jalahalli M.; Delafontaine, Patrice; Siebenlist, Ulrich; Gardner, Jason D.; Chandrasekar, Bysani

2016-01-01

Aberrant activation of the renin-angiotensin-aldosterone system (RAAS) contributes to adverse cardiac remodeling and eventual failure. Here we investigated whether TRAF3-interacting Protein 2 (TRAF3IP2), a redox-sensitive cytoplasmic adaptor molecule and an upstream regulator of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), mediates aldosterone-induced cardiac hypertrophy and fibrosis. Wild type (WT) and TRAF3IP2-null mice were infused with aldosterone (0.2mg/kg/day) for 4 weeks along with 1%NaCl in drinking water. Aldosterone/salt, but not salt alone, upregulated TRAF3IP2 expression in WT mouse hearts. Aldosterone elevated blood pressure to a similar extent in both WT and TRAF3IP2-null groups. Importantly, TRAF3IP2 gene deletion attenuated aldosterone/salt-induced (i) p65 and c-Jun activation, (ii) extracellular matrix (collagen Iα1 and collagen 3α1), matrix metalloproteinase (MMP2), lysyl oxidase (LOX), inflammatory cytokine (IL-6 and IL-18), chemokine (CXCL1 and CXCL2), and adhesion molecule (ICAM1) gene expression in hearts, (iii) IL-6, IL-18, and MMP2 protein levels, (iv) systemic IL-6 and IL-18 levels, and (iv) cardiac hypertrophy and fibrosis. These results indicate that TRAF3IP2 is a critical signaling intermediate in aldosterone/salt-induced myocardial hypertrophy and fibrosis, and thus a potential therapeutic target in hypertensive heart disease. PMID:27040306

TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis.

PubMed

Sakamuri, Siva S V P; Valente, Anthony J; Siddesha, Jalahalli M; Delafontaine, Patrice; Siebenlist, Ulrich; Gardner, Jason D; Bysani, Chandrasekar

2016-07-05

Aberrant activation of the renin-angiotensin-aldosterone system (RAAS) contributes to adverse cardiac remodeling and eventual failure. Here we investigated whether TRAF3 Interacting Protein 2 (TRAF3IP2), a redox-sensitive cytoplasmic adaptor molecule and an upstream regulator of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), mediates aldosterone-induced cardiac hypertrophy and fibrosis. Wild type (WT) and TRAF3IP2-null mice were infused with aldosterone (0.2 mg/kg/day) for 4 weeks along with 1%NaCl in drinking water. Aldosterone/salt, but not salt alone, upregulated TRAF3IP2 expression in WT mouse hearts. Further, aldosterone elevated blood pressure to a similar extent in both WT and TRAF3IP2-null groups. However, TRAF3IP2 gene deletion attenuated aldosterone/salt-induced (i) p65 and c-Jun activation, (ii) extracellular matrix (collagen Iα1 and collagen IIIα1), matrix metalloproteinase (MMP2), lysyl oxidase (LOX), inflammatory cytokine (IL-6 and IL-18), chemokine (CXCL1 and CXCL2), and adhesion molecule (ICAM1) mRNA expression in hearts, (iii) IL-6, IL-18, and MMP2 protein levels, (iv) systemic IL-6 and IL-18 levels, and (iv) cardiac hypertrophy and fibrosis. These results indicate that TRAF3IP2 is a critical signaling intermediate in aldosterone/salt-induced myocardial hypertrophy and fibrosis, and thus a potential therapeutic target in hypertensive heart disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Immunohistochemical, morphological and histometrical analyses of follicular development in Astyanax bimaculatus (Teleostei: Characidae) exposed to an organochlorine insecticide.

PubMed

Marcon, Lucas; Thomé, Ralph Gruppi; Mounteer, Ann Honor; Bazzoli, Nilo; Rizzo, Elizete; Benjamin, Laércio Dos Anjos

2017-09-01

Thiodan ® is an organochlorine insecticide used in agriculture that can reach aquatic ecosystems where it can affect fish reproduction. This research aimed to evaluate follicular development and the expression of integrin β1, collagen type IV and caspase 3 by morphological, histometrical and immunohistochemical analyses of Astyanax bimaculatus exposed to Thiodan ® . Treatments included three sublethal concentrations of Thiodan ® (1.15, 2.30, and 5.60μgL -1 ) for 96h and a control without the insecticide. Mature females with ovaries in advanced follicular development were chosen for study (average weight: 11.52 ± 2.0g; average total length: 9.12 ± 0.64cm). The follicles of A. bimaculatus exhibited normal morphology, while the diameters of secondary follicles showed an increase (P< 0.05) in all concentrations when compared to the control group; a characteristic of follicles undergoing the initial stages of intoxication. Immunohistochemical analysis revealed secondary follicles with greater expression of integrin β1 and collagen type IV in cytoplasm of follicular cells than in the primary follicles in all treatments and in the control. Immunolocalization of caspase 3 was detected in follicular cells during secondary development in all tested concentrations of Thiodan ® and the control. These analyses demonstrate positive immunolocalization throughout the course of follicular development, even in fish exposed to varying concentrations of Thiodan ® for 96h, demonstrating that follicular cells retain their physiological integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of the NC1 domain of {alpha}3 chain as critical for {alpha}3{alpha}4{alpha}5 type IV collagen network assembly.

PubMed

LeBleu, Valerie; Sund, Malin; Sugimoto, Hikaru; Birrane, Gabriel; Kanasaki, Keizo; Finan, Elizabeth; Miller, Caroline A; Gattone, Vincent H; McLaughlin, Heather; Shield, Charles F; Kalluri, Raghu

2010-12-31

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

Directing Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Impact of Medium Composition and Culture Condition.

PubMed

Petry, L; Kippenberger, S; Meissner, M; Kleemann, J; Kaufmann, R; Rieger, U M; Wellenbrock, S; Reichenbach, G; Zöller, N; Valesky, E

2018-04-28

Adipose-derived stem cells (ASC) are known to transdifferentiate into a wide range of different cell species in vitro including along the epidermal lineage. This property makes them a promising tool for regenerative medicine in order to restore the epidermal barrier. The present study is dedicated to identify in vitro conditions enabling transdifferentiation to a keratinocyte-like phenotype. Especially, the impact of different culture conditions (media compositions, 2D-, 3D-cultures) and extracellular matrix (ECM) molecules was evaluated. ASC derived from subcutaneous abdominal fat were characterized by stemness associated markers and subjected to different media. Epithelial differentiation in 2D cultures was monitored by pan-cytokeratin expression using flow cytometry and immunocytochemistry. In order to evaluate the impact of different ECM molecules on epidermal stratification, 3D cultures were produced, lifted to the air-liquid-interface (ALI) and examined by histological analysis and quantitative real-time RT-PCR. We identified a medium composition containing retinoic acid, hydrocortisone, ascorbic acid and BMP-4 enabling maximum pan-cytokeratin expression in 2D cultures. Moreover, adhesion to type IV collagen further promotes the pan-cytokeratin expression. When cultures were lifted to the ALI, significant stratification was observed, particularly in supports coated with type IV collagen or fibronectin. Moreover, epidermal differentiation markers (involucrin, cytokeratin 1 and 14) become induced. Conditions with hampered wound healing such as non-healing ulcers demand new treatment regimes. The here introduced optimized protocols for transdifferentiation of ASC into keratinocyte-like cells may help to establish more effective treatment procedures. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

PubMed

Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

A novel taspine derivative, HMQ1611, suppresses adhesion, migration and invasion of ZR-75-30 human breast cancer cells.

PubMed

Zhan, Yingzhuan; Wang, Nan; Liu, Cuicui; Chen, Yinnan; Zheng, Lei; He, Langchong

2014-05-01

Taspine was screened for the first time from Radix et Rhizoma leonticis (Hong Mao Qi in Chinese) using cell membrane chromatography in our laboratory. Its anticancer and antiangiogenic properties were demonstrated, and it could serve as a lead compound in anticancer agent development. Here, we investigated the role of one of the derivatives, HMQ1611, with increased activity and solubility, on the regulation of breast cancer cell ZR-75-30 adhesion, migration and invasion. The effect of HMQ1611 on adhesion, invasion and migration of human breast cancer cells ZR-75-30 was examined. The migration and invasive potential of ZR-75-30 cells were examined by wound-healing assays and matrigel invasion chamber assays. The adhesion to type IV collagen and laminin were evaluated by MTT assay. The expression and proteinase activity of two matrix metalloproteinases (MMPs), matrix metalloproteinases 2 (MMP-2) and matrix metalloproteinases 9 (MMP-9), were analyzed by Western blot analysis and gelatin zymography, respectively. HMQ1611 effectively inhibited ZR-75-30 cell invasion and significantly suppressed adhesion to type IV collagen and laminin-coated substrate in a dose-dependent manner. Western blot and gelatin zymography analysis showed that HMQ1611 significantly inhibited the expression and secretion of MMP-2 and MMP-9 in ZR-75-30 cells. Additionally, treatment of ZR-75-30 cells with HMQ1611 downregulated the expression of MMP-2 and MMP-9. HMQ1611 had potential to suppress the adhesion, migration and invasion of ZR-75-30 cancer cells, and it could serve as a potential novel therapeutic candidate for the treatment of metastatic breast cancer.

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression.

PubMed

Wierer, Michael; Prestel, Matthias; Schiller, Herbert B; Yan, Guangyao; Schaab, Christoph; Azghandi, Sepiede; Werner, Julia; Kessler, Thorsten; Malik, Rainer; Murgia, Marta; Aherrahrou, Zouhair; Schunkert, Heribert; Dichgans, Martin; Mann, Matthias

2018-02-01

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Compartment-resolved Proteomic Analysis of Mouse Aorta during Atherosclerotic Plaque Formation Reveals Osteoclast-specific Protein Expression*

PubMed Central

Wierer, Michael; Prestel, Matthias; Schiller, Herbert B.; Yan, Guangyao; Schaab, Christoph; Azghandi, Sepiede; Werner, Julia; Kessler, Thorsten; Malik, Rainer; Murgia, Marta; Aherrahrou, Zouhair; Schunkert, Heribert; Dichgans, Martin; Mann, Matthias

2018-01-01

Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)-based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function. PMID:29208753

A mouse collagen4α4 mutation causing Alport glomerulosclerosis with abnormal collagen α3α4α5(IV) trimers

PubMed Central

Korstanje, Ron; Caputo, Christina; Doty, Rosalinda; Cook, Susan; Bronson, Roderick; Davisson, Muriel; Miner, Jeffrey H.

2013-01-01

A spontaneous mutation termed bilateral wasting kidneys (bwk) was identified in a colony of NONcNZO recombinant inbred mice. These mice exhibit a rapid increase of urinary albumin at an early age associated with glomerulosclerosis, interstitial nephritis, and tubular atrophy. The mutation was mapped to a location on Chromosome 1 containing the Col4a3 and Col4a4 genes, for which mutations in the human orthologs cause the hereditary nephritis Alport syndrome. DNA sequencing identified a G to A mutation in the conserved GT splice donor of Col4a4 intron 30, resulting in skipping of exon 30 but maintaining the mRNA reading frame. Protein analyses showed that mutant collagen α3α4α5(IV) trimers were secreted and incorporated into the glomerular basement membrane (GBM), but levels were low, and GBM lesions typical of Alport syndrome were observed. Moving the mutation into the more renal damage-prone DBA/2J and 129S1/SvImJ backgrounds revealed differences in albuminuria and its rate of increase, suggesting an interaction between the Col4a4 mutation and modifier genes. This novel mouse model of Alport syndrome is the only one shown to accumulate abnormal collagen α3α4α5(IV) in the GBM, as also found in a subset of Alport patients. These mice will be valuable for testing potential therapies, for understanding abnormal collagen IV structure and assembly, for gaining better insights into the mechanisms leading to Alport syndrome and to the variability in the age of onset and associated phenotypes. PMID:24522496

Apoptotic cell infusion treats ongoing collagen-induced arthritis, even in the presence of methotrexate, and is synergic with anti-TNF therapy.

PubMed

Bonnefoy, Francis; Daoui, Anna; Valmary-Degano, Séverine; Toussirot, Eric; Saas, Philippe; Perruche, Sylvain

2016-08-11

Apoptotic cell-based therapies have been proposed to treat chronic inflammatory diseases. The aim of this study was to investigate the effect of intravenous (i.v.) apoptotic cell infusion in ongoing collagen-induced arthritis (CIA) and the interaction of this therapy with other treatments used in rheumatoid arthritis (RA), including methotrexate (MTX) or anti-TNF therapy. The effects of i.v. apoptotic cell infusion were evaluated in a CIA mouse model in DBA/1 mice immunized with bovine type II collagen. The number and functions of antigen-presenting cells (APC), regulatory CD4(+) T cells (Treg), and circulating anti-collagen auto-antibodies were analyzed in CIA mice. Treatment of arthritic mice with i.v. apoptotic cell infusion significantly reduced the arthritis clinical score. This therapeutic approach modified T cell responses against the collagen auto-antigen with selective induction of collagen-specific Treg. In addition, we observed that APC from apoptotic-cell-treated animals were resistant to toll-like receptor ligand activation and favored ex vivo Treg induction, indicating APC reprogramming. Apoptotic cell injection-induced arthritis modulation was dependent on transforming growth factor (TGF)-β, as neutralizing anti-TGF-β antibody prevented the effects of apoptotic cells. Methotrexate did not interfere, while anti-TNF therapy was synergic with apoptotic-cell-based therapy. Overall, our data demonstrate that apoptotic-cell-based therapy is efficient in treating ongoing CIA, compatible with current RA treatments, and needs to be evaluated in humans in the treatment of RA.

Matrix Metalloproteinase Dysregulation in the Stria Vascularis of Mice with Alport Syndrome

PubMed Central

Gratton, Michael Anne; Rao, Velidi H.; Meehan, Daniel T.; Askew, Charles; Cosgrove, Dominic

2005-01-01

Alport syndrome results from mutations in genes encoding collagen α3(IV), α4(IV), or α5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism. PMID:15855646

Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices.

PubMed

Katow, H

1986-02-01

The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes. These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate. The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer. The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM). The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably. However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel. This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells. These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.

The matricellular protein periostin contributes to proper collagen function and is downregulated during skin aging.

PubMed

Egbert, M; Ruetze, M; Sattler, M; Wenck, H; Gallinat, S; Lucius, R; Weise, J M

2014-01-01

Periostin is a secreted 90kDa matricellular protein, which is predominantly expressed in collagen-rich tissues. Collagen is the most abundant protein in mammals and has great tensile strength. Recent investigations have shown that periostin influences collagen fibrillogenesis and biomechanical properties of murine connective tissues. We investigated the function of periostin concerning collagen homeostasis during intrinsic and extrinsic skin aging. For this purpose, human skin samples of young and old donors as well as samples of photoaged and sun-protected skin areas were analyzed for periostin expression. Using in vitro models, we determined the cell types responsible for periostin expression and performed functional analyses with periostin knockdown cells. TaqMan Real-Time PCR, UV irradiation, knockdown experiments, immunostaining, electron microscopy, collagen degradation assay, collagen crosslink analysis. Periostin expression is highest in the papillary dermis and downregulated during skin aging. Fibroblasts and non-follicular skin derived precursors were identified as main source for periostin expression in human skin. Periostin knockdown in fibroblasts has no effect on collagen expression, but results in an increased fibril diameter and aberrant collagen structure. This leads to an increased susceptibility of collagen toward proteases, whereas recombinant periostin protects collagen fibrils from degradation. Our data show that periostin plays an important role for proper collagen assembly and homeostasis. During skin aging periostin expression decreases and contributes to the phenotype of aged skin. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Biological Differences between Hanwoo longissimus dorsi and semimembranosus Muscles in Collagen Synthesis of Fibroblasts.

PubMed

Subramaniyan, Sivakumar Allur; Hwang, Inho

2017-01-01

Variations in physical toughness between muscles and animals are a function of growth rate and extend of collagen type I and III. The current study was designed to investigate the ability of growth rate, collagen concentration, collagen synthesizing and degrading genes on two different fibroblast cells derived from Hanwoo m. longissimus dorsi (LD) and semimembranosus (SM) muscles. Fibroblast cell survival time was determined for understanding about the characteristics of proliferation rate between the two fibroblasts. We examined the collagen concentration and protein expression of collagen type I and III between the two fibroblasts. The mRNA expression of collagen synthesis and collagen degrading genes to elucidate the molecular mechanisms on toughness and tenderness through collagen production between the two fibroblast cells. From our results the growth rate, collagen content and protein expression of collagen type I and III were significantly higher in SM than LD muscle fibroblast. The mRNA expressions of collagen synthesized genes were increased whereas the collagen degrading genes were decreased in SM than LD muscle. Results from confocal microscopical investigation showed increased fluorescence of collagen type I and III appearing stronger in SM than LD muscle fibroblast. These results implied that the locomotion muscle had higher fibroblast growth rate, leads to produce more collagen, and cause tougher than positional muscle. This in vitro study mirrored that background toughness of various muscles in live animal is likely associated with fibroblast growth pattern, collagen synthesis and its gene expression.

[Effects of pirfenidone on hepatic fibrosis in mice induced by carbon tetrachloride].

PubMed

Xiao, Min; Qu, Xiao-Hu; Lv, Jv-Ping; Shi, Yang; Li, Chang-Xi; Xie, Ke-Jian

2016-04-08

To investigate the effects of pirfenidone on CCl4-induced liver fibrosis in mice. After 8-week feeding, 40 healthy male SPF ICR mice were randomly divided into 4 groups:liver fibrosis group (CCL 4 group), low doses of Pirfenidone group (PFD-L group), high doses of Pirfenidone group (PFD-H group) and control group. The mice in CCL 4 group, low doses of Pirfenidone group (PFD-L group), high doses of Pirfenidone group (PFD-H group) were injected intraperitoneally with 0.4 ml 10% CCL 4 solution dissolved in soybean oil. Then the PFD-L and PFD-H groups were treated with 120 mg and 240 mg PFD via gastric gavage, respectively. Control group was injected with same volume of saline. Alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP) in serum were tested with automatic biochemistry analyzer and the pathologic changes of liver tissue were examined by HE staining. Furthermore, we identi-fied hyaluronic acids(HA), laminin(LN), collagentype IV(IV-C) in serum using radioimmunoassay and the expression of smooth muscle acti-nalpha(α-SMA) related gene in liver was tested by real-time fluorescence quantitative PCR. Compared with control group, hepatic lobules in CCL 4 mice were damaged significantly, collagenous fiber was deposited obviously, and counterfeit hepatic lobules formed. The serum levels of ALT, AST, ALP were increased obviously ( P <0.05) with the enhancement of HA, LN, IV-C in serum ( P <0.05) and the ex-pression of α-SMA related gene ( P <0.05). Compared to CCL 4 -treated mice, the serum levels of ALT, AST, ALP in PFD-L and PFD-H groups were decreased, HA, LN, IV-C in PFD-L and PFD-H mice went down obviously,and the expression of α-SMA related gene was con-trolled ( P <0.05). From pathological observation, we found the degree of liver fibrosis in PFD-L mice was reduced and collagenous fiber was decreased, only a little counterfeit hepatic lobule could be found. Cell arrangement in PFD-H mice recovered, the structural of hepatic lobules disordered and no obvious counterfeit hepatic lobules were found. Therefore, the recovery of PFD-H group was better than PFD-L group. Pirfenidone has a protective role in improving the outcome of the liver fibrosis and it may become a new direction of early intervention in liver fibrosis.

Diagnostic efficiency of amylase and type IV collagen in predicting chronic pancreatitis.

PubMed

Das, Subir Kumar; Varadhan, Sowmya; Dhanya, L; Mukherjee, Sukhes; Mohana, S; Balakrishnan, V; Vasudevan, D M

2009-01-01

Chronic pancreatitis, an irreversible inflammatory disease of the pancreas, is associated with the replacement of the destroyed parenchyma by extended development of fibrosis. Despite marked progress in diagnostic tools, no consensus has been reached in diagnosis of chronic pancreatitis. In this study we examined the hematological and biochemical parameters among 40 chronic pancreatitis patients within 18 to 67 yrs. ESR level and ALP activity was elevated in 40% cases. Serum amylase activity increased in 32 patients and it showed significant correlation with ALP (r=0.458, p=0.003), CA-19.9 (r=0.556, p<0.001), and calcium level (r=-0.472, p=0.002). Type IV collagen level in chronic pancreatitis also elevated (164.4 ± 55.5 ng/ml) and showed negative significant correlation with calcium level (r= -0.505, p=0.001). However, no significant correlation was observed between amylase activity and type IV collagen (r=0.289, p= 0.07).

Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells.

PubMed

Filla, Mark S; Dimeo, Kaylee D; Tong, Tiegang; Peters, Donna M

2017-12-01

Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a pre-formed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids.

PubMed

Oktem, G; Vatansever, S; Ayla, S; Uysal, A; Aktas, S; Karabulut, B; Bilir, A

2006-02-01

Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the Docetaxel group.

Immunohistochemical diagnosis of Alport's syndrome in paraffin-embedded renal sections: antigen retrieval with autoclave heating.

PubMed

Naito, Ichiro; Ninomiya, Yoshifumi; Nomura, Shinsuke

2003-03-01

Alport's syndrome (AS) is a hereditary renal disease caused by mutations in the genes encoding collagen type IV. Immunohistochemical analysis of the alpha chains of collagen type IV has been found to be useful for the diagnosis of this disease. The monoclonal antibodies (mAbs) generated by us recognize alpha 1(IV) through alpha 6(IV) chains of collagen type IV on fresh-frozen sections but not on paraffin-embedded sections. Antigen retrieval by autoclave heating has been found to restore the epitopes recognized by the mAbs; however the heating conditions had not been well established. In this study, the heating conditions were carefully examined using renal sections obtained from AS and non-AS patients. The heating was performed in an autoclave, at 105 degrees -127 degrees C for 6-8 min. During the heating, the sections were immersed in 0.2 N HCl solution (pH 0.9). Then, the mAbs were applied for 30 min, and the bound mAbs were detected using the LSAB kit. The optimal temperature for the antigen retrieval varied among specimens, and was dependent on the type of basement membrane examined. Thus, it was considered that heating at two or three different temperatures could be helpful for the precise diagnosis of AS. Adopting the antigen retrieval method could extend the possibility of immunohistochemical diagnosis of AS to cases without using fresh-frozen sections.

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36

PubMed Central

Munkonda, Mercedes N.; Akbari, Shareef; Landry, Chloe; Sun, Suzy; Xiao, Fengxia; Turner, Maddison; Holterman, Chet E.; Nasrallah, Rania; Hébert, Richard L.; Kennedy, Christopher R. J.; Burger, Dylan

2018-01-01

ABSTRACT Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100–1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-β) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease. PMID:29435202

A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts.

PubMed

Dupont-Deshorgue, A; Oudart, J B; Brassart, B; Deslee, G; Perotin, J M; Diebold, M D; Monboisse, J C; Ramont, L; Brassart-Pasco, S

2015-08-01

Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression. Copyright © 2015 Elsevier Inc. All rights reserved.

Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

PubMed

Hsu, Han-Hsiu; Uemura, Toshimasa; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

2016-08-01

Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mechanical stretching stimulates collagen synthesis via down-regulating SO2/AAT1 pathway

PubMed Central

Liu, Jia; Yu, Wen; Liu, Yan; Chen, Selena; Huang, Yaqian; Li, Xiaohui; Liu, Cuiping; Zhang, Yanqiu; Li, Zhenzhen; Du, Jie; Tang, Chaoshu; Du, Junbao; Jin, Hongfang

2016-01-01

The aim of the study was to investigate the role of endogenous sulfur dioxide (SO2)/ aspartate aminotransferase 1 (AAT1) pathway in stretch-induced excessive collagen expression and its mechanism. The mechanical stretch downregulated SO2/AAT1 pathway and increased collagen I and III protein expression. Importantly, AAT1 overexpression blocked the increase in collagen I and III expression, transforming growth factor-β1 (TGF- β1) expression and phosphorylation of Smad2/3 induced by stretch, but AAT1 knockdown mimicked the increase in collagen I and III expression, TGF- β1 expression and phosphorylation of Smad2/3 induced by stretch. Mechanistically, SB431542, a TGF-β1/Smad2/3 inhibitor, eliminated excessive collagen I and III accumulation induced by AAT1 knockdown, stretch or stretch plus AAT1 knockdown. In a rat model of high pulmonary blood flow-induced pulmonary vascular collagen accumulation, AAT1 expression and SO2 content in lung tissues of rat were reduced in shunt rats with high pulmonary blood flow. Supplement of SO2 derivatives inhibited activation of TGF- β1/Smad2/3 pathway and alleviated the excessive collagen accumulation in lung tissues of shunt rats. The results suggested that deficiency of endogenous SO2/AAT1 pathway mediated mechanical stretch-stimulated abnormal collagen accumulation via TGF-β1/Smad2/3 pathway. PMID:26880260

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

A PTEN-COL17A1 fusion gene and its novel regulatory role in Collagen XVII expression and GBM malignance.

PubMed

Yan, Xiaoyan; Zhang, Chuanbao; Liang, Tingyu; Yang, Fan; Wang, Haoyuan; Wu, Fan; Wang, Wen; Wang, Zheng; Cheng, Wen; Xu, Jiangnan; Jiang, Tao; Chen, Jing; Ding, Yaozhong

2017-10-17

Collagen XVII expression has recently been demonstrated to be correlated with the tumor malignance. While Collagen XVII is known to be widely distributed in neurons of the human brain, its precise role in pathogenesis of glioblastoma multiforme (GBM) is unknown. In this study, we identified and characterized a new PTEN-COL17A1 fusion gene in GMB using transcriptome sequencing. Although fusion gene did not result in measurable fusion protein production, its presence is accompanied with high levels of COL17A1 expression, revealed a novel regulatory mechanism of Collagen XVII expression by PTEN-COL17A1 gene fusion. Knocked down Collagen XVII expression in glioma cell lines resulted in decreased tumor invasiveness, along with significant reduction of MMP9 expression, while increased Collagen XVII expression promotes invasive activities of glioma cells and associated with GBM recurrences. Together, our results uncovered a new PTEN-COL17A1 fusion gene and its novel regulatory role in Collagen XVII expression and GBM malignance, and demonstrated that COL17A1 could serve as a useful prognostic biomarker and therapeutic targets for GBM.

Collagen crosslinks in chondromalacia of the patella.

PubMed

Väätäinen, U; Kiviranta, I; Jaroma, H; Arokosi, J; Tammi, M; Kovanen, V

1998-02-01

The aim of the study was to determine collagen concentration and collagen crosslinks in cartilage samples from chondromalacia of the patella. To study the extracellular matrix alterations associated to chondromalacia, we determined the concentration of collagen (hydroxyproline) and its hydroxylysylpyridinoline and lysylpyridinoline crosslinks from chondromalacia foci of the patellae in 12 patients and 7 controls from apparently normal cadavers. The structure of the collagen network in 8 samples of grades II-IV chondromalacia was examined under polarized light microscopy. The full-thickness cartilage samples taken with a surgical knife from chondromalacia lesions did not show changes in collagen, hydroxylysylpyridinoline and lysylpyridinoline concentration as compared with the controls. Polarized light microscopy showed decreased birefringence in the superficial cartilage of chondromalacia lesions, indicating disorganization or disappearance of collagen fibers in this zone. It is concluded that the collagen network shows gradual disorganization with the severity of chondromalacia lesion of the patella without changes in the concentration or crosslinks of collagen.

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis.

PubMed

Habgood, Anthony N; Tatler, Amanda L; Porte, Joanne; Wahl, Sharon M; Laurent, Geoffrey J; John, Alison E; Johnson, Simon R; Jenkins, Gisli

2016-06-01

Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

Postnatal changes and sexual dimorphism in collagen expression in mouse skin

PubMed Central

Arai, Koji Y.; Hara, Takuya; Nagatsuka, Toyofumi; Kudo, Chikako; Tsuchiya, Sho; Nomura, Yoshihiro; Nishiyama, Toshio

2017-01-01

To investigate sexual dimorphism and postnatal changes in skin collagen expression, mRNA levels of collagens and their regulatory factors in male and female skin were examined during the first 120 days of age by quantitative realtime PCR. Levels of mRNAs encoding extracellular matrices did not show any differences between male and female mice until day 15. Col1a1 and Col1a2 mRNAs noticeably increased at day 30 and remained at high levels until day 120 in male mice, while those in female mice remained at low levels during the period. Consistent with the mRNA expression, pepsin-soluble type I collagen contents in skin was very high in mature male as compared to female. Col3a1 mRNA in male mice also showed significantly high level at day 120 as compared to female. On the other hand, expression of mRNAs encoding TGF-ßs and their receptors did not show apparent sexual dimorphism although small significant differences were observed at some points. Castration at 60 days of age resulted in a significant decrease in type I collagen mRNA expression within 3 days, and noticeably decreased expression of all fibril collagen mRNAs examined within 14 days, while administration of testosterone tube maintained the mRNA expression at high levels. Despite the in vivo effect of testosterone, administration of physiological concentrations of testosterone did not affect fibril collagen mRNA expression in either human or mouse skin fibroblasts in vitro, suggesting that testosterone does not directly affect collagen expression in fibroblasts. In summary, present study demonstrated dynamic postnatal changes in expression of collagens and their regulatory factors, and suggest that testosterone and its effects on collagen expression are responsible for the skin sexual dimorphism but the effects of testosterone is not due to direct action on dermal fibroblasts. PMID:28494009

Losartan attenuates paraquat-induced pulmonary fibrosis in rats.

PubMed

Guo, F; Sun, Y B; Su, L; Li, S; Liu, Z F; Li, J; Hu, X T; Li, J

2015-05-01

Paraquat (PQ) is one of the most widely used herbicides in the world and can cause pulmonary fibrosis in the cases with intoxication. Losartan, an angiotensin II type 1 receptor antagonist, has beneficial effects on the treatment of fibrosis. The aim of this study was to examine the effect of losartan on pulmonary fibrosis in PQ-intoxicated rats. Adult male Sprague Dawley rats (n = 32, 180-220 g) were randomly assigned to four groups: (i) control group; (ii) PQ group; (iii) PQ + losartan 7d group; and (iv) PQ + losartan 14d group. Losartan treatment (intragastrically (i.g.), 10 mg/kg) was performed for 7 and 14 days after a single i.g. dose of 40 mg/kg PQ. All rats were killed on the 16th day, and hematoxylin-eosin and Masson's trichrome staining were used to examine lung injury and fibrosis. The levels of hydroxyproline and transforming growth factor β1 (TGF-β1), matrix metallopeptidase 9 (Mmp9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) messenger RNA (mRNA) expression and relative expression levels of collagen type I and III were also detected. PQ caused a significant increase in hydroxyproline content, mRNA expression of TGF-β1, Mmp9, and TIMP-1, and relative expression levels of collagen type I and III ( p < 0.05), while losartan significantly decreased the amount of hydroxyproline and downregulated TGF-β1, Mmp9, and TIMP-1 mRNA and collagen type I and III expressions ( p < 0.05). Histological examination of PQ-treated rats showed lung injury and widespread inflammatory cell infiltration in the alveolar space and pulmonary fibrosis, while losartan could markedly reduce such damage and prevent pulmonary fibrosis. The results of this study indicated that losartan could reduce lung damage and prevent pulmonary fibrosis induced by PQ. © The Author(s) 2014.

Lipo-PGE1 suppresses collagen production in human dermal fibroblasts via the ERK/Ets-1 signaling pathway.

PubMed

Yang, Yoolhee; Kim, Hee Jung; Woo, Kyong-Je; Cho, Daeho; Bang, Sa Ik

2017-01-01

Dysregulation of collagen production contributes to various pathological processes, including tissue fibrosis as well as impaired wound healing. Lipo-prostaglandin E1 (Lipo-PGE1), a lipid microsphere-incorporated prostaglandin E1, is used as a vasodilator for the treatment of peripheral vascular diseases. Lipo-PGE1 was recently shown to enhance human dermal fibroblast (HDF) migration and in vivo wound healing. No published study has characterized the role of Lipo-PGE1 in collagen regulation in HDFs. Here, we investigated the cellular signaling mechanism by which Lipo-PGE1 regulates collagen in HDFs. Collagen production was evaluated by the Sircol collagen assay, Western blot analysis of type I collagen and real time PCR. Unexpectedly, Lipo-PGE1 decreased mRNA expression of collagen 1A1, 1A2, and 3A1. Lipo-PGE1 markedly inhibited type I collagen and total soluble collagen production. In addition, Lipo-PGE1 inhibited transforming growth factor-β-induced collagen expression via Smad2 phosphorylation. To further investigate whether extracellular signal-regulated kinase (ERK)/Ets-1 signaling, a crucial pathway in collagen regulation, is involved in Lipo-PGE1-inhibited collagen production, cells were pretreated with an ERK-specific inhibitor, PD98059, prior to the addition of Lipo-PGE1. Lipo-PGE1-inhibited collagen mRNA expression and total soluble collagen production were recovered by pretreatment with PD98059. Moreover, Lipo-PGE1 directly induced the phosphorylation of ERK. Furthermore, silencing of Ets-1 recovered Lipo-PGE1-inhibited collagen production and PD98059 blocked Lipo-PGE1-enhanced Ets-1 expression. The present study reveals an important role for Lipo-PGE1 as a negative regulator of collagen gene expression and production via ERK/Ets-1 signaling. These results suggest that Lipo-PGE1 could potentially be a therapeutic target in diseases with deregulated collagen turnover.

Alport syndrome and Pierson syndrome: Diseases of the glomerular basement membrane.

PubMed

Funk, Steven D; Lin, Meei-Hua; Miner, Jeffrey H

2018-04-16

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5β2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Molecular cloning, expression and characterization of albolamin: a type P-IIa snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris).

PubMed

Jangprasert, Panchalee; Rojnuckarin, Ponlapat

2014-03-01

Snake venom metalloproteinases (SVMPs) can damage vessel wall, degrade clotting factors, inhibit integrins and block platelet functions. Studying them not only gives us deeper insights in pathogenesis of snakebites, but also potentially yields novel therapeutic agents. Here, we discovered a clone of an RGD-containing SVMP from the green pit viper (Cryptelytrops albolabris) venom gland cDNA library. Sequence analysis revealed that it belonged to the P-IIa subclass of SVMP comprising signal peptide, prodomain, metalloproteinase and disintegrin. Compared with other P-II SVMPs, it contained 2 additional conserved cysteines that were predicted to prevent the release of disintegrin from the metalloproteinase domain in the mature protein. The N-terminal histidine-tagged construct of metalloproteinase and disintegrin domains of albolamin was inserted into the pPICZαA vector and expressed in Pichia pastoris. The recombinant protein molecular weight was approximately 35 kDa on Western blot probed with anti-polyhistidine antibody. The recombinant albolamin could digest human type IV collagen starting within 15 min after incubation. In addition, it dose-dependently inhibited collagen-induced platelet aggregation with the IC50 of 1.8 μM. However, there was no effect on ADP-induced platelet aggregation. Therefore, the inhibition mechanism is probably through blocking collagen receptor(s). Albolamin activities probably contributed to pathology of green pit viper bites. Its disintegrin domain deserves further studies for the potential to be a useful agent affecting platelet functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

PubMed

Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

2014-12-01

The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Collagen XIX Is Expressed by Interneurons and Contributes to the Formation of Hippocampal Synapses

PubMed Central

Su, Jianmin; Gorse, Karen; Ramirez, Francesco; Fox, Michael A.

2010-01-01

Extracellular matrix (ECM) molecules contribute to the formation and maintenance of synapses in the mammalian nervous system. We previously discovered a family of nonfibrillar collagens that organize synaptic differentiation at the neuromuscular junction (NMJ). Although many NMJ-organizing cues contribute to central nervous system (CNS) synaptogenesis, whether similar roles for collagens exist at central synapses remained unclear. In the present study we discovered that col19a1, the gene encoding nonfibrillar collagen XIX, is expressed by subsets of hippocampal neurons. Colocalization with the interneuron-specific enzyme glutamate decarboxylase 67 (Gad67), but not other cell-type-specific markers, suggests that hippocampal expression of col19a1 is restricted to interneurons. However, not all hippocampal interneurons express col19a1 mRNA; subsets of neuropeptide Y (NPY)-, somatostatin (Som)-, and calbindin (Calb)-immunoreactive interneurons express col19a1, but those containing parvalbumin (Parv) or calretinin (Calr) do not. To assess whether collagen XIX is required for the normal formation of hippocampal synapses, we examined synaptic morphology and composition in targeted mouse mutants lacking collagen XIX. We show here that subsets of synaptotagmin 2 (Syt2)-containing hippocampal nerve terminals appear malformed in the absence of collagen XIX. The presence of Syt2 in inhibitory hippocampal synapses, the altered distribution of Gad67 in collagen XIX-deficient subiculum, and abnormal levels of gephyrin in collagen XIX-deficient hippocampal extracts all suggest inhibitory synapses are affected by the loss of collagen XIX. Together, these data not only reveal that collagen XIX is expressed by central neurons, but show for the first time that a nonfibrillar collagen is necessary for the formation of hippocampal synapses. PMID:19937713

Endoplasmic Reticulum Chaperon Tauroursodeoxycholic Acid Attenuates Aldosterone-Infused Renal Injury

PubMed Central

Guo, Honglei; Li, Hongmei; Ling, Lilu

2016-01-01

Aldosterone (Aldo) is critically involved in the development of renal injury via the production of reactive oxygen species and inflammation. Endoplasmic reticulum (ER) stress is also evoked in Aldo-induced renal injury. In the present study, we investigated the role of ER stress in inflammation-mediated renal injury in Aldo-infused mice. C57BL/6J mice were randomized to receive treatment for 4 weeks as follows: vehicle infusion, Aldo infusion, vehicle infusion plus tauroursodeoxycholic acid (TUDCA), and Aldo infusion plus TUDCA. The effect of TUDCA on the Aldo-infused inflammatory response and renal injury was investigated using periodic acid-Schiff staining, real-time PCR, Western blot, and ELISA. We demonstrate that Aldo leads to impaired renal function and inhibition of ER stress via TUDCA attenuates renal fibrosis. This was indicated by decreased collagen I, collagen IV, fibronectin, and TGF-β expression, as well as the downregulation of the expression of Nlrp3 inflammasome markers, Nlrp3, ASC, IL-1β, and IL-18. This paper presents an important role for ER stress on the renal inflammatory response to Aldo. Additionally, the inhibition of ER stress by TUDCA negatively regulates the levels of these inflammatory molecules in the context of Aldo. PMID:27721575

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

PubMed Central

2012-01-01

Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition. PMID:22694981

[Immunohistochemistry contribution in Alport syndrome diagnosis].

PubMed

Krichen Makni, S; Kharrat, M; Ben Hmida, M; Chaker, H; Gubler, M-C; Antignac, C; Jlidi, R; Hachicha, J; Sellami Boudwara, T

2005-07-01

Alport syndrome (AS) is an hereditary disease characterised by the association of progressive hematuria nephritis. The diagnosis is based on clinical genetic and ultrastructural findings. Nowadays, immunohistochemical technique is of great interest. It enables us to analyze the distribution of the different chains of the type IV collagen in renal basement membrane (RBM) and epidermal basement membrane (EBM) which appeared to be abnormal in 70% of cases. We report a prospective study of five families affected with AS. Six patients were investigated by immunohistochemical studies of kidney (3 cases) and skin (6 cases) frozen specimens. Monoclonal antibodies recognizing the collagenous domain of alpha1 (MAB1), alpha3 (MAB3) and alpha5 (MAB5) chains of type IV collagen were used. Two methods were performed: direct immunofluorescence and immunohistochemical (ultravision) analysis. The different chains distribution of type IV collagen in the EBM and RBM was normal in four cases (4 men), abnormal in two patients (1 man and woman). Based on the clinical, genetical and immunohistochemical findings we established three transmission modes: autosomal recessive in two families, dominant X linked in two other familiales, and autosomal dominant in one family. Immunohistochemical studies is a simple technique of an easy interpretation accomplished on kidney frozen specimen, or even on a simple cutaneous biopsy. It could be very useful for the diagnosis and enables us in addition to determine the mode of transmission of AS.

Spatiotemporal distribution of extracellular matrix changes during mouse duodenojejunal flexure formation.

PubMed

Onouchi, Sawa; Ichii, Osamu; Nakamura, Teppei; Elewa, Yaser Hosny Ali; Kon, Yasuhiro

2016-08-01

Although gut flexures characterize gut morphology, the mechanisms underlying flexure formation remain obscure. Previously, we analyzed the mouse duodenojejunal flexure (DJF) as a model for its formation and reported asymmetric morphologies between the inner and outer bending sides of the fetal mouse DJF, implying their contribution to DJF formation. We now present the extracellular matrix (ECM) as an important factor for gut morphogenesis. We investigate ECM distribution during mouse DJF formation by histological techniques. In the intercellular space of the gut wall, high Alcian-Blue positivity for proteoglycans shifted from the outer to the inner side of the gut wall during DJF formation. Immunopositivity for fibronectin, collagen I, or pan-tenascin was higher at the inner than at the outer side. Collagen IV and laminins localized to the epithelial basement membrane. Beneath the mesothelium at the pre-formation stage, collagen IV and laminin immunopositivity showed inverse results, corresponding to the different cellular characteristics at this site. At the post-formation stage, however, laminin positivity beneath the mesothelium was the reverse of that observed during the pre-formation stage. High immunopositivity for collagen IV and laminins at the inner gut wall mesenchyme of the post-formation DJF implied a different blood vessel distribution. We conclude that ECM distribution changes spatiotemporally during mouse DJF formation, indicating ECM association with the establishment of asymmetric morphologies during this process.

Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells*

PubMed Central

Shah, Anu; Xia, Ling; Goldberg, Howard; Lee, Ken W.; Quaggin, Susan E.; Fantus, I. George

2013-01-01

Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN. PMID:23329835

Inhibition of leptin-induced vascular extracellular matrix remodelling by adiponectin.

PubMed

Zhang, Zhi; Wang, Fang; Wang, Bing-Jian; Chu, Guang; Cao, Qunan; Sun, Bao-Gui; Dai, Qiu-Yan

2014-10-01

Vascular extracellular matrix (ECM) remodelling, which is the result of disruption in the balance of ECM synthesis and degradation, induces vessel fibrosis and thereby leads to hypertension. Leptin is known to promote tissue fibrosis, while adiponectin has recently been demonstrated to be anti-fibrogenic in tissue fibrosis. In this study, we aimed to evaluate the leptin-antagonist function of adiponectin and to further elucidate the mechanisms through which adiponectin dampens leptin signalling in vascular smooth muscle cells, thus preventing excess ECM production, in our already established 3D co-culture vessel models. Our 3D co-culture vessel model, which mimics true blood vessels, is composed of vascular endothelial cells, vascular smooth muscle cells and collagen type I. We validated the profibrogenic effects of leptin and analysed matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase 1 (TIMP1) and collagen types II/IV secretion in 3D vessel models. The protective/inhibitory effects of adiponectin were re-analysed by inhibiting adiponectin receptor 1 (AdipoR) and AdipoR2 expression in endothelial cells using RNAi technology. In the 3D vessel models, adiponectin blocked the leptin-stimulated secretion of collagen types II/IV and TIMP1 while significantly increasing MMP2/9 activity. In endothelial cells, adiponectin induced phosphorylation of AMPK, thereby suppressing leptin-mediated STAT3 phosphorylation through induction of SOCS3 in smooth muscle cells. Our findings indicate that adiponectin disrupted the leptin-induced vascular ECM remodelling via AdipoR1 and enhanced AMPK signalling in endothelial cells, which, in turn, promoted SOCS3 up-regulation in smooth muscle cells to repress leptin-stimulated phosphorylation of STAT3. © 2014 The authors.

Inhibition of leptin-induced vascular extracellular matrix remodelling by adiponectin

PubMed Central

Zhang, Zhi; Wang, Fang; Wang, Bing-jian; Chu, Guang; Cao, Qunan; Sun, Bao-Gui; Dai, Qiu-Yan

2014-01-01

Vascular extracellular matrix (ECM) remodelling, which is the result of disruption in the balance of ECM synthesis and degradation, induces vessel fibrosis and thereby leads to hypertension. Leptin is known to promote tissue fibrosis, while adiponectin has recently been demonstrated to be anti-fibrogenic in tissue fibrosis. In this study, we aimed to evaluate the leptin-antagonist function of adiponectin and to further elucidate the mechanisms through which adiponectin dampens leptin signalling in vascular smooth muscle cells, thus preventing excess ECM production, in our already established 3D co-culture vessel models. Our 3D co-culture vessel model, which mimics true blood vessels, is composed of vascular endothelial cells, vascular smooth muscle cells and collagen type I. We validated the profibrogenic effects of leptin and analysed matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase 1 (TIMP1) and collagen types II/IV secretion in 3D vessel models. The protective/inhibitory effects of adiponectin were re-analysed by inhibiting adiponectin receptor 1 (AdipoR) and AdipoR2 expression in endothelial cells using RNAi technology. In the 3D vessel models, adiponectin blocked the leptin-stimulated secretion of collagen types II/IV and TIMP1 while significantly increasing MMP2/9 activity. In endothelial cells, adiponectin induced phosphorylation of AMPK, thereby suppressing leptin-mediated STAT3 phosphorylation through induction of SOCS3 in smooth muscle cells. Our findings indicate that adiponectin disrupted the leptin-induced vascular ECM remodelling via AdipoR1 and enhanced AMPK signalling in endothelial cells, which, in turn, promoted SOCS3 up-regulation in smooth muscle cells to repress leptin-stimulated phosphorylation of STAT3. PMID:24982243

Upregulation of distinct collagen transcripts in post-surgery scar tissue: a study of conjunctival fibrosis.

PubMed

Seet, Li-Fong; Toh, Li Zhen; Chu, Stephanie W L; Finger, Sharon N; Chua, Jocelyn L L; Wong, Tina T

2017-06-01

Excessive accumulation of collagen is often used to assess the development of fibrosis. This study aims to identify collagen genes that define fibrosis in the conjunctiva following glaucoma filtration surgery (GFS). Using the mouse model of GFS, we have identified collagen transcripts that were upregulated in the fibrotic phase of wound healing via RNA-seq. The collagen transcripts that were increased the most were encoded by Col8a1 , Col11a1 and Col8a2 Further analysis of the Col8a1 , Col11a1 and Col8a2 transcripts revealed their increase by 67-, 54- and 18-fold, respectively, in the fibrotic phase, compared with 12-fold for Col1a1 , the most commonly evaluated collagen gene for fibrosis. However, only type I collagen was significantly upregulated at the protein level in the fibrotic phase. Type VIII and type I collagens colocalized in fibrous structures and in ACTA2-positive pericytes, and appeared to compensate for each other in expression levels. Type XI collagen showed low colocalization with both type VIII and type I collagens but can be found in association with macrophages. Furthermore, we show that both mouse and human conjunctival fibroblasts expressed elevated levels of the most highly expressed collagen genes in response to TGFβ2 treatment. Importantly, conjunctival tissues from individuals whose GF surgeries have failed due to scarring showed 3.60- and 2.78-fold increases in type VIII and I collagen transcripts, respectively, compared with those from individuals with no prior surgeries. These data demonstrate that distinct collagen transcripts are expressed at high levels in the conjunctiva after surgery and their unique expression profiles may imply differential influences on the fibrotic outcome. © 2017. Published by The Company of Biologists Ltd.

Alcohol modulation of cardiac matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs favors collagen accumulation.

PubMed

El Hajj, Elia C; El Hajj, Milad C; Voloshenyuk, Tetyana G; Mouton, Alan J; Khoutorova, Elena; Molina, Patricia E; Gilpin, Nicholas W; Gardner, Jason D

2014-02-01

Chronic alcohol consumption has been shown in human and animal studies to result in collagen accumulation, myocardial fibrosis, and heart failure. Cardiac fibroblasts produce collagen and regulate extracellular matrix (ECM) homeostasis through the synthesis and activity of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), with the balance of MMPs/TIMPs determining the rate of collagen turnover. Dynamic changes of MMP and TIMP expression were reported in alcohol-induced hepatic fibrosis; however, the effect of alcohol on MMP/TIMP balance in the heart and cardiac fibroblasts is unknown. We hypothesized that alcohol exposure alters cardiac fibroblast MMP and TIMP expression to promote collagen accumulation in the heart. Cardiac fibroblasts isolated from adult rats were cultured in the presence of alcohol (12.5 to 200 mM) for 48 hours. MMP, TIMP, and collagen type I and III expression were assayed by Western blot analysis. Hydroxyproline (HPro) was used as a marker of collagen production. The in vivo cardiac effects of ethanol (EtOH) were determined using rats exposed to EtOH vapor for 2 weeks, resulting in blood alcohol levels of 150 to 200 mg/dl. Cardiac collagen volume fraction (CVF), as well as MMP, TIMP, and collagen expression, was assessed. EtOH-exposed rats exhibited up-regulation of TIMP-1, TIMP-3 and TIMP-4 in the heart, with no significant increases in MMPs. Cardiac fibroblasts exhibited transformation to a profibrotic phenotype following exposure to alcohol. These changes were reflected by increased α-smooth muscle actin and collagen I and III expression, as well as increased collagen secretion. In vivo EtOH exposure also produced fibrosis, indicated by increased CVF and expression of collagens. Alcohol exposure modulates cardiac fibroblast MMP/TIMP expression favoring a profile associated with collagen accumulation. Our data suggest that this disrupted MMP/TIMP profile may contribute to the development of myocardial fibrosis and cardiac dysfunction resulting from chronic alcohol abuse. Copyright © 2013 by the Research Society on Alcoholism.

Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

PubMed

Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

2016-01-01

During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

Enhanced osteoprogenitor elongated collagen fiber matrix formation by bioactive glass ionic silicon dependent on Sp7 (osterix) transcription.

PubMed

Varanasi, Venu G; Odatsu, Tetsurou; Bishop, Timothy; Chang, Joyce; Owyoung, Jeremy; Loomer, Peter M

2016-10-01

Bioactive glasses release ions, those enhance osteoblast collagen matrix synthesis and osteogenic marker expression during bone healing. Collagen matrix density and osteogenic marker expression depend on osteogenic transcription factors, (e.g., Osterix (OSX)). We hypothesize that enhanced expression and formation of collagen by Si(4+) depends on enhanced expression of OSX transcription. Experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were dissolved in basal medium to make glass conditioned medium (GCM). ICP-MS analysis was used to measure bioactive glass ion release rates. MC3T3-E1 cells were cultured for 20 days, and gene expression and extracellular matrix collagen formation was analyzed. In a separate study, siRNA was used to determine the effect of OSX knockdown on impacting the effect of Si(4+) on osteogenic markers and matrix collagen formation. Each bioactive glass exhibited similar ion release rates for all ions, except Mg(2+) released by 6P53-b. Gene expression results showed that GCM markedly enhanced many osteogenic markers, and 45S5 GCM showed higher levels of expression and collagen matrix fiber bundle density than 6P53-b GCM. Upon knockdown of OSX transcription, collagen type 5, alkaline phosphatase, and matrix density were not enhanced as compared to wild type cells. This study illustrates that the enhancement of elongated collagen fiber matrix formation by Si(±) depends on OSX transcription. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2604-2615, 2016. © 2016 Wiley Periodicals, Inc.

Detection of microinvasion in vulvar and cervical intraepithelial neoplasia using double immunostaining for cytokeratin and basement membrane components.

PubMed

Rush, Demaretta; Hyjek, Elizabeth; Baergen, Rebecca N; Ellenson, Lora H; Pirog, Edyta C

2005-06-01

Identification of early invasion in vulvar intraepithelial neoplasia 3 (VIN 3) and cervical intraepithelial neoplasia 3 (CIN 3) may be difficult with the use of routine hematoxylin-eosin staining. Presence of obscuring inflammation and tangential tissue sectioning are the most common diagnostic pitfalls. To examine the utility of double immunostaining for cytokeratin-collagen IV or cytokeratin-laminin in the detection of early invasion in VIN 3 and CIN 3. The study group consisted of 10 cases of "VIN 3, suspicious for invasion" and 10 cases of "CIN 3, suspicious for invasion." The negative control group consisted of VIN 3 (n = 15) and CIN 3 (n = 10). The positive control group consisted of cases of invasive vulvar carcinoma (n = 11) and invasive cervical carcinoma (n = 25). All cases were double immunostained for cytokeratin and collagen IV and, in a separate reaction, for cytokeratin and laminin. The continuity of the basement membrane and the presence of stromal invasion were assessed in the stained sections. The staining for collagen IV and laminin yielded identical results. A well-defined, continuous basement membrane was visualized in all cases of VIN 3 and CIN 3. A discontinuous or absent basement membrane was observed around the malignant cells on the invasive tumor front in all cases of vulvar and cervical carcinoma. In 2 of 10 cases of VIN 3, suspicious for invasion and in 4 of 10 cases of CIN 3, suspicious for invasion definitive foci of microinvasion were identified with the use of double immunostaining. A well-defined, continuous basement membrane was present in the remaining cases "suspicious for invasion." Double immunostaining for cytokeratin- collagen IV or cytokeratin-laminin is useful for evaluation of early invasion in equivocal cases of VIN 3 and CIN 3.

New lethal disease involving type I and III collagen defect resembling geroderma osteodysplastica, De Barsy syndrome, and Ehlers-Danlos syndrome IV.

PubMed

Jukkola, A; Kauppila, S; Risteli, L; Vuopala, K; Risteli, J; Leisti, J; Pajunen, L

1998-06-01

We describe the clinical findings and biochemical features of a male child suffering from a so far undescribed lethal connective tissue disorder characterised by extreme hypermobility of the joints, lax skin, cataracts, severe growth retardation, and insufficient production of type I and type III procollagens. His features are compared with Ehlers-Danlos type IV, De Barsy syndrome, and geroderma osteodysplastica, as these disorders show some symptoms and signs shared with our patient. The child died because of failure of the connective tissue structures joining the skull and the spine, leading to progressive spinal stenosis. The aortic valve was translucent and insufficient. The clinical symptoms and signs, together with histological findings, suggested a collagen defect. Studies on both skin fibroblast cultures and the patient's serum showed reduced synthesis of collagen types I and III at the protein and RNA levels. The sizes of the mRNAs and newly synthesised proteins were normal, excluding gross structural abnormalities. These findings are not in accordance with any other collagen defect characterised so far.

Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components.

PubMed

Berasaín, P; Goñi, F; McGonigle, S; Dowd, A; Dalton, J P; Frangione, B; Carmona, C

1997-02-01

The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.

[Immunohistochemical study of the specific features of expression of matrix metalloproteinases 1, 9 in the photoaged skin, the foci of actinic keratosis and basal cell carcinoma].

PubMed

Kuznetsova, E V; Snarskaya, E S; Zavalishina, L E; Tkachenko, S B

Matrix metalloproteinases (MMPs) mediate the degradation of all types of collagens and other extracellular matrix components (elastin, proteoglycans, and laminin), their synthesis and accumulation play a key role in the hydrolysis of basement membrane. MMPs are involved in a wide range of proteolytic processes in the presence of different physiological and pathological changes, including inflammation, wound healing, angiogenesis, and carcinogenesis. to study the specific features of MMP-1 and MMP-9 expression in different stages of skin photoaging, in the foci of actinic keratosis and basal cell carcinoma by immunohistochemical examination. 12 samples of the healthy skin (6 samples of the eyelid skin with Glogau grade II photoaging; 6 ones of eyelid skin with Glogau grades III-IV photoaging) and biopsies from 8 foci of actinic keratosis and from 8 ones of basal cell carcinoma were examined. A positive reaction to MMPs was shown as different brown staining intensity in the cytoplasm of keratinocytes/tumor cells. MMP-1 and MMP-9 expression was recorded in 67% of the histological specimens of the Glogau grade III photoaged skin and in 100% of those of Glogau grade IV. In the foci of actinic keratosis, the expression of MMP-1 was observed in 62.5% of cases and that of MMP-9 was seen in 87.5%. In basal cell carcinoma, the expression of MMP-1 and MMP-9 was detected in all investigated samples. The immunomorphological findings are indicative of the important role of the level of MMP-1 and MMP-9 expression that is associated with the degree of progression of skin photoaging processes. Minimal MMP-1 and MMP-9 expression was recorded even in grades III-IV photoaging and in the foci of actinic keratosis. Intense MMP-1 and MMP-9 expression was detected in malignant skin epithelial neoplasms as different clinicomorphological types of basal cell carcinoma.

Hemothorax in vascular Ehlers-Danlos syndrome.

PubMed

Álvarez, Kevin; Jordi, López; Jose Angel, Hernández

2017-10-16

Vascular Ehlers-Danlos syndrome (EDS IV) is a rare genetic disorder characterized by an alteration in the COL3A1 gene which encodes type III collagen. It is the most common type of collagen in vessels of medium size and certain organs such as the intestines and the uterus. The alteration of this type of collagen produces aneurisms and ruptures of vessels and organs. A high level of clinical suspicion is required for diagnosis. It is a complex disease whose management requires a multidisciplinary team to treat the different complications that may occur. We report the case of a 50-year-old man diagnosed with EDS IV detected incidentally after hemothorax secondary to a coughing spell. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Clinical presentations of Ehlers Danlos syndrome type IV.

PubMed Central

Pope, F M; Narcisi, P; Nicholls, A C; Liberman, M; Oorthuys, J W

1988-01-01

Ehlers Danlos syndrome type IV is an often lethal disease caused by various mutations of type III collagen genes. It presents in infancy and childhood in several ways, and the symptoms and signs include low birth weight, prematurity, congenital dislocation of the hips, easy inappropriate bruising (sometimes suspected as child battering), and a diagnostic facial phenotype. These features predict a lethal adult disease often complicated by fatal arterial rupture in early or middle adult life. Most affected patients can be diagnosed from radiolabelled collagen protein profiles by polyacrylamide gel electrophoresis. Prenatal diagnosis by specific type III collagen restriction fragment length polymorphisms is possible in some families, and will become increasingly important. Prenatal diagnosis and prevention of the disease in selected families is already possible and will be widely available in the future. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 Fig 9 Fig 10 Fig 11 PMID:3178263

Tissue factor activity and ECM-related gene expression in human aortic endothelial cells grown on electrospun biohybrid scaffolds.

PubMed

Han, Jingjia; Gerstenhaber, Jonathan A; Lazarovici, Philip; Lelkes, Peter I

2013-05-13

All blood vessels are lined with a quiescent endothelium, which aids in regulating regular blood flow and avoiding thrombus formation. Current attempts at replacing diseased blood vessels frequently fail due to the intrinsic thrombogenicity of the materials used as vascular grafts. In extending our previous work where we introduced a new candidate scaffolds for vascular grafts electrospun from a blend solution of PLGA, gelatin, and elastin (PGE), this study aimed to evaluate the potential of PGE scaffolds to support nonthrombogenic monolayers of primary isolates of human aortic endothelial cells (HAECs), as assessed by a combination of biochemical, molecular, and bioinformatics-based analyses. After 24 h of culture on 3-D fibrous PGE scaffolds, HAECs formed a confluent, nonthrombogenic, and physiologically competent monolayer, as assessed by tissue factor (TF) gene expression and protein activity assays. The levels of TF mRNA/protein activity in HAECs grown on PGE scaffolds were similar to those on gelatin or collagen IV-coated 2-D surfaces. In addition, bioinformatics-based analysis of a focused microarray containing 84 ECM-related cDNA probes demonstrated that HAECs essentially expressed a histotypic ECM-related "transcriptome" on PGE scaffolds, where cells were more quiescent than cells cultured on 2-D coverslips coated with gelatin (a well-known "inert" substrate for conventional EC culture), but less so than on 2-D PGE films. These data suggest an important role for nanorough substrates (PGE films) in passivating endothelial cells and confirm the crucial effect of substrate composition in this process. Principal component analysis of microarray data on the above substrates (including collagen IV) implied that substrate composition plays a greater role than surface topography in affecting the endothelial ECM-related "transcriptome". Taken together, our findings suggest that electrospun PGE scaffolds are potentially suitable for application in small diameter vascular tissue engineering.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

PubMed Central

Baldo, Cristiani; Jamora, Colin; Yamanouye, Norma; Zorn, Telma M.; Moura-da-Silva, Ana M.

2010-01-01

Background Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology. PMID:20614020

Qualitative and quantitative assessment of collagen and elastin in annulus fibrosus of the physiologic and scoliotic intervertebral discs.

PubMed

Kobielarz, Magdalena; Szotek, Sylwia; Głowacki, Maciej; Dawidowicz, Joanna; Pezowicz, Celina

2016-09-01

The biophysical properties of the annulus fibrosus of the intervertebral disc are determined by collagen and elastin fibres. The progression of scoliosis is accompanied by a number of pathological changes concerning these structural proteins. This is a major cause of dysfunction of the intervertebral disc. The object of the study were annulus fibrosus samples excised from intervertebral discs of healthy subjects and patients treated surgically for scoliosis in the thoracolumbar or lumbar spine. The research material was subjected to structural analysis by light microscopy and quantitative analysis of the content of collagen types I, II, III and IV as well as elastin by immunoenzymatic test (ELISA). A statistical analysis was conducted to assess the impact of the sampling site (Mann-Whitney test, α=0.05) and scoliosis (Wilcoxon matched pairs test, α=0.05) on the obtained results. The microscopic studies conducted on scoliotic annulus fibrosus showed a significant architectural distortion of collagen and elastin fibres. Quantitative biochemical assays demonstrated region-dependent distribution of only collagen types I and II in the case of healthy intervertebral discs whereas in the case of scoliotic discs region-dependent distribution concerned all examined proteins of the extracellular matrix. Comparison of scoliotic and healthy annulus fibrosus revealed a significant decrease in the content of collagen type I and elastin as well as a slight increase in the proportion of collagen types III and IV. The content of collagen type II did not differ significantly between both groups. The observed anomalies are a manifestation of degenerative changes affecting annulus fibrosus of the intervertebral disc in patients suffering from scoliosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

PubMed

Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

2017-02-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

PubMed Central

Chapman, Mark A.; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David

2017-01-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173–183, 2009; Kjaer M. Physiol Rev 84: 649–98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins—fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. PMID:27881411

A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts.

PubMed

Engel, U; Pertz, O; Fauser, C; Engel, J; David, C N; Holstein, T W

2001-06-15

The smallest known collagens with only 14 Gly-X-Y repeats referred to as minicollagens are the main constituents of the capsule wall of nematocysts. These are explosive organelles found in Hydra, jellyfish, corals and other Cnidaria. Minicollagen-1 of Hydra recombinantly expressed in mammalian 293 cells contains disulfide bonds within its N- and C-terminal Cys-rich domains but no interchain cross-links. It is soluble and self-associates through non-covalent interactions to form 25-nm-long trimeric helical rod-like molecules. We have used a polyclonal antibody prepared against the recombinant protein to follow the maturation of minicollagens from soluble precursors present in the endoplasmic reticulum and post-Golgi vacuoles to the disulfide-linked insoluble assembly form of the wall. The switch from intra- to intermolecular disulfide bonds is associated with 'hardening' of the capsule wall and provides an explanation for its high tensile strength and elasticity. The process is comparable to disulfide reshuffling between the NC1 domains of collagen IV in mammalian basement membranes.

α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

PubMed

Cattavarayane, Sandhanakrishnan; Palovuori, Riitta; Tanjore Ramanathan, Jayendrakishore; Manninen, Aki

2015-02-27

The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.

Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts.

PubMed

Xu, Wen-Xiao; Liu, Sheng-Zhi; Wu, Di; Qiao, Guo-Fen; Yan, Jinglong

2015-01-01

Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

PubMed

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

PubMed Central

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295

Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineering.

PubMed

Donegan, Gail C; Hunt, John A; Rhodes, Nicholas

2010-02-01

Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space.

Effect of Sea buckthorn on liver fibrosis: A clinical study

PubMed Central

Gao, Ze-Li; Gu, Xiao-Hong; Cheng, Feng-Tao; Jiang, Fo-Hu

2003-01-01

AIM: To appraise the effect of sea buckthorn (Hippophae rhamnoides) on cirrhotic patients. METHODS: Fifty cirrhotic patients of Child-Pugh grade A and B were randomly divided into two groups: Group A as the treated group (n = 30), taking orally the sea buckthorn extract, 15 g 3 times a day for 6 mo. Group B as the control group (n = 18), taking vitamin B complex one tablet, 3 times a day for 6 mo. The following tests were performed before and after the treatment in both groups to determine LN, HA, collagens types III and IV, cytokines IL-6 and TNFα, liver serum albumin, total bile acid, ALT, AST and prothrombin time. RESULTS: The serum levels of TNFα, IL-6, laminin and type IV collagen in group A were significantly higher than those in the control group. After a course of sea buckthorn treatment, the serum levels of LN, HA, collagen types III and IV, total bile acid (TBA) decreased significantly as compared with those before and after treatment in the control group. The sea buckthorn notably shortened the duration for normalization of aminotransferases. CONCLUSION: Sea buckthorn may be a hopeful drug for prevention and treatment of liver fibrosis. PMID:12854177

Human eccrine sweat gland cells reconstitute polarized spheroids when subcutaneously implanted with Matrigel in nude mice.

PubMed

Li, Haihong; Zhang, Mingjun; Chen, Liyun; Li, Xuexue; Zhang, Bingna

2016-10-01

Increasing evidence indicates that maintenance of cell polarity plays a pivotal role in the regulation of glandular homeostasis and function. We examine the markers for polarity at different time points to investigate the formation of cell polarity during 3D reconstitution of eccrine sweat glands. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 2, 3, 4, 5 and 6 weeks post-implantation, Matrigel plugs were removed and immunostained for basal collagen IV, lateral β-catenin, lateroapical ZO-1 and apical F-actin. The results showed that the cell polarity of the spheroids appeared in sequence. Formation of basal polarity was prior to lateral, apical and lateroapical polarity. Collagen IV was detected basally at 2 weeks, β-catenin laterally and ZO-1 lateroapically at 3 weeks, and F-actin apically at 4 weeks post-implantation. At week 5 and week 6, the localization and the positive percentage of collagen IV, β-catenin, ZO-1 or F-actin in spheroids was similar to that in native eccrine sweat glands. We conclude that the reconstituted 3D eccrine sweat glands are functional or potentially functional.

In vitro reconstruction of human junctional and sulcular epithelium

PubMed Central

Dabija-Wolter, G; Bakken, V; Cimpan, M R; Johannessen, A C; Costea, D E

2013-01-01

BACKGROUND The aim of this study was to develop and characterize standardized in vitro three-dimensional organotypic models of human junctional epithelium (JE) and sulcular epithelium (SE). METHODS Organotypic models were constructed by growing human normal gingival keratinocytes on top of collagen matrices populated with gingival fibroblasts (GF) or periodontal ligament fibroblasts (PLF). Tissues obtained were harvested at different time points and assessed for epithelial morphology, proliferation (Ki67), expression of JE-specific markers (ODAM and FDC-SP), cytokeratins (CK), transglutaminase, filaggrin, and basement membrane proteins (collagen IV and laminin1). RESULTS The epithelial component in 3- and 5-day organotypics showed limited differentiation and expressed Ki-67, ODAM, FDC-SP, CK 8, 13, 16, 19, and transglutaminase in a similar fashion to control JE samples. PLF supported better than GF expression of CK19 and suprabasal proliferation, although statistically significant only at day 5. Basement membrane proteins started to be deposited only from day 5. The rate of proliferating cells as well as the percentage of CK19-expressing cells decreased significantly in 7- and 9-day cultures. Day 7 organotypics presented higher number of epithelial cell layers, proliferating cells in suprabasal layers, and CK expression pattern similar to SE. CONCLUSION Both time in culture and fibroblast type had impact on epithelial phenotype. Five-day cultures with PLF are suggested as JE models, 7-day cultures with PLF or GF as SE models, while 9-day cultures with GF as gingival epithelium (GE) models. Such standard, reproducible models represent useful tools to study periodontal bacteria–host interactions in vitro. PMID:22947066

Outcomes of Corneal Cross-Linking Correlate With Cone-Specific Lysyl Oxidase Expression in Patients With Keratoconus.

PubMed

Shetty, Rohit; Rajiv Kumar, Nimisha; Pahuja, Natasha; Deshmukh, Rashmi; Vunnava, KrishnaPoojita; Abilash, Valsala Gopalakrishnan; Sinha Roy, Abhijit; Ghosh, Arkasubhra

2018-03-01

To evaluate the correlation of visual and keratometry outcomes after corneal cross-linking (CXL) in patients with keratoconus with cone epithelium-specific gene expression levels. Corneal epithelium was obtained from 35 eyes that underwent accelerated CXL (KXLII, 9 mW/cm for 10 min). Using corneal topography, epithelium over the cone and periphery was obtained separately from each subject. The ratio of gene expression for lysyl oxidase (LOX), matrix metalloproteinase 9 (MMP9), bone morphogenic protein 7, tissue inhibitor of metalloproteinase 1, collagen, type I, alpha 1, and collagen, type IV, alpha 1 (COL IVA1) from the cone and peripheral cornea was correlated with the outcome of cross-linking surgery. Patients were assessed for visual acuity, keratometry, refraction, and corneal densitometry before and 6 months after surgery. Based on the change in corneal flattening indicated by ΔKmax, the outcomes were classified as a higher response or lower response. Reduction in keratometric indices correlated with improved spherical equivalent after CXL. Preoperative levels of cone-specific LOX expression in cases with a higher response were significant (P = 0.001). COL IVA1, bone morphogenic protein 7, and tissue inhibitor of metalloproteinase 1 gene expressions were reduced in the cones of the subjects with a lower response. MMP9 levels were relatively lower in cases with a higher response compared with those with a lower response. Our study demonstrates that preoperative levels of molecular factors such as LOX, MMP9, and COL IVA1 aid in understanding CXL outcomes at the tissue level.

Developmental changes in skin collagen biosynthesis pathway in posthatch male and female chickens

NASA Technical Reports Server (NTRS)

Pines, M.; Schickler, M.; Hurwitz, S.; Yamauchi, M.

1996-01-01

The developmental changes in skin collagen biosynthesis pathway in male and female chickens were evaluated. Concentration of collagen, levels of mRNA for collagen type I subunits and for lysyl hydroxylase, and the level of three lysyl oxidase-derived cross-links: dehydro-dihydroxylysinonorleucine (DHLNL), dehydro-hydroxylysinonorleucine (HLNL), and dehydro-histidinohydroxymerodesmosine (HHMD) were determined during 4 wk posthatching. Skin collagen content increased with age and was higher in males than in females. In both sexes, the expression of the genes coding for alpha 1 and alpha 2 of collagen type I decreased with age: alpha 1(I) gene expression decreased from Day 3 onwards, whereas the reduction in alpha 2(I) gene expression started 1 wk later. At all ages examined, the expression of both genes was higher in male than in female skin. Males and females lysyl hydroxylase gene expression remained low until Day 16, after which an increase in the enzyme gene expression was observed. An increase in skin HLNL content was observed from Day 3 in both sexes reaching a peak in males at Day 9 and in females 1 wk later. The DHLNL content, which was higher in males than in females at all ages tested, dramatically decreased in both male and female skin from 3 d of age, reaching its lowest level at Day 16, and remained at that low level thereafter. The skin content of HHMD in males and females followed an oscillatory behavior with higher peaks in the male skin. The results suggest that the higher tensile strength of male skin than female skin may be due to the elevated skin collagen content that resulted from increased expression in collagen type I genes on the one hand, and from the higher amounts of various collagen cross-links on the other.

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro

NASA Technical Reports Server (NTRS)

Uzawa, K.; Grzesik, W. J.; Nishiura, T.; Kuznetsov, S. A.; Robey, P. G.; Brenner, D. A.; Yamauchi, M.

1999-01-01

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

Expression of collagen in ovular membranes of pregnant smokers and non-smokers: a pilot study.

PubMed

Negrini, Romulo; Araujo Júnior, Edward; Piato, Sebastião; Chade, Milca Cezar; Rios, Adriana Ribeiro Santos; Silva, Maria Antonieta Galvão; Aldrighi, José Mendes

2015-09-01

Our study compared the amount of total collagen and type I collagen in ovular membranes of pregnant smokers and non-smokers. The study group consisted of 14 pregnant smokers at 24-36 weeks of gestation; 39 pregnant non-smokers between 24-36 weeks of gestation comprised the control group. The expressions of total collagen and type I collagen were analyzed using two histological sections of the fetal membranes. The assessment of total collagen was performed using the Picro-Cirius red stain, and type I collagen expression was determined by means of immunohistochemistry The Mann-Whitney test was applied to verify possible differences between the groups. The average area covered by total collagen was lower in smokers (20630.45 microm2) as compared to non-smokers (24058.61 microm2), although the difference was not statistically significant (p = 0.454). Comparison involving collagen type I deemed similar results (20001.33 microm2 vs. 25328.29 microm2, p = 0.158). The amount of total collagen and type I collagen was lower in ovular membranes of pregnant smokers as compared to non-smokers, although the difference was not statistically significant.

Osteogenesis Imperfecta

PubMed Central

Sam, Justin Easow; Dharmalingam, Mala

2017-01-01

Osteogenesis imperfecta is a common heritable connective tissue disorder. Nearly ninety percent are due to Type I collagen mutations. Type I-IV are autosomal dominant, and Type VI–XIII are autosomal recessive. They are Graded 1-5 based on severity. Genomic testing is done by collagen analysis from fibroblasts. The mainstay of treatment is bisphosphonate therapy. The prognosis is variable. PMID:29285457

Regulation of COX-2–mediated signaling by α3 type IV noncollagenous domain in tumor angiogenesis

PubMed Central

Boosani, Chandra Shekhar; Mannam, Arjuna P.; Cosgrove, Dominic; Silva, Rita; Hodivala-Dilke, Kairbaan M.; Keshamouni, Venkateshwar G.

2007-01-01

Human α3 chain, a noncollagenous domain of type IV collagen [α3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of α3(IV)NC1 to αVβ3 and α3β1 integrins. α3(IV)NC1 binds αVβ3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of α3β1 and αVβ3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on α3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by α3(IV)NC1 in endothelial cells, only α3β1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of α3(IV)NC1 to α3β1 integrin leads to inhibition of COX-2–mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IκBα/NFκB axis, and is independent of αVβ3 integrin. Furthermore, β3 integrin–null endothelial cells, when treated with α3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in α3 integrin–null endothelial cells, indicating that regulation of COX-2 by α3(IV)NC1 is mediated by integrin α3β1. Our in vitro and in vivo findings demonstrate that α3β1 integrin is critical for α3(IV)NC1-mediated inhibition of COX-2–dependent angiogenic signaling and inhibition of tumor progression. PMID:17426256

Cigarette smoke extract induces select matrix metalloproteinases and integrin expression in periodontal ligament fibroblasts.

PubMed

Bulmanski, Zachary; Brady, Matthew; Stoute, Diana; Lallier, Thomas E

2012-06-01

The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. Treatment with CSE-induced cell death at concentrations of ≥5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Vα3 and decreased collagen XIα1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins α1, α2, and α10 (collagen receptors) and α9 (a tenascin receptor). This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion.

Inhibitory effect of gallic acid on CCl4-mediated liver fibrosis in mice.

PubMed

Wang, Jing; Tang, Long; White, James; Fang, Jing

2014-05-01

The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl4). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl4-induced liver injury control group (group B), and CCl4 induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl4 induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl4-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl4 in mice, which might be through the inhibition of hepatic stellate cell activity.

Assessment of Cpa, Scl1 and Scl2 in clinical group A streptococcus isolates and patients from north India: an evaluation of the host pathogen interaction.

PubMed

Chaudhary, Priyanka; Kumar, Rajesh; Sagar, Vivek; Sarkar, Subendu; Singh, Rupneet; Ghosh, Sujata; Singh, Surjit; Chakraborti, Anuradha

2018-01-01

Group A streptococcus (GAS) infection remains a major concern due to multiple diseases including pharyngitis, impetigo, acute rheumatic fever (ARF) and rheumatic heart disease (RHD). It uses different adhesins and virulence factors like Cpa (collagen binding protein) and Scl (collagen-like protein) in its pathogenicity. Scl having similarities with human collagen may contribute to inducing autoimmunity in the host. Here we assessed gene expression, antibody titer of Cpa, Scl1 and Scl2 in both clinical GAS isolates (n = 45) and blood (n = 45) obtained from pharyngitis, ARF (acute rheumatic fever) and RHD respectively. Skin isolates (n = 30) were obtained from impetigo patients. The study revealed a total of 27 GAS emm types. Frequency of cpa, scl1, scl2 was high in ARF isolates. The antibody titer of these proteins was high in all isolates, and also in patients with pharyngitis and ARF. All isolates showed high binding affinity toward collagen I and IV, which further indicates a potential host pathogen interaction. Our study reflects a strong association of Cpa and Scls in early and post-GAS pathogenicity. However, the increased antibody titer of Scl1 and Scl2 during ARF may be attributed to a cogent immune response in the host. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis.

PubMed

Li, Yanning; Zhong, Yan; Gong, Wenjian; Gao, Xuehan; Qi, Huanli; Liu, Kun; Qi, Jinsheng

2018-07-01

Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains ( Col4a1-a5 ), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. Col4a1-a5 expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of Col4a1a2 , Col4a3a4 and Col4a5 were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal Col4a1 -a5 mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit Col4a1-a5 mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide. © 2018 Society for Endocrinology.

Intraglomerular pressure and mesangial stretching stimulate extracellular matrix formation in the rat.

PubMed Central

Riser, B L; Cortes, P; Zhao, X; Bernstein, J; Dumler, F; Narins, R G

1992-01-01

To define the interplay of glomerular hypertension and hypertrophy with mesangial extracellular matrix (ECM) deposition, we examined the effects of glomerular capillary distention and mesangial cell stretching on ECM synthesis. The volume of microdissected rat glomeruli (Vg), perfused ex vivo at increasing flows, was quantified and related to the proximal intraglomerular pressure (PIP). Glomerular compliance, expressed as the slope of the positive linear relationship between PIP and Vg was 7.68 x 10(3) microns 3/mmHg. Total Vg increment (PIP 0-150 mmHg) was 1.162 x 10(6) microns 3 or 61% (n = 13). A 16% increase in Vg was obtained over the PIP range equivalent to the pathophysiological limits of mean transcapillary pressure difference. A similar effect of renal perfusion on Vg was also noted histologically in tissue from kidneys perfused/fixed in vivo. Cultured mesangial cells undergoing cyclic stretching increased their synthesis of protein, total collagen, and key components of ECM (collagen IV, collagen I, laminin, fibronectin). Synthetic rates were stimulated by cell growth and the degree of stretching. These results suggest that capillary expansion and stretching of mesangial cells by glomerular hypertension provokes increased ECM production which is accentuated by cell growth and glomerular hypertrophy. Mesangial expansion and glomerulosclerosis might result from this interplay of mechanical and metabolic forces. Images PMID:1430216

FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program

PubMed Central

Ionescu, Andreia; Kozhemyakina, Elena; Nicolae, Claudia; Kaestner, Klaus H.; Olsen, Bjorn R.; Lassar, Andrew B.

2012-01-01

During endochondral ossification small immature chondrocytes enlarge to form hypertrophic chondrocytes, which express collagen X. In this work, we demonstrate that FoxA factors are induced during chondrogenesis, bind to conserved binding sites in the collagen X enhancer, and can promote the expression of a collagen X-luciferase reporter in both chondrocytes and fibroblasts. In addition, we demonstrate by both gain and loss of function analyses that FoxA factors play a crucial role driving the expression of both endogenous collagen X and other hypertrophic chondrocyte-specific genes. Mice engineered to lack expression of both FoxA2 and FoxA3 in their chondrocytes display defects in chondrocyte hypertrophy, alkaline phosphatase expression, and mineralization in their sternebrae and in addition exhibit postnatal dwarfism that is coupled to significantly decreased expression of both collagen X and MMP13 in their growth plates. Together, our findings indicate that FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program. PMID:22595668

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies.

PubMed

Girkontaite, I; Frischholz, S; Lammi, P; Wagner, K; Swoboda, B; Aigner, T; Von der Mark, K

1996-09-01

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder

PubMed Central

Miyake, Makito; Hori, Shunta; Morizawa, Yosuke; Tatsumi, Yoshihiro; Toritsuka, Michihiro; Ohnishi, Sayuri; Shimada, Keiji; Furuya, Hideki; Khadka, Vedbar S.; Deng, Youping; Ohnishi, Kenta; Iida, Kota; Gotoh, Daisuke; Nakai, Yasushi; Inoue, Takeshi; Anai, Satoshi; Torimoto, Kazumasa; Aoki, Katsuya; Tanaka, Nobumichi; Konishi, Noboru; Fujimoto, Kiyohide

2017-01-01

Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder. PMID:28415608

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

PubMed

Davidenko, Natalia; Hamaia, Samir; Bax, Daniel V; Malcor, Jean-Daniel; Schuster, Carlos F; Gullberg, Donald; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

2018-01-01

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.

PubMed

El Ghalbzouri, Abdoelwaheb; Jonkman, Marcel F; Dijkman, Remco; Ponec, Maria

2005-01-01

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae).

PubMed

Pallela, Ramjee; Bojja, Sreedhar; Janapala, Venkateswara Rao

2011-07-01

Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries. Copyright © 2011 Elsevier B.V. All rights reserved.

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase.

PubMed

Baumann, Stephan; Hennet, Thierry

2016-08-26

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase*

PubMed Central

Baumann, Stephan

2016-01-01

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1–2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability. PMID:27402836

Collagen mimetic peptide engineered M13 bacteriophage for collagen targeting and imaging in cancer.

PubMed

Jin, Hyo-Eon; Farr, Rebecca; Lee, Seung-Wuk

2014-11-01

Collagens are over-expressed in various human cancers and subsequently degraded and denatured by proteolytic enzymes, thus making them a target for diagnostics and therapeutics. Genetically engineered bacteriophage (phage) is a promising candidate for the development of imaging or therapeutic materials for cancer collagen targeting due to its promising structural features. We genetically engineered M13 phages with two functional peptides, collagen mimetic peptide and streptavidin binding peptide, on their minor and major coat proteins, respectively. The resulting engineered phage functions as a therapeutic or imaging material to target degraded and denatured collagens in cancerous tissues. We demonstrated that the engineered phages are able to target and label abnormal collagens expressed on A549 human lung adenocarcinoma cells after the conjugation with streptavidin-linked fluorescent agents. Our engineered collagen binding phage could be a useful platform for abnormal collagen imaging and drug delivery in various collagen-related diseases. Published by Elsevier Ltd.

C-type natriuretic peptide suppresses mesangial proliferation and matrix expression via a MMPs/TIMPs-independent pathway in vitro.

PubMed

Huang, Bao Yu; Hu, Peng; Zhang, Dong Dong; Jiang, Guang Mei; Liu, Si Yan; Xu, Yao; Wu, Yang Fang; Xia, Xun; Wang, Ya

2017-08-01

C-type natriuretic peptide (CNP) acts mainly in a local, paracrine fashion to regulate vascular tone and cell proliferation. Although several in vivo studies have demonstrated that CNP exerts an inhibitory effect on mesangial matrix generation, a limited number of reports exist about the anti-extracellular matrix (ECM) accumulation effect of CNP and its underlying mechanisms in mesangial cells (MCs) in vitro. In this study, human MCs were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100 pM) for 24, 48 and 72 h, respectively. CNP administration significantly suppresses MCs proliferation and collagen (Col)-IV expression in a time- and dose-dependent manner. In addition, the study presented herein was designed as a first demonstration of the regulative effects of CNP on the metabolisms of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in MCs in vitro, and found that: (1) CNP administration significantly decreased the secretion and expression of MMP-2 and MMP-9 in the cultured MCs; (2) the secretion and expression of TIMP-1 progressively elevated after treatment with CNP for 24 and 48 h, whereas declined at later time point; (3) CNP expression was negatively correlated with MMP-2 and MMP-9 expression; (4) the balance of MMPs/TIMPs was shifted toward the reduction in MMP-2 and MMP-9 activity and/or the increment in TIMP-1 expression, which could not account for the down-regulation of Col-IV expression in CNP-treated MCs. In conclusion, CNP suppresses mesangial proliferation and ECM expression via a MMPs/TIMPs-independent pathway in vitro.

Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an in vitro study.

PubMed

Eslami, Hosein; Motahari, Paria; Safari, Ebrahim; Seyyedi, Maryam

2017-06-30

production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm 2 , G3-irradiated at 0.15 J/cm 2 , G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm 2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm 2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm 2 ) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies.

Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an in vitro study

PubMed Central

Eslami, Hosein; Motahari, Paria; Safari, Ebrahim; Seyyedi, Maryam

2017-01-01

Back ground and aim production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Methods and materials Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm2, G3-irradiated at 0.15 J/cm2, G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. Results The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm2) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. Conclusion LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies. PMID:28785130

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

PubMed Central

2013-01-01

Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair. PMID:23899094

Age-related modifications of type I collagen impair DDR1-induced apoptosis in non-invasive breast carcinoma cells.

PubMed

Charles, Saby; Hassan, Rammal; Kevin, Magnien; Emilie, Buache; Sylvie, Brassart-Pasco; Laurence, Van-Gulick; Pierre, Jeannesson; Erik, Maquoi; Hamid, Morjani

2018-05-07

Type I collagen and DDR1 axis has been described to decrease cell proliferation and to initiate apoptosis in non-invasive breast carcinoma in three-dimensional cell culture matrices. Moreover, MT1-MMP down-regulates these effects. Here, we address the effect of type I collagen aging and MT1-MMP expression on cell proliferation suppression and induced-apoptosis in non-invasive MCF-7 and ZR-75-1 breast carcinoma. We provide evidence for a decrease in cell growth and an increase in apoptosis in the presence of adult collagen when compared to old collagen. This effect involves a differential activation of DDR1, as evidenced by a higher DDR1 phosphorylation level in adult collagen. In adult collagen, inhibition of DDR1 expression and kinase function induced an increase in cell growth to a level similar to that observed in old collagen. The impact of aging on the sensitivity of collagen to MT1-MMP has been reported recently. We used the MT1-MMP expression strategy to verify whether, by degrading adult type I collagen, it could lead to the same phenotype observed in old collagen 3D matrix. MT1-MMP overexpression abrogated the proliferation suppression and induced-apoptosis effects only in the presence of adult collagen. This suggests that differential collagen degradation by MT1-MMP induced a structural disorganization of adult collagen and inhibits DDR1 activation. This could in turn impair DDR1-induced cell growth suppression and apoptosis. Taken together, our data suggest that modifications of collagen structural organization, due to aging, contribute to the loss of the growth suppression and induced apoptosis effect of collagen in luminal breast carcinoma. MT1-MMP-dependent degradation and aging of collagen have no additive effects on these processes.

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels.

PubMed

Unemori, E N; Werb, Z

1986-09-01

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.

Hyaluronic acid increases tendon derived cell viability and collagen type I expression in vitro: Comparative study of four different Hyaluronic acid preparations by molecular weight.

PubMed

Osti, Leonardo; Berardocco, Martina; di Giacomo, Viviana; Di Bernardo, Graziella; Oliva, Francesco; Berardi, Anna C

2015-10-06

Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate in human rotator cuff tendon derived cells the effects of four different HA on cell viability, proliferation, apoptosis and the expression of collagen type I and collagen type III. An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of four different HA preparations (Ps) (sodium hyaluronate MW: 500-730 KDa - Hyalgan®, 1000 kDa Artrosulfur HA®, 1600 KDa Hyalubrix® and 2200 KDa Synolis-VA®) at various concentrations. Tendon derived cells morphology were evaluated after 0, 7 and 14 d of culture. Viability, proliferation, apoptosis were evaluated after 0, 24 and 48 h of culture. The expression and deposition of collagen type I and collagen type III were evaluated after 1, 7 and 14 d of culture. All HAPs tested increased viability and proliferation, in dose dependent manner. HAPs already reduce apoptosis at 24 h compared to control cells (without HAPs). Furthermore, HAPs stimulated the synthesis of collagen type I in a dose dependent fashion over 14 d, without increase in collagen type III; moreover, in the presence of Synolis-VA® the expression and deposition of collagen type I was significantly higher as compare with the other HAPs. HAPs enhanced viability, proliferation and expression of collagen type I in tendon derived cells.

Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes.

PubMed

Fong, Gloria; Backman, Ludvig J; Hart, David A; Danielson, Patrik; McCormack, Bob; Scott, Alex

2013-01-01

The loss of collagen organization is considered a hallmark histopathologic feature of tendinosis. At the cellular level, tenocytes have been shown to produce signal substances that were once thought to be restricted to neurons. One of the main neuropeptides implicated in tendinosis, substance P (SP), is known to influence collagen organization, particularly after injury. The aim of this study was to examine the influence of SP on collagen remodeling by primary human tendon cells cultured in vitro in three-dimensional collagen lattices. We found that SP stimulation led to an increased rate of collagen remodeling mediated via the neurokinin-1 receptor (NK-1 R), the preferred cell receptor for SP. Gene expression analysis showed that SP stimulation resulted in significant increases in MMP3, COL3A1 and ACTA2 mRNA levels in the collagen lattices. Furthermore, cyclic tensile loading of tendon cell cultures along with the administration of exogenous SP had an additive effect on MMP3 expression. Immunoblotting confirmed that SP increased MMP3 protein levels via the NK-1 R. This study indicates that SP, mediated via NK-1 R, increases collagen remodeling and leads to increased MMP3 mRNA and protein expression that is further enhanced by cyclic mechanical loading. Copyright © 2012 Orthopaedic Research Society.

Cadherin-11 modulates cell morphology and collagen synthesis in periodontal ligament cells under mechanical stress.

PubMed

Feng, Lishu; Zhang, Yimei; Kou, Xiaoxing; Yang, Ruili; Liu, Dawei; Wang, Xuedong; Song, Yang; Cao, Haifeng; He, Danqing; Gan, Yehua; Zhou, Yanheng

2017-03-01

To examine the role of cadherin-11, an integral membrane adhesion molecule, in periodontal ligament cells (PDLCs) under mechanical stimulation. Human PDLCs were cultured and subjected to mechanical stress. Cadherin-11 expression and cell morphology of PDLCs were investigated via immunofluorescence staining. The mRNA and protein expressions of cadherin-11 and type I collagen (Col-I) of PDLCs were evaluated by quantitative real-time polymerase chain reaction and Western blot, respectively. Small interfering RNA was used to knock down cadherin-11 expression in PDLCs. The collagen matrix of PDLCs was examined using toluidine blue staining. Cadherin-11 was expressed in PDLCs. Mechanical stress suppressed cadherin-11 expression in PDLCs with prolonged force treatment time and increased force intensity, accompanied by suppressed β-catenin expression. Simultaneously, mechanical stress altered cell morphology and repressed Col-I expression in a time- and dose-dependent manner in PDLCs. Moreover, knockdown of cadherin-11 with suppressed β-catenin expression resulted in altered PDLC morphology and repressed collagen expression, which were consistent with the changes observed under mechanical stress. Results of this study suggest that cadherin-11 is expressed in PDLCs and modulates PDLC morphology and collagen synthesis in response to mechanical stress, which may play an important role in the homeostasis and remodeling of the PDL under mechanical stimulation.

Pregnancy complicated with Alport syndrome: a good obstetric outcome and failure to diagnose an infant born to a mother with Alport syndrome by umbilical cord immunofluorescence staining.

PubMed

Matsubara, Shigeki; Ueda, Yoshihiko; Takahashi, Hisako; Nagai, Takashi; Kuwata, Tomoyuki; Muto, Shigeaki; Yamaguchi, Takehiko; Takizawa, Toshihiro; Suzuki, Mitsuaki

2009-12-01

Alport syndrome is a familial progressive nephritis. The most frequent type is X-linked Alport syndrome, caused by genetic abnormalities in the alpha 5 chain of type IV collagen. Skin biopsy is a useful tool for diagnosing this disease. It is not well known how this syndrome affects pregnancy and how it is affected by pregnancy, or whether the umbilical cord may provide material for detecting this collagen abnormality. We report a primigravida with Alport syndrome with mild proteinuria who gave birth abdominally to a term male infant without deteriorating renal function during pregnancy. The umbilical cord from not only this infant but also from an Alport (-) control infant showed negative immunofluorescence staining for the alpha 5 chain of type IV collagen. Women with Alport syndrome without renal dysfunction may follow an uneventful obstetrical course until term. The cord may not be suitable for diagnosing Alport syndrome with immunofluorescence staining.

Bone tissue ultrastructural defects in a mouse model for osteogenesis imperfecta: a Raman spectroscopy study

NASA Astrophysics Data System (ADS)

Chen, Tsoching; Kozloff, Kenneth M.; Goldstein, Steven A.; Morris, Michael D.

2004-07-01

Osteogenesis imperfecta (OI) is genetic defect in which the genes that code for the α1(I) or α2(I) chains of type I collagen are defective. The defects often result in substitution of a bulky amino acid for glycine, causing formation of collagen that can not form the normal triple helix. Depending on the details of the defects, the outcomes range from controllable to lethal. This study focuses on OI type IV, a more common and moderately severe form of the disease. People with the disease have a substantial increase in the risk and rate of fracture. We examine the spectroscopic consequences of these defects, using a mouse model (BRTL) that mimics OI type IV. We compare Raman images from tibial cortical tissue of wild-type mice and BRTL mice with single copy of mutation and show that both mineral to matrix ratios and collagen inter-fibril cross-links are different in wild-type and mutant mice.

Compound effects of aging and experimental FSGS on glomerular epithelial cells.

PubMed

Schneider, Remington R S; Eng, Diana G; Kutz, J Nathan; Sweetwyne, Mariya T; Pippin, Jeffrey W; Shankland, Stuart J

2017-02-17

Advanced age portends a poorer prognosis in FSGS. To understand the impact of age on glomerular podocytes and parietal epithelial cells (PECs), experimental FSGS was induced in 3m-old mice (20-year old human age) and 27m-old mice (78-year old human age) by abruptly depleting podocytes with a cytopathic anti-podocyte antibody. Despite similar binding of the disease-inducing antibody, podocyte density was lower in aged FSGS mice compared to young FSGS mice. Activated PEC density was higher in aged versus young FSGS mice, as was the percentage of total activated PECs. Additionally, the percentage of glomeruli containing PECs with evidence of phosphorylated ERK and EMT was higher in aged FSGS mice. Extracellular matrix, measured by collagen IV and silver staining, was higher in aged FSGS mice along Bowman's capsule. However, collagen IV accumulation in the glomerular tufts alone and in glomeruli with both tuft and Bowman's capsule accumulation were similar in young FSGS and aged FSGS mice. Thus, the major difference in collagen IV staining in FSGS was along Bowman's capsule in aged mice. The significant differences in podocytes, PECs and extracellular matrix accumulation between young mice and old mice with FSGS might explain the differences in outcomes in FSGS based on age.

Compound effects of aging and experimental FSGS on glomerular epithelial cells

PubMed Central

Kutz, J. Nathan; Sweetwyne, Mariya T.; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

Advanced age portends a poorer prognosis in FSGS. To understand the impact of age on glomerular podocytes and parietal epithelial cells (PECs), experimental FSGS was induced in 3m-old mice (20-year old human age) and 27m-old mice (78-year old human age) by abruptly depleting podocytes with a cytopathic anti-podocyte antibody. Despite similar binding of the disease-inducing antibody, podocyte density was lower in aged FSGS mice compared to young FSGS mice. Activated PEC density was higher in aged versus young FSGS mice, as was the percentage of total activated PECs. Additionally, the percentage of glomeruli containing PECs with evidence of phosphorylated ERK and EMT was higher in aged FSGS mice. Extracellular matrix, measured by collagen IV and silver staining, was higher in aged FSGS mice along Bowman's capsule. However, collagen IV accumulation in the glomerular tufts alone and in glomeruli with both tuft and Bowman's capsule accumulation were similar in young FSGS and aged FSGS mice. Thus, the major difference in collagen IV staining in FSGS was along Bowman's capsule in aged mice. The significant differences in podocytes, PECs and extracellular matrixaccumulation between young mice and old mice with FSGS might explain the differences in outcomes in FSGS based on age. PMID:28222042

Long-term treatment with EGFR inhibitor erlotinib attenuates renal inflammatory cytokines but not nephropathy in Alport syndrome mouse model.

PubMed

Omachi, Kohei; Miyakita, Rui; Fukuda, Ryosuke; Kai, Yukari; Suico, Mary Ann; Yokota, Tsubasa; Kamura, Misato; Shuto, Tsuyoshi; Kai, Hirofumi

2017-12-01

Alport syndrome (AS) is a hereditary kidney disease caused by mutation of type IV collagen. Loss of collagen network induces collapse of glomerular basement membrane (GBM) structure. The previous studies showed that upregulation of some tyrosine kinase receptors signaling accompanied GBM disorder in AS mouse model. EGFR signaling is one of the well-known receptor kinase signaling that is involved in glomerular diseases. However, whether EGFR signaling is relevant to AS progression is still uninvestigated. Here, we determined the involvement of EGFR in AS and the effect of suppressing EGFR signaling by erlotinib treatment on AS progression. Phosphorylated EGFR expression was investigated by Western blotting analysis and immunostaining of kidney tissues of Col4a5 mutant mice (a mouse model of X-linked AS). To check the effect of blocking EGFR signaling in AS, we administered erlotinib to AS mice once a day (10 mg/kg/day) orally for 18 weeks. Renal function parameters (proteinuria, serum creatinine, and BUN) and renal histology were assessed, and the gene expressions of inflammatory cytokines were analyzed in renal tissues. Phosphorylated EGFR expression was upregulated in AS mice kidney tissues. Erlotinib slightly reduced the urinary protein and suppressed the expression of renal injury markers (Lcn2, Lysozyme) and inflammatory cytokines (Il-6, Il-1β and KC). Erlotinib did not improve renal pathology, such as glomerular sclerosis and fibrosis. These findings suggest that EGFR signaling is upregulated in kidney, but although inhibiting this signaling pathway suppressed renal inflammatory cytokines, it did not ameliorate renal dysfunction in AS mouse model.

A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Assembly.

PubMed

Knüppel, Larissa; Ishikawa, Yoshihiro; Aichler, Michaela; Heinzelmann, Katharina; Hatz, Rudolf; Behr, Jürgen; Walch, Axel; Bächinger, Hans Peter; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 μM) or pirfenidone (100-1,000 μM) in the absence or presence of transforming growth factor-β1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.

Renal myofibroblasts contract collagen I matrix lattices in vitro.

PubMed

Kelynack, K J; Hewitson, T D; Pedagogos, E; Nicholls, K M; Becker, G J

1999-01-01

Myofibroblasts, cells with both fibroblastic and smooth muscle cell features, have been implicated in renal scarring. In addition to synthetic properties, contractile features and integrin expression may allow myofibroblasts to rearrange and contract interstitial collagenous proteins. Myofibroblasts from normal rat kidneys were grown in cell-populated collagen lattices to measure cell generated contraction. Following detachment of cell populated collagen lattices, myofibroblasts progressively contracted collagen lattices, reducing lattice diameter by 42% at 24 h. Alignment of myofibroblasts, rearrangement of fibrils and beta(1) integrin expression were observed within lattices. We postulate that interstitial myofibroblasts contribute to renal scarring through manipulation of collagenous proteins. Copyright 1999 S. Karger AG, Basel

Propolis Modifies Collagen Types I and III Accumulation in the Matrix of Burnt Tissue.

PubMed

Olczyk, Pawel; Wisowski, Grzegorz; Komosinska-Vassev, Katarzyna; Stojko, Jerzy; Klimek, Katarzyna; Olczyk, Monika; Kozma, Ewa M

2013-01-01

Wound healing represents an interactive process which requires highly organized activity of various cells, synthesizing cytokines, growth factors, and collagen. Collagen types I and III, serving as structural and regulatory molecules, play pivotal roles during wound healing. The aim of this study was to compare the propolis and silver sulfadiazine therapeutic efficacy throughout the quantitative and qualitative assessment of collagen types I and III accumulation in the matrix of burnt tissues. Burn wounds were inflicted on pigs, chosen for the evaluation of wound repair because of many similarities between pig and human skin. Isolated collagen types I and III were estimated by the surface plasmon resonance method with a subsequent collagenous quantification using electrophoretic and densitometric analyses. Propolis burn treatment led to enhanced collagens and its components expression, especially during the initial stage of the study. Less expressed changes were observed after silver sulfadiazine (AgSD) application. AgSD and, with a smaller intensity, propolis stimulated accumulation of collagenous degradation products. The assessed propolis therapeutic efficacy, throughout quantitatively and qualitatively analyses of collagen types I and III expression and degradation in wounds matrix, may indicate that apitherapeutic agent can generate favorable biochemical environment supporting reepithelization.

Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes.

PubMed

Bader, Hannah L; Keene, Douglas R; Charvet, Benjamin; Veit, Guido; Driever, Wolfgang; Koch, Manuel; Ruggiero, Florence

2009-01-01

Connective tissues ensure the cohesion of the tissues of the body, but also form specialized structures such as tendon and bone. Collagen XII may enhance the stability of connective tissues by bridging collagen fibrils, but its function is still unclear. Here, we used the zebrafish model to visualize its expression pattern in the whole organism. The zebrafish col12a1 gene is homologous to the small isoform of the tetrapod col12a1 gene. In agreement with the biochemical data reported for the small isoform, the zebrafish collagen XII alpha1 chain was characterized as a collagenase sensitive band migrating at approximately 200 kDa. Using newly generated polyclonal antibodies and anti-sense probes, we performed a comprehensive analysis of its expression in developing zebrafish. Collagen XII exhibited a much broader expression pattern than previously thought: it was ubiquitously expressed in the connective tissue sheaths (fascia) that encase the tissues and organs of the body. For example, it was found in sclera, meninges, epimysia and horizontal and vertical myosepta. Collagen XII was also detected in head mesenchyme, pharyngeal arches and within the spinal cord, where it was first expressed within and then at the lateral borders of the floor plate and at the dorsal midline. Furthermore, double immunofluorescence staining with laminin and immunogold electron microscopy revealed that collagen XII is associated with basement membranes. These data suggest that collagen XII is implicated in tissue cohesion by stabilizing fascia and by linking fascia to basement membranes.

Regeneration of cervix after excisional treatment for cervical intraepithelial neoplasia: a study of collagen distribution.

PubMed

Phadnis, S V; Atilade, A; Bowring, J; Kyrgiou, M; Young, M P A; Evans, H; Paraskevaidis, E; Walker, P

2011-12-01

To study the distribution of collagen in the regenerated cervical tissue after excisional treatment for cervical intraepithelial neoplasia (CIN). Cohort study. A large tertiary teaching hospital in London. Women who underwent repeat excisional treatment for treatment failure or persistent CIN. Eligible women who underwent a repeat excisional treatment for treatment failure, including hysterectomy, between January 2002 and December 2007 in our colposcopy unit were identified by the Infoflex(®) database and SNOMED encoded histopathology database. Collagen expression was assessed using picro-Sirius red stain and the intensity of staining was compared in paired specimens from the first and second treatments. Differences in collagen expression were examined in the paired excisional treatment specimens. A total of 17 women were included. Increased collagen expression in the regenerated cervical tissue of the second cone compared with the first cone was noted in six women, decreased expression was noted in five women, and the pattern of collagen distribution was equivocal in six women. There is no overall change in collagen distribution during regeneration following excisional treatment for CIN. © 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2011 RCOG.

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes.

PubMed

Yu, Seon-Mi; Kim, Song-Ja

2013-11-01

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined. We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes. © 2013 Published by Elsevier Inc.

Increased oxygen exposure alters collagen expression and tissue architecture during ligature-induced periodontitis.

PubMed

Gajendrareddy, P K; Junges, R; Cygan, G; Zhao, Y; Marucha, P T; Engeland, C G

2017-06-01

The aim of this study was to evaluate the effects of increased oxygen availability on gene expression and on collagen deposition/maturation in the periodontium following disease. Male Wistar rats had ligatures placed around their molars to induce periodontal disease, and a subset of animals underwent hyperbaric oxygen (HBO) treatment for 2 h twice per day. At 15 and 28 d, tissue gene expression of COL1A1, transforming growth factor-β1 and alkaline phosphatase was determined; other histological samples were stained with Picrosirius red to evaluate levels of collagen deposition, maturation and thickness. In animals that underwent HBO treatment, type I collagen expression was higher and collagen deposition, maturation and thickness were more robust. Reduced mRNA levels of transforming growth factor-beta1 and alkaline phosphatase in HBO-treated rats on day 28 suggested that a quicker resolution in both soft tissue and bone remodeling occurred following oxygen treatment. No differences in inflammation were observed between groups. The extracellular matrix regenerated more quickly in the HBO-treated group as evidenced by higher collagen expression, deposition and maturation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

PubMed Central

Watt, Stephen A.; Dayal, Jasbani H. S.; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R.; Kimble, Roy M.; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W. H. Irwin; Leigh, Irene M.; McGrath, John A.; Salas-Alanis, Julio C.; Tolar, Jakub; South, Andrew P.

2015-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention. PMID:26380979

Evaluation of Anterior Vertebral Interbody Fusion Using Osteogenic Mesenchymal Stem Cells Transplanted in Collagen Sponge.

PubMed

Yang, Wencheng; Dong, Youhai; Hong, Yang; Guang, Qian; Chen, Xujun

2016-05-01

The study used a rabbit model to achieve anterior vertebral interbody fusion using osteogenic mesenchymal stem cells (OMSCs) transplanted in collagen sponge. We investigated the effectiveness of graft material for anterior vertebral interbody fusion using a rabbit model by examining the OMSCs transplanted in collagen sponge. Anterior vertebral interbody fusion is commonly performed. Although autogenous bone graft remains the gold-standard fusion material, it requires a separate surgical procedure and is associated with significant short-term and long-term morbidity. Recently, mesenchymal stem cells from bone marrow have been studied in various fields, including posterolateral spinal fusion. Thus, we hypothesized that cultured OMSCs transplanted in porous collagen sponge could be used successfully even in anterior vertebral interbody fusion. Forty mature male White Zealand rabbits (weight, 3.5-4.5 kg) were randomly allocated to receive one of the following graft materials: porous collagen sponge plus cultured OMSCs (group I); porous collagen sponge alone (group II); autogenous bone graft (group III); and nothing (group IV). All animals underwent anterior vertebral interbody fusion at the L4/L5 level. The lumbar spine was harvested en bloc, and the new bone formation and spinal fusion was evaluated using radiographic analysis, microcomputed tomography, manual palpation test, and histologic examination at 8 and 12 weeks after surgery. New bone formation and bony fusion was evident as early as 8 weeks in groups I and III. And there was no statistically significant difference between 8 and 12 weeks. At both time points, by microcomputed tomography and histologic analysis, new bone formation was observed in both groups I and III, fibrous tissue was observed and there was no new bone in both groups II and IV; by manual palpation test, bony fusion was observed in 40% (4/10) of rabbits in group I, 70% (7/10) of rabbits in group III, and 0% (0/10) of rabbits in both groups II and IV. These findings suggest that mesenchymal stem cells that have been cultured with osteogenic differentiation medium and loaded with collagen sponge could induce bone formation and anterior vertebral interbody fusion. And the rabbit model we developed will be useful in evaluating the effects of graft materials for anterior vertebral interbody fusion. Further study is needed to determine the most appropriate carrier for OMSCs and the feasibility in the clinical setting.

RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells.

PubMed

Tsutsumida, Hideaki; Swanson, Benjamin J; Singh, Pankaj K; Caffrey, Thomas C; Kitajima, Shinichi; Goto, Masamichi; Yonezawa, Suguru; Hollingsworth, Michael A

2006-05-15

MUC1 is a highly glycosylated, type I transmembrane protein expressed by normal ductal epithelial cells of the pancreas, breast, lung, and gastrointestinal tract, and overexpressed in many cases of adenocarcinoma. We down-regulated MUC1 expression by RNA interference and investigated the effects on malignant and metastatic potential of a human pancreatic cancer cell line, S2-013. MUC1-suppressed clones, S2-013.MTII.C1 and S2-013.MTII.C2, were established by targeting a sequence 3,151 bp from the initiation codon and characterized in vitro for proliferation, invasion, and adhesion. We evaluated the effects of MUC1 suppression in vivo on tumor growth and metastatic properties following implantation into the cecum or pancreas of athymic mice. MUC1-suppressed clones showed significantly decreased proliferation in vitro and in vivo. Global gene expression was evaluated by oligonucleotide microarray analysis. Surprisingly, genes predicted to increase doubling times (cyclin B1 and cyclin D3) were overexpressed in MUC1-suppressed clones. There were alterations in expression of several genes that may affect the malignant properties of pancreatic cancer. Adhesion of MUC1-suppressed cells in vitro to type IV collagen and fibronectin was slightly increased, and adhesion was slightly decreased to type I collagen and laminin. Results of implantation to cecum and pancreas showed significant reduction of metastasis to lymph nodes, lung, or peritoneal sites compared with S2-013.gfp-neo control cells. These results support the hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of MUC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma.

Emodin alleviates bleomycin-induced pulmonary fibrosis in rats.

PubMed

Guan, Ruijuan; Zhao, Xiaomei; Wang, Xia; Song, Nana; Guo, Yuhong; Yan, Xianxia; Jiang, Liping; Cheng, Wenjing; Shen, Linlin

2016-11-16

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with few treatment options and poor prognosis. Emodin, extracted from Chinese rhubarb, was found to be able to alleviate bleomycin (BLM)-induced pulmonary fibrosis, yet the underlying mechanism remains largely unknown. This study aimed to further investigate the effects of emodin on the inflammation and fibrosis of BLM-induced pulmonary fibrosis and the mechanism involved in rats. Our results showed that emodin improved pulmonary function, reduced weight loss and prevented death in BLM-treated rats. Emodin significantly relieved lung edema and fibrotic changes, decreased collagen deposition, and suppressed the infiltration of myofibroblasts [characterized by expression of α-smooth muscle actin (α-SMA)] and inflammatory cells (mainly macrophages and lymphocytes). Moreover, emodin reduced levels of TNF-α, IL-6, TGF-β1 and heat shock protein (HSP)-47 in the lungs of BLM-treated rats. In vitro, emodin profoundly inhibited TGF-β1-induced α-SMA, collagen IV and fibronectin expression in human embryo lung fibroblasts (HELFs). Emodin also inhibited TGF-β1-induced Smad2/3 and STAT3 activation, indicating that Smad2/3 and STAT3 inactivation mediates emodin-induced effects on TGF-β1-induced myofibroblast differentiation. These results suggest that emodin can exert its anti-fibrotic effect via suppression of TGF-β1 signaling and subsequently inhibition of inflammation, HSP-47 expression, myofibroblast differentiation and extracellular matrix (ECM) deposition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Osthole inhibits the expressions of collagen I and III through Smad signaling pathway after treatment with TGF-β1 in mouse cardiac fibroblasts.

PubMed

Liu, Jin-Cheng; Wang, Feng; Xie, Mei-Lin; Cheng, Zong-Qi; Qin, Qiong; Chen, Lin; Chen, Rong

2017-02-01

Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-β1 (TGF-β1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-β1. The mouse CFs stimulated with TGF-β1 were cultured and treated with osthole 1.25-5μg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-β receptor I (TβRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively. After treatment with TGF-β1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TβRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole. The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-β1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages

PubMed Central

Bao, Zhang; Chen, Ran; Zhang, Pei; Lu, Shan; Chen, Xing; Yao, Yake; Jin, Xiaozheng; Sun, Yilan; Zhou, Jianying

2016-01-01

Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in RAW264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of RAW264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the RAW264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy. PMID:27432120

Blockade of AT1 Receptors Protects the Blood–Brain Barrier and Improves Cognition in Dahl Salt-Sensitive Hypertensive Rats

PubMed Central

Pelisch, Nicolas; Hosomi, Naohisa; Ueno, Masaki; Nakano, Daisuke; Hitomi, Hirofumi; Mogi, Masaki; Shimada, Kenji; Kobori, Hiroyuki; Horiuchi, Masatsugu; Sakamoto, Haruhiko; Matsumoto, Masayasu; Kohno, Masakazu; Nishiyama, Akira

2011-01-01

BACKGROUND The present study tested the hypothesis that inappropriate activation of the brain renin–angiotensin system (RAS) contributes to the pathogenesis of blood–brain barrier (BBB) disruption and cognitive impairment during development of salt-dependent hypertension. Effects of an angiotensin II (AngII) type-1 receptor blocker (ARB), at a dose that did not reduce blood pressure, were also examined. METHODS Dahl salt-sensitive (DSS) rats at 6 weeks of age were assigned to three groups: low-salt diet (DSS/L; 0.3% NaCl), high-salt diet (DSS/H; 8% NaCl), and high-salt diet treated with ARB, olmesartan at 1 mg/kg. RESULTS DSS/H rats exhibited hypertension, leakage from brain microvessels in the hippocampus, and impaired cognitive functions, which were associated with increased brain AngII levels, as well as decreased mRNA levels of tight junctions (TJs) and collagen-IV in the hippocampus. In DSS/H rats, olmesartan treatment, at a dose that did not alter blood pressure, restored the cognitive decline, and ameliorated leakage from brain microvessels. Olmesartan also decreased brain AngII levels and restored mRNA expression of TJs and collagen-IV in DSS/H rats. CONCLUSIONS These results suggest that during development of salt-dependent hypertension, activation of the brain RAS contributes to BBB disruption and cognitive impairment. Treatment with an ARB could elicit neuroprotective effects in cognitive disorders by preventing BBB permeability, which is independent of blood pressure changes. PMID:21164491

TCDD disrupts posterior palatogenesis and causes cleft palate.

PubMed

Yamada, Tomohiro; Hirata, Azumi; Sasabe, Eri; Yoshimura, Tomohide; Ohno, Seiji; Kitamura, Naoya; Yamamoto, Tetsuya

2014-01-01

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Memantine mediates neuroprotection via regulating neurovascular unit in a mouse model of focal cerebral ischemia.

PubMed

Chen, Zheng-Zhen; Yang, Dan-Dan; Zhao, Zhan; Yan, Hui; Ji, Juan; Sun, Xiu-Lan

2016-04-01

Memantine is a low-moderate affinity and uncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist, which is also a potential neuroprotectant in acute ischemic stroke for its particular action profiles. The present study was to reveal the mechanisms involved in the neuroprotection of memantine. We used a mouse model of permanent focal cerebral ischemia via middle cerebral artery occlusion to verify our hypothesis. 2,3,5-Triphenyltetrazolium chloride staining was used to compare infarct size. The amount of astrocytes and the somal volume of the microglia cell body were analyzed by immunohistochemistry and stereological estimates. Western blotting was used to determine the protein expressions. Memantine prevented cerebral ischemia-induced brain infarct and neuronal injury, and reduced oxygen-glucose deprivation-induced cortical neuronal apoptosis. Moreover, memantine reduced the amount of the damaged astrocytes and over activated microglia after 24h of ischemia. In the early phase of ischemia, higher production of MMP-9 was observed, and thereby collagen IV was dramatically disrupted. Meanwhile, the post-synaptic density protein 95(PSD-95) was also severely cleavaged. Memantine decreased MMP-9 secretion, prevented the degradation of collagen IV in mouse brain. PSD-95 cleavage was also inhibited by memantine. These results suggested that memantine exerted neuroprotection effects in acute ischemic brain damage, partially via improving the functions of neurovascular unit. Taking all these findings together, we consider that memantine might be a promising protective agent against ischemic stroke. Copyright © 2016 Elsevier Inc. All rights reserved.

EPAC expression and function in cardiac fibroblasts and myofibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olmedo, Ivonne; Muñoz, Claudia; Guzmán, Nancy

In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor β1 (TGF-β1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction. Method: Rat neonatal CF and CMF weremore » treated with TGF-β1 at different times and concentrations. EPAC-1 protein levels and Rap1 activation were measured by western blot and pull down assay respectively. EPAC cellular functions were determined by adhesion, migration and collagen gel contraction assay; and collagen expression was determined by western blot. Results: TGF-β1 through Smad and JNK significantly reduced EPAC-1 expression in CF, while in CMF this cytokine increased EPAC-1 expression through ERK1/2, JNK, p38, AKT and Smad3. EPAC activation was able to induce higher Rap1-GTP levels in CMF than in CF. EPAC and PKA, both cAMP effectors, promoted CF and CMF adhesion on fibronectin, as well as CF migration; however, this effect was not observed in CMF. EPAC but not PKA activation mediated collagen gel contraction in CF, while in CMF both PKA and EPAC mediated collagen gel contraction. Finally, the EPAC and PKA activation reduced collagen synthesis in CF and CMF. Conclusion: TGF-β1 differentially regulates the expression of EPAC in CF and CMF; and EPAC regulates differentially CF and CMF functions associated with cardiac remodeling. - Highlights: • TGF-β1 regulates EPAC-1 expression in cardiac fibroblast and myofibroblast. • Rap-1GTP levels are higher in cardiac myofibroblast than fibroblast. • EPAC-1 controls adhesion, migration and collagen synthesis in cardiac fibroblast. • PKA regulates collagen gel contraction in cardiac myofibroblast.« less

Impaired Collagen Biosynthesis and Cross‐linking in Aorta of Patients With Bicuspid Aortic Valve

PubMed Central

Wågsäter, Dick; Paloschi, Valentina; Hanemaaijer, Roeland; Hultenby, Kjell; Bank, Ruud A.; Franco‐Cereceda, Anders; Lindeman, Jan H. N.; Eriksson, Per

2013-01-01

Background Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysm. In the present study, collagen homeostasis in nondilated and dilated aorta segments from patients with BAV was studied, with normal and dilated aortas from tricuspid aortic valve (TAV) patients as reference. Methods and Results Ascending aortas from 56 patients were used for biochemical and morphological analyses of collagen. mRNA expression was analyzed in 109 patients. Collagen turnover rates were similar in nondilated and dilated aortas of BAV patients, showing that aneurysmal formation in BAV is, in contrast to TAV, not associated with an increased collagen turnover. However, BAV in general was associated with an increased aortic collagen turnover compared with nondilated aortas of TAV patients. Importantly, the ratio of hydroxylysyl pyridinoline (HP) to lysyl pyridinoline (LP), 2 distinct forms of collagen cross‐linking, was lower in dilated aortas from patients with BAV, which suggests that BAV is associated with a defect in the posttranslational collagen modification. This suggests a deficiency at the level of lysyl hydroxylase (PLOD1), which was confirmed by mRNA and protein analyses that showed reduced PLOD1 expression but normal lysyl oxidase expression in dilated aortas from patients with BAV. This suggests that impaired collagen cross‐linking in BAV patients may be attributed to changes in the expression and/or activity of PLOD1. Conclusions Our results demonstrate an impaired biosynthesis and posttranslational modification of collagen in aortas of patients with BAV, which may explain the increased aortic aneurysm formation in BAV patients. PMID:23525417

In Vitro Expression of the Extracellular Matrix Components Aggrecan, Collagen Types I and II by Articular Cartilage-Derived Chondrocytes.

PubMed

Schneevoigt, J; Fabian, C; Leovsky, C; Seeger, J; Bahramsoltani, M

2017-02-01

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan-based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long-term results of therapeutic procedures including cell-based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage-derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT-qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells. © 2016 Blackwell Verlag GmbH.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

PubMed

Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

2018-06-01

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

Hybrid pig versus Gottingen minipig-derived cartilage and chondrocytes show pig line-dependent differences.

PubMed

Müller, Claudia; Marzahn, Ulrike; Kohl, Benjamin; El Sayed, Karym; Lohan, Anke; Meier, Carola; Ertel, Wolfgang; Schulze-Tanzil, Gundula

2013-11-01

Minipigs are widely used as a large animal model for cartilage repair. However, many in vitro studies are based on porcine chondrocytes derived from abundantly available premature hybrid pigs. It remains unclear whether pig line-dependent differences exist which could limit the comparability between in vitro and in vivo results using either hybrid or miniature pig articular chondrocytes. Porcine knee joint femoral cartilage was isolated from 3- to 5-month-old hybrid pigs and Göttingen minipigs. Cartilage from both pig lines was analysed for thickness, zonality, cell content, size and proteoglycan deposition. Cultured articular chondrocytes from both pig lines were investigated for gene and/or protein expression of cartilage-specific proteins such as type II collagen, aggrecan, the chondrogenic transcription factor Sox9, non-specific type I collagen and the cell-matrix receptor β1-integrin. Cartilage was significantly thinner in the miniature pig compared to the hybrid pig, but the differences between the medial and lateral femur condyles did not reach a significant level. Knee joint cartilage zone formation started only in the minipig, whereas cellularity and cell diameters were comparable in both pig lines. Blood vessels could be detected in the hybrid pig but not the minipig cartilage. Sulphated proteoglycan deposition was more pronounced in cartilage zones II-IV of both pig lines. Minipig chondrocytes expressed type II and I collagen, Sox9 and β1-integrin at a higher level than hybrid pig chondrocytes. These distinct line-dependent differences should be considered when using hybrid pig-derived chondrocytes for tissue engineering and Göttingen minipigs as a large animal model.

Collagen Membranes Adsorb the Transforming Growth Factor-β Receptor I Kinase-Dependent Activity of Enamel Matrix Derivative.

PubMed

Stähli, Alexandra; Miron, Richard J; Bosshardt, Dieter D; Sculean, Anton; Gruber, Reinhard

2016-05-01

Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)-β signaling in oral fibroblasts. Three commercially available collagen products were exposed to EMD or recombinant TGF-β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF-β target genes interleukin (IL)-11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. EMD or TGF-β1 provoked a significant increase of IL-11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF-β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin-coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL-11 and PRG4. These in vitro findings suggest that collagen products adsorb a TGF-β receptor I kinase-dependent activity of EMD and make it available for potential target cells.

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

PubMed

Shoji, Atsushi; Suenaga, Yumiko; Hosaka, Atsushi; Ishida, Yuuki; Yanagida, Akio; Sugawara, Masao

2017-10-25

We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly. Copyright © 2017 Elsevier B.V. All rights reserved.

A Sulfilimine Bond Identified in Collagen IV

PubMed Central

Vanacore, Roberto; Ham, Amy-Joan L.; Voehler, Markus; Sanders, Charles R.; Conrads, Thomas P.; Veenstra, Timothy D.; Sharpless, K. Barry; Dawson, Philip E.; Hudson, Billy G.

2010-01-01

Collagen IV networks are ancient proteins of basement membranes that underlie epithelia in metazoa from sponge to human. The networks provide structural integrity to tissues and serve as ligands for integrin cell-surface receptors. They are assembled by oligomerization of triple-helical protomers and are covalently cross-linked, a key reinforcement that stabilizes networks. We used Fourier-transform ion cyclotron resonance mass spectrometry and nuclear magnetic resonance spectroscopy to show that a sulfilimine bond (-S=N-) crosslinks hydroxylysine-211 and methionine-93 of adjoining protomers, a bond not previously found in biomolecules. This bond, the nitrogen analog of a sulfoxide, appears to have arisen at the divergence of sponge and cnidaria, an adaptation of the extracellular matrix in response to mechanical stress in metazoan evolution. PMID:19729652

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

PubMed

Manduca, P; Abelmoschi, M L

1992-01-01

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.

Fragments of Citrullinated and MMP-degraded Vimentin and MMP-degraded Type III Collagen Are Novel Serological Biomarkers to Differentiate Crohn's Disease from Ulcerative Colitis.

PubMed

Mortensen, Joachim Høg; Godskesen, Line Elbjerg; Jensen, Michael Dam; Van Haaften, Wouter Tobias; Klinge, Lone Gabriels; Olinga, Peter; Dijkstra, Gerard; Kjeldsen, Jens; Karsdal, Morten Asser; Bay-Jensen, Anne-Christine; Krag, Aleksander

2015-10-01

A hallmark of inflammatory bowel disease [IBD] is chronic inflammation, which leads to excessive extracellular matrix [ECM] remodelling and release of specific protein fragments, called neoepitopes. We speculated that the biomarker profile panel for ulcerative colitis [UC] and Crohn's disease [CD] represent a heterogeneous expression pattern, and may be applied as a tool to aid in the differentiation between UC and CD. Serum biomarkers of degraded collagens I, III-IV [C1M, C3M, and C4M], collagen type 1 and IV formation [P1NP, P4NP], and citrullinated and MMP-degraded vimentin [VICM] were studied with a competitive ELISA assay system in a cohort including 164 subjects [CD n = 72, UC n = 60, and non-IBD controls n = 32] and a validation cohort of 61 subjects [CD n = 46, and UC n = 15]. Receiver operating characteristic curve analysis and logistic regression modelling were carried out to evaluate the discriminative power of the biomarkers. All biomarkers were corrected for confounding factors. VICM and C3M demonstrated the highest diagnostic power, alone, to differentiate CD from UC with an area under the curve [AUC] of 0.77 and 0.69, respectively. Furthermore, the biomarkers C1M [AUC = 0.81], C3M [AUC = 0.83], VICM [AUC = 0.83], and P1NP [AUC = 0.77] were best to differentiate UC from non-IBD. The best combinations of biomarkers to differentiate CD from UC and UC from non-IBD were VICM, C3M, C4M [AUC = 0.90] and VICM, C3M [AUC = 0.98] respectively. Specific extracellular matrix degradation markers are elevated in IBD and can discriminate CD from UC and UC from non-IBD controls with a high diagnostic accuracy. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Aniridia-related keratopathy: Structural changes in naïve and transplanted corneal buttons

PubMed Central

Stenevi, Ulf; Pedrosa Domellöf, Fátima

2018-01-01

Background To study structural changes in naïve and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK). Methods and findings Two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor α11 integrin and laminin α3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, α-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naïve ARK, KLAL ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous. Conclusions Given the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run. PMID:29889891

Aniridia-related keratopathy: Structural changes in naïve and transplanted corneal buttons.

PubMed

Vicente, André; Byström, Berit; Lindström, Mona; Stenevi, Ulf; Pedrosa Domellöf, Fátima

2018-01-01

To study structural changes in naïve and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK). Two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor α11 integrin and laminin α3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, α-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naïve ARK, KLAL ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous. Given the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run.

Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

NASA Technical Reports Server (NTRS)

Hardman, P.; Spooner, B. S.

1992-01-01

The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

[Relationship between the changes in ischemia/reperfusion cerebro-microvessel basement membrane injury and gelatinase system in senile rat].

PubMed

Li, Jian-sheng; Liu, Ke; Liu, Jing-xia; Wang, Ming-hang; Zhao, Yue-wu; Liu, Zheng-guo

2008-11-01

To study the relationship of cerebro-microvessel basement membrane injury and gelatinase system after cerebral ischemia/reperfusion (I/R) in aged rats. Cerebral I/R injury model was reproduced by intraluminal silk ligature thrombosis of the middle cerebral artery occlusion (MCAO). Rats were divided randomly into sham control and I/R groups in young rats [ischemia 3 hours (I 3 h) and reperfusion 6 hours (I/R 6 h), 12 hours (I/R 12 h), 24 hours (I/R 24 h), 3 days (I/R 3 d), 6 days (I/R 6 d)], and sham control group and I/R group in aged rats (I 3 h and I/R 6 h, I/R 12 h, I/R 24 h , I/R 3 d, I/R 6 d). The change in cerebro-cortex microvessel basement membrane structure, basement membrane type IV collagen (Col IV) and laminin (LN) contents, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) expression in every group were determined with immunohistochemical method and zymogram analysis. With the increase in age, Col IV and LN contents of the microvessel basement membrane were increased, and MMP-2 and MMP-9 expressions were stronger. With prolongation of I/R, the degradation of microvessel basement membrane components (Col IV and LN) was positively correlated with the duration of cerebral I/R. MMP-2 expression was increased gradually, and MMP-9 and TIMP-1 expression increased at the beginning and decreased subsequently. Col IV(I 3 h, I/R 6 h , I/R 12 h), LN (I 3 h, I/R 6-24 h), MMP-2 (I 3 h, I/R 6 h-6 d) and MMP-9 (I 3 h, I/R 6-24 h) expression level in aged rats with I/R injury were higher, and TIMP-1 (I/R 24 h) expression was lower than those in young rats (P<0.05 or P<0.01). In addition, changes in MMP-2 and MMP-9 contents as determined by zymogram analysis method coincided with their immunoexpression. With the increase of age, alteration in membrane components of cerebro-microvessel basement membrane in rats is related with MMPs and TIMP. Cerebro-microvessel basement membrane injury is more serious in aged rats than that of young rats. Changes in cerebro-microvessel basement membrane injury in aged rats is related with gelatinase system change.

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types

PubMed Central

Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

2007-01-01

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle–tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-β-1 (TGF-β-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague–Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7–9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-β-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-β-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-β-1 in loading-induced collagen synthesis in the muscle–tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus. PMID:17540706

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types.

PubMed

Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

2007-08-01

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF-beta-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-beta-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-beta-1 in loading-induced collagen synthesis in the muscle-tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland.

PubMed

Syaidah, Rahimi; Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

2013-11-01

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

LOXL1 deficiency in the lamina cribrosa as candidate susceptibility factor for a pseudoexfoliation-specific risk of glaucoma.

PubMed

Schlötzer-Schrehardt, Ursula; Hammer, Christian M; Krysta, Anita W; Hofmann-Rummelt, Carmen; Pasutto, Francesca; Sasaki, Takako; Kruse, Friedrich E; Zenkel, Matthias

2012-09-01

To test the hypothesis that a primary disturbance in lysyl oxidase-like 1 (LOXL1) and elastin metabolism in the lamina cribrosa of eyes with pseudoexfoliation syndrome constitutes an independent risk factor for glaucoma development and progression. Observational, consecutive case series. Posterior segment tissues obtained from 37 donors with early and late stages of pseudoexfoliation syndrome without glaucoma, 37 normal age-matched control subjects, 5 eyes with pseudoexfoliation-associated open-angle glaucoma, and 5 eyes with primary open-angle glaucoma (POAG). Protein and mRNA expression of major elastic fiber components (elastin, fibrillin-1, fibulin-4), collagens (types I, III, and IV), and lysyl oxidase crosslinking enzymes (LOX, LOXL1, LOXL2) were assessed in situ by quantitative real-time polymerase chain reaction, (immuno)histochemistry, and light and electron microscopy. Lysyl oxidase-dependent elastin fiber assembly was assessed by primary optic nerve head astrocytes in vitro. Expression levels of elastic proteins, collagens, and lysyl oxidases in the lamina cribrosa. Lysyl oxidase-like 1 proved to be the major lysyl oxidase isoform in the normal lamina cribrosa in association with a complex elastic fiber network. Compared with normal and POAG specimens, lamina cribrosa tissues obtained from early and late stages of pseudoexfoliation syndrome without and with glaucoma consistently revealed a significant coordinated downregulation of LOXL1 and elastic fiber constituents on mRNA and protein level. In contrast, expression levels of collagens and other lysyl oxidase isoforms were not affected. Dysregulated expression of LOXL1 and elastic proteins was associated with pronounced (ultra)structural alterations of the elastic fiber network in the laminar beams of pseudoexfoliation syndrome eyes. Inhibition of LOXL1 interfered with elastic fiber assembly by optic nerve head astrocytes in vitro. The findings provide evidence for a pseudoexfoliation-specific elastinopathy of the lamina cribrosa resulting from a primary disturbance in LOXL1 regulation and elastic fiber homeostasis, possibly rendering pseudoexfoliation syndrome eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development and progression. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Combining 2D angiogenesis and 3D osteosarcoma microtissues to improve vascularization.

PubMed

Chaddad, Hassan; Kuchler-Bopp, Sabine; Fuhrmann, Guy; Gegout, Hervé; Ubeaud-Sequier, Geneviève; Schwinté, Pascale; Bornert, Fabien; Benkirane-Jessel, Nadia; Idoux-Gillet, Ysia

2017-11-15

Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG-63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well-organized network, and that tubule-like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Liver fibrosis score predicts mortality in heart failure patients with preserved ejection fraction.

PubMed

Yoshihisa, Akiomi; Sato, Yu; Yokokawa, Tetsuro; Sato, Takamasa; Suzuki, Satoshi; Oikawa, Masayoshi; Kobayashi, Atsushi; Yamaki, Takayoshi; Kunii, Hiroyuki; Nakazato, Kazuhiko; Saitoh, Shu-Ichi; Takeishi, Yasuchika

2018-04-01

Heart failure with preserved ejection fraction (HFpEF) has several pathophysiological aspects, including stiffness and/or congestion of multiple organs. Poor prognosis is expected in heart failure patients with liver stiffness, which has recently been assessed by non-alcoholic fatty liver disease fibrosis score (NFS; based on aspartate aminotransferase to alanine aminotransferase ratio, platelet counts, and albumin). We aimed to investigate the impact of NFS on prognosis of HFpEF patients, with consideration for the peripheral collagen markers such as procollagen type III peptide (PIIIP), type IV collagen 7S, and hyaluronic acid. We performed a prospective observational study. Consecutive 492 hospitalized HFpEF patients were divided into four groups based on their NFS: first-fourth quartiles (n = 123). The fourth quartile group had the highest levels of PIIIP, type IV collagen 7S, hyaluronic acid, and B-type natriuretic peptide (P<0.001 each). In addition, there were significant positive correlations between PIIIP, type IV collagen 7S, hyaluronic acid, B-type natriuretic peptide, and NFS (P < 0.001 each). In the follow-up period (mean 1107 days), 93 deaths occurred. All-cause mortality increased in all four quartiles (8.1%, 12.2%, 23.6%, and 31.7%, P < 0.001). In the multivariable Cox proportional hazard analysis, NFS was an independent predictor of all-cause mortality in the HFpEF patients. NFS, a novel indicator of liver fibrosis, correlates with circulating systemic markers of fibrosis and congestion and is associated with higher all-cause mortality in HFpEF patients. NFS can be calculated simply and may be a useful tool to assess liver stiffness and prognosis in HFpEF patients. © 2017 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.

Tissue-engineered cartilaginous constructs for the treatment of caprine cartilage defects, including distribution of laminin and type IV collagen.

PubMed

Jeng, Lily; Hsu, Hu-Ping; Spector, Myron

2013-10-01

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration.

Tissue-Engineered Cartilaginous Constructs for the Treatment of Caprine Cartilage Defects, Including Distribution of Laminin and Type IV Collagen

PubMed Central

Jeng, Lily; Hsu, Hu-Ping

2013-01-01

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration. PMID:23672504

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

PubMed

Dawson, R A; Goberdhan, N J; Freedlander, E; MacNeil, S

1996-03-01

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

Quinapril decreases myocardial accumulation of extracellular matrix components in spontaneously hypertensive rats.

PubMed

Panizo, A; Pardo, J; Hernández, M; Galindo, M F; Cenarruzabeitia, E; Díez, J

1995-08-01

In genetic and acquired hypertension, a structural remodeling of the nonmyocyte compartment of myocardium, including the accumulation of fibrillar collagen and other components of the extracellular matrix (ECM) within the interstitium, represents a determinant of pathologic hypertrophy that leads to ventricular dysfunction. Therefore, to evaluate the potential benefit of the angiotensin converting enzyme (ACE) inhibitor quinapril in reversing the interstitial remodeling in spontaneously hypertensive rats (SHR) with established left ventricular hypertrophy (LVH), we treated 16-week-old male SHR with oral quinapril (average dose, 10 mg/kg body weight/day) for 20 weeks. Interstitial fibrosis was determined morphometrically using an automatic image analyzer. The amount of collagen was evaluated by measuring myocardial hydroxyproline concentration. Myocardial deposition of collagen molecules (types I, III, and IV) and other ECM components (fibronectin, laminin) was analyzed by immunohistochemical techniques using specific monoclonal antibodies. The activity of ACE was measured in left ventricular tissue by a fluorometric assay. In quinapril-treated SHR compared with 36-week-old untreated SHR and age- and sex-matched Wistar-Kyoto (WKY) controls, we found 1) a lesser degree of LVH and a lesser level of blood pressure, 2) a lesser degree of interstitial fibrosis, represented by less interstitial collagen volume fraction (5.73 +/- 0.45% v 3.42 +/- 0.28%, P < .05; WKY, 3.44 +/- 0.66%), 3) a lower hydroxyproline concentration (1.09 +/- 0.05 mumol/L/g dry weight/100 g body weight to 0.81 +/- 0.05 mumol/L/g dry weight/100 g body weight, P < .05; WKY, 0.96 +/- 0.06 mumol/L/g dry weight/100 g body weight), 4) a lesser presence of collagen fibers, and 5) a lesser presence of collagen IV, fibronectin, and laminin.(ABSTRACT TRUNCATED AT 250 WORDS)

Early Diagnosis and Intervention Strategies for Post-Traumatic Heterotopic Ossification in Severely Injured Extremities

DTIC Science & Technology

2015-10-01

collagen, type X, alpha 1 COL11A1 collagen, type XI, alpha 1 COL1A1 collagen, type I, alpha 1 COL2A1 collagen, type II, alpha 1 COL4A3 collagen, type IV...Symbol  Fold Regulation (8  days post injury)  Gene Symbol  Fold Regulation (15  days post injury)  Spp1  172.4535 Spp1  1105.6776 Col1a1   137.7958 Tac1...130.0625 ll1b  67.3332 Cxcl5  86.3414 ll10  49.9631 Col1a1   84.2834 Has1  45.5771 ll1b  74.488 Tac1  42.9908 Mmp9  69.8933 Cxcl1  25.259 ll10  40.5062

Comparison of histological structure and biocompatibility between human acellular dermal matrix (ADM) and porcine ADM.

PubMed

Ge, Liangpeng; Zheng, Shuquan; Wei, Hong

2009-02-01

The present study was to compare the difference of histological structure and biocompatibility between human ADM and porcine ADM. The scaffold structure, collagen arrangement and collagen structure of human ADM and those of porcine ADM were very similar except for a slight difference in their black and white bands assessed by both light microscopy and electron microscopy. The positive immunohistochemical staining results of porcine ADM using human antibodies of collagen I, collagen III, collagen IV, fibronectin, laminin and vimentin and the result of SDS-PAGE implied a strong homology between the main proteins of human ADM and porcine ADM. In addition, statistical analysis indicated that there was no significant difference (P<0.05) between the biocompatibility of the two ADMs. Based on these results, we conclude that porcine ADM bears a strong similarity to human ADM, and might be a substitute for human ADM in the future.

Slit2 promotes tumor growth and invasion in chemically induced skin carcinogenesis.

PubMed

Qi, Cuiling; Lan, Haimei; Ye, Jie; Li, Weidong; Wei, Ping; Yang, Yang; Guo, Simei; Lan, Tian; Li, Jiangchao; Zhang, Qianqian; He, Xiaodong; Wang, Lijing

2014-07-01

Slit, a neuronal guidance cue, binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit has been reported to have an important effect on tumor growth and metastasis. In the current study, we evaluated the role of Slit2 in skin tumor growth and invasion in mice using a two-step chemical carcinogenesis protocol. We found that Slit2 expression correlated with the loss of basement membrane in the samples of human skin squamous cell carcinoma at different stages of disease progression. Slit2-Tg mice developed significantly more skin tumors than wild-type mice. Furthermore, the skin tumors that occurred in Slit2-Tg mice were significantly larger than those in the wild-type mice 10 weeks after 7,12-dimethylbenz[a]anthracene initiation until the end of the experiment. We also found that pathological development of the wild-type mice was delayed compared with that of Slit2-Tg mice. To further investigate the mechanism of increasing tumors in Slit2-Tg mice, we analyzed the expression of 5-bromo-2'-deoxyuridine (BrdU) in mouse skin lesions and found that the number of BrdU-positive cells and microvessel density in skin lesions were significantly higher in Slit2-Tg mice than in wild-type mice. Histological staining of PAS and type IV collagen and the colocalization of Slit2 and type IV collagen demonstrated varying degrees of loss of the basement membrane in the skin lesions from Slit2-Tg mice that were at the stage of carcinoma in situ. However, the basement membrane was well defined in the wild-type mice. In addition, MMP2, but not MMP9, was upregulated in the skin tissue of Slit2-Tg mice. Interruption of Slit2-Robo1 signaling by the antibody R5 significantly repressed the invasive capability of the squamous cell carcinoma cell line A431. Taken together, our findings reveal that Slit2 promotes DMBA/TPA-induced skin tumorigenesis by increasing cell proliferation, microvessel density, and invasive behavior of cutaneous squamous cell carcinoma, along with loss of basement membrane, by upregulation of MMP2 expression.

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model.

PubMed

Koskimaki, Jacob E; Karagiannis, Emmanouil D; Tang, Benjamin C; Hammers, Hans; Watkins, D Neil; Pili, Roberto; Popel, Aleksander S

2010-02-01

Angiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays. One family of peptides with high activity is derived from the alpha-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the alpha5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line. Pentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model. The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.

The 2014International Workshop on Alport Syndrome.

PubMed

Miner, Jeffrey H; Baigent, Colin; Flinter, Frances; Gross, Oliver; Judge, Parminder; Kashtan, Clifford E; Lagas, Sharon; Savige, Judith; Blatt, Dave; Ding, Jie; Gale, Daniel P; Midgley, Julian P; Povey, Sue; Prunotto, Marco; Renault, Daniel; Skelding, Jules; Turner, A Neil; Gear, Susie

2014-10-01

Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5000-10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin-converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK, from January 3-5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities-patient families, physicians, geneticists, researchers, Pharma, and funding organizations-were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function.

Stress failure of pulmonary capillaries: role in lung and heart disease

NASA Technical Reports Server (NTRS)

West, J. B.; Mathieu-Costello, O.

1992-01-01

Pulmonary capillaries have extremely thin walls to allow rapid exchange of respiratory gases across them. Recently it has been shown that the wall stresses become very large when the capillary pressure is raised, and in anaesthetised rabbits, ultrastructural damage to the walls is seen at pressures of 40 mm Hg and above. The changes include breaks in the capillary endothelial layer, alveolar epithelial layer, and sometimes all layers of the wall. The strength of the thin part of the capillary wall can be attributed to the type IV collagen in the extracellular matrix. Stress failure of pulmonary capillaries results in a high-permeability form of oedema, or even frank haemorrhage, and is apparently the mechanism of neurogenic pulmonary oedema and high-altitude pulmonary oedema. It also explains the exercise-induced pulmonary haemorrhage that occurs in all racehorses. Several features of mitral stenosis are consistent with stress failure. Overinflation of the lung also leads to stress failure, a common cause of increased capillary permeability in the intensive care environment. Stress failure also occurs if the type IV collagen of the capillary wall is weakened by autoantibodies as in Goodpasture's syndrome. Neutrophil elastase degrades type IV collagen and this may be the starting point of the breakdown of alveolar walls that is characteristic of emphysema. Stress failure of pulmonary capillaries is a hitherto overlooked and potentially important factor in lung and heart disease.

The effect of amphiphilic siloxane oligomers on fibroblast and keratinocyte proliferation and apoptosis.

PubMed

Lynam, Emily C; Xie, Yan; Loli, Bree; Dargaville, Tim R; Leavesley, David I; George, Graeme A; Upton, Zee

2010-11-01

The formation of hypertrophic scars (HSF) is a frequent medical outcome of wound repair and often requires further therapy with treatments such as silicone gel sheets (SGS) or apoptosis-inducing agents, including bleomycin. Although widely used, knowledge regarding SGS and their mode of action is limited. Preliminary research has shown that small amounts of amphiphilic silicone present in SGS have the ability to move into skin during treatment. We demonstrate herein that a commercially available analogue of these amphiphilic siloxane species, the rake copolymer GP226, decreases collagen synthesis on exposure to cultures of fibroblasts derived from HSF. By size exclusion chromatography, GP226 was found to be a mixture of siloxane species, containing five fractions of different molecular weight. By studies of collagen production, cell viability and proliferation, it was revealed that a low molecular weight fraction (fraction IV) was the most active, reducing the number of viable cells present after treatment and thereby reducing collagen production as a result. On exposure of fraction IV to human keratinocytes, viability and proliferation were also significantly affected. HSF undergoing apoptosis after application of fraction IV were also detected via real-time microscopy and by using the TUNEL assay. Taken together, these data suggests that these amphiphilic siloxanes could be potential non-invasive substitutes to apoptotic-inducing chemical agents that are currently used as scar treatments.

The 2014 International Workshop on Alport Syndrome

PubMed Central

Miner, Jeffrey H; Baigent, Colin; Flinter, Frances; Gross, Oliver; Judge, Parminder; Kashtan, Clifford E; Lagas, Sharon; Savige, Judith; Blatt, Dave; Ding, Jie; Gale, Daniel P; Midgley, Julian P; Povey, Sue; Prunotto, Marco; Renault, Daniel; Skelding, Jules; Turner, A Neil; Gear, Susie

2014-01-01

Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5000–10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin-converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK, from January 3–5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities—patient families, physicians, geneticists, researchers, Pharma, and funding organizations—were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function. PMID:24988067

Regulation of extracellular matrix elements and sarcomerogenesis in response to different periods of passive stretching in the soleus muscle of rats.

PubMed

Peviani, Sabrina M; Guzzoni, Vinicius; Pinheiro-Dardis, Clara M; Silva, Yara P da; Fioravante, Alisson C R; Sagawa, Adriana H; Delfino, Gabriel B; Durigan, João L Q; Salvini, Tania F

2018-06-13

Stretching is a common method used to prevent muscle shortening and improve limited mobility. However, the effect of different time periods on stretching-induced adaptation of the extracellular matrix and its regulatory elements have yet to be investigated. We aimed to evaluate the expression of fibrillar collagens, sarcomerogenesis, metalloproteinase (MMP) activity and gene expression of the extracellular matrix (ECM) regulators in the soleus (SOL) muscle of rats submitted to different stretching periods. The soleus muscles were submitted to 10 sets of passive stretching over 10 (St 10d) or 15 days (St 15d) (1 min per set, with 30 seconds' rest between sets). Sarcomerogenesis, muscle cross-sectional area (CSA), and MMP activity and mRNA levels in collagen (type I, III and IV), connective tissue growth factor (CTGF), growth factor-beta (TGF-β), and lysyl oxidase (LOX) were analyzed. Passive stretching over both time periods mitigated COL-I deposition in the SOL muscle of rats. Paradoxically, 10 days of passive stretching induced COL-I and COL-III synthesis, with concomitant upregulation of TGF-β1 and CTGF at a transcriptional level. These responses may be associated with lower LOX mRNA levels in SOL muscles submitted to 10 passive stretching sessions. Moreover, sarcomerogenesis was observed after 15 days of stretching, suggesting that stretching-induced muscle adaptations are time-dependent responses.

Low molecular weight proteins in urines from healthy subjects as well as diabetic, nephropathic and diabetic-nephropathic patients: a MALDI study.

PubMed

Lapolla, Annunziata; Seraglia, Roberta; Molin, Laura; Williams, Katherine; Cosma, Chiara; Reitano, Rachele; Sechi, Annalisa; Ragazzi, Eugenio; Traldi, Pietro

2009-03-01

Urine samples from healthy subjects as well as diabetic, nephropathic and diabetic-nephropathic patients were analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry in order to establish evidence of some possible differences in the peptide profile related to the pathological states. Multivariate analysis suggested the possibility of a distinction among the considered groups of patients. Some differences have been found, in particular, in the relative abundances of three ions at m/z 1912, 1219 and 2049. For these reasons, further investigation was carried out by MALDI/TOF/TOF to determine the sequence of these peptides and, consequently, to individuate their possible origin. By this approach, the peptide at m/z 1912 was found to originate from uromodulin, and its lower expression in the case of nephropathy can be well related to the pathological condition. Ions at m/z 2049 and 1219 originate from the collagen alpha-1(I) chain precursor and from the collagen alpha-5 (IV) chain precursor, respectively, and, also in this case, their different expressions can be related to the pathologies under investigation. The obtained data seem to indicate that urine is an interesting biological fluid to investigate on the peptide profile and to obtain, consequently, information on the dismetabolism activated by specific pathologies. Copyright (c) 2009 John Wiley & Sons, Ltd.

Strain-Specific Induction of Endometrial Periglandular Fibrosis in Mice Exposed During Adulthood to the Endocrine Disrupting Chemical Bisphenol A

PubMed Central

Kendziorski, Jessica A.; Belcher, Scott M.

2015-01-01

The aim of this study was to compare effects of bisphenol A (BPA) on collagen accumulation in uteri of two mouse strains. Adult C57Bl/6N and CD-1 mice were exposed to dietary BPA (0.004–40 mg/kg/day) or 17α-ethinyl estradiol (0.00002–0.001 mg/kg/day) as effect control. An equine endometrosis-like phenotype with increased gland nesting and periglandular collagen accumulation was characteristic of unexposed C57Bl/6N, but not CD-1, endometrium. BPA non-monotonically increased gland nest density and periglandular collagen accumulation in both strains. Increased collagen I and III expression, decreased matrix metalloproteinase 2 (MMP2) and MMP14 expression, and increased immune response were associated with the endometrosis phenotype in the C57Bl/6N strain and the 30 ppm BPA CD-1 group. The association between the pro-collagen shift in increased collagen expression and decreased MMP2 expression and activity implies that strain differences and BPA exposure salter regulation of endometrial remodeling and contributes to increased fibrosis, a component of several human uterine diseases. PMID:26307436

Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease.

PubMed

Mak, Ki M; Mei, Rena

2017-08-01

Basement membranes provide structural support to epithelium, endothelium, muscles, fat cells, Schwann cells, and axons. Basement membranes are multifunctional: they modulate cellular behavior, regulate organogenesis, promote tissue repair, form a barrier to filtration and tumor metastasis, bind growth factors, and mediate angiogenesis. All basement membranes contain type IV collagen (Col IV), laminin, nidogen, and perlecan. Col IV and laminin self-assemble into two independent supramolecular networks that are linked to nidogen and perlecan to form a morphological discernable basement membrane/basal lamina. The triple helical region, 7S domain and NCI domain of Col IV, laminin and laminin fragment P1 have been evaluated as noninvasive fibrosis biomarkers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. Elevated serum Col IV and laminin are related to degrees of fibrosis and severity of hepatitis, and may reflect hepatic basement membrane metabolism. But the serum assays have not been linked to disclosing the anatomical sites and lobular distribution of perisinusoidal basement membrane formation in the liver. Hepatic sinusoids normally lack a basement membrane, although Col IV is a normal matrix component of the space of Disse. In liver disease, laminin deposits in the space of Disse and codistributes with Col IV, forming a perisinusoidal basement membrane. Concomitantly, the sinusoidal endothelium loses its fenestrae and is transformed into vascular type endothelium. These changes lead to capillarization of hepatic sinusoids, a significant pathology that impairs hepatic function. Accordingly, codistribution of Col IV and laminin serves as histochemical marker of perisinusoidal basement membrane formation in liver disease. Anat Rec, 300:1371-1390, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

PubMed

Håkansson, Joakim; Xian, Xiaojie; He, Liqun; Ståhlberg, Anders; Nelander, Sven; Samuelsson, Tore; Kubista, Mikael; Semb, Henrik

2005-01-01

To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.

Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model.

PubMed

Rubio-Araiz, Ana; Porcu, Francesca; Pérez-Hernández, Mercedes; García-Gutiérrez, Mª Salud; Aracil-Fernández, María Auxiliadora; Gutierrez-López, María Dolores; Guerri, Consuelo; Manzanares, Jorge; O'Shea, Esther; Colado, María Isabel

2017-07-01

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice. © 2016 Society for the Study of Addiction.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

PubMed

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-11-19

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

PubMed Central

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-01-01

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides. PMID:28952593

Losartan inhibits collagen I synthesis and improves the distribution and efficacy of nanotherapeutics in tumors

PubMed Central

Diop-Frimpong, Benjamin; Chauhan, Vikash P.; Krane, Stephen; Boucher, Yves; Jain, Rakesh K.

2011-01-01

The dense collagen network in tumors significantly reduces the penetration and efficacy of nanotherapeutics. We tested whether losartan—a clinically approved angiotensin II receptor antagonist with noted antifibrotic activity—can enhance the penetration and efficacy of nanomedicine. We found that losartan inhibited collagen I production by carcinoma-associated fibroblasts isolated from breast cancer biopsies. Additionally, it led to a dose-dependent reduction in stromal collagen in desmoplastic models of human breast, pancreatic, and skin tumors in mice. Furthermore, losartan improved the distribution and therapeutic efficacy of intratumorally injected oncolytic herpes simplex viruses. Finally, it also enhanced the efficacy of i.v. injected pegylated liposomal doxorubicin (Doxil). Thus, losartan has the potential to enhance the efficacy of nanotherapeutics in patients with desmoplastic tumors. PMID:21282607

Catalytic site of human protein-glucosylgalactosylhydroxylysine glucosidase: Three crucial carboxyl residues were determined by cloning and site-directed mutagenesis.

PubMed

Hamazaki, Hideaki; Hamazaki, Michiko Horikawa

2016-01-15

Protein-glucosylgalactosylhydroxylysine glucosidase (PGGHG; EC3.2.1.107) cleaves glucose from disaccharide unit (Glc-α1,2-Gal) linked to hydroxylysine residues of collagen. In the present paper we first show that PGGHG is the product of ATHL1 gene as follows. (1) PGGHG was purified from chick embryos and digested with trypsin. LC-MS/MS analysis suggested the tryptic-peptides were from the ATHL1 gene product. (2) Chick embryo ATHL1 cDNA was cloned to a cloning and expression vector and two plasmid clones with different ATHL1 CDS insert were obtained. (3) Each plasmid DNA was transformed into Escherichia coli cells for expression and two isoforms of chicken PGGHG were obtained. (4) Both isoforms effectively released glucose from type IV collagen. Next, we searched for carboxyl residues crucial for catalytic activity as follows; human ATHL1 cDNA was cloned into a cloning and expression vector and 18 mutants were obtained by site-directed mutagenesis for 15 carboxyl residues conserved in ATHL1 of jawed vertebrates. The expression analysis indicated that substitutions of Asp301, Glu430 and Glu574 with sterically conservative (D301N, E430Q, E574Q) or functionally conservative (D301E, E430D, E574D) residues led to the complete elimination of enzyme activity. These findings lead us to the conclusion that PGGHG is encoded by ATHL1 and three carboxyl residues (corresponding to Asp301, Glu430 and Glu574 of human PGGHG) might be involved in the catalytic site of PGGHG. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of losartan on hepatic expression of nonphagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C.

PubMed

Colmenero, Jordi; Bataller, Ramón; Sancho-Bru, Pau; Domínguez, Marlene; Moreno, Montserrat; Forns, Xavier; Bruguera, Miquel; Arroyo, Vicente; Brenner, David A; Ginès, Pere

2009-10-01

Angiotensin II promotes liver fibrogenesis by stimulating nonphagocytic NADPH oxidase (NOX)-induced oxidative stress. Angiotensin II type 1 (AT1) receptor blockers attenuate experimental liver fibrosis, yet their effects in human liver fibrosis are unknown. We investigated the effects of losartan on hepatic expression of fibrogenic, inflammatory, and NOX genes in patients with chronic hepatitis C (CHC). Fourteen patients with CHC and liver fibrosis received oral losartan (50 mg/day) for 18 mo. Liver biopsies were performed at baseline and after treatment. The degree of inflammation and fibrosis was evaluated by histological analysis (METAVIR). Collagen content was measured by morphometric quantification of Sirius red staining. Overall collagen content and fibrosis stage remained stable in the whole series, yet the fibrosis stage decreased in seven patients. Inflammatory activity improved in seven patients. The effect of losartan on hepatic expression of 31 profibrogenic and inflammatory genes and components of the NOX complex was assessed by quantitative PCR. Losartan treatment was associated with a significant decrease in the expression of several profibrogenic and NOX genes including procollagen alpha1(I) and alpha1(IV), urokinase-type plasminogen activator, metalloproteinase type 2, NOX activator 1 (NOXA-1) and organizer 1 (NOXO-1), and Rac-1. Losartan was well tolerated in all patients and was effective in attenuating the activity of the systemic renin-angiotensin system. No effects on serum liver tests or viral load were observed. We conclude that prolonged administration of losartan, an oral AT1 receptor blocker, is associated with downregulation of NOX components and fibrogenic genes in patients with CHC. Controlled studies are warranted to assess the effect of AT1 receptor blockers in chronic liver injury.

Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

PubMed Central

Parra, E.R.; Pincelli, M.S.; Teodoro, W.R.; Velosa, A.P.P.; Martins, V.; Rangel, M.P.; Barbas-Filho, J.V.; Capelozzi, V.L.

2014-01-01

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis. PMID:24919172

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

PubMed Central

Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

2012-01-01

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

PubMed Central

Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

2016-01-01

Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes

PubMed Central

Dayal, Jasbani H. S.; Cole, Clare L.; Pourreyron, Celine; Watt, Stephen A.; Lim, Yok Zuan; Salas-Alanis, Julio C.; Murrell, Dedee F.; McGrath, John A.; Stieger, Bruno; Jahoda, Colin; Leigh, Irene M.; South, Andrew P.

2014-01-01

ABSTRACT Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332. PMID:24357722

Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Hua-Yu; Li, Chao; Zheng, Zhao

The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at themore » gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.« less

Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

PubMed

Sugioka, Koji; Kodama-Takahashi, Aya; Yoshida, Koji; Aomatsu, Keiichi; Okada, Kiyotaka; Nishida, Teruo; Shimomura, Yoshikazu

2017-03-01

Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

Immunolocalization of Collagens (I and III) and Cartilage Oligomeric Matrix Protein in the Normal and Injured Equine Superficial Digital Flexor Tendon

PubMed Central

2013-01-01

This is a descriptive study of tendon pathology with different structural appearances of repair tissue correlated to immunolocalization of cartilage oligomeric matrix protein (COMP) and type I and III collagens and expression of COMP mRNA. The material consists of nine tendons from seven horses (5–25 years old; mean age of 10 years) with clinical tendinopathy and three normal tendons from horses (3, 3, and 13 years old) euthanized for non-orthopedic reasons. The injured tendons displayed different repair-tissue appearances with organized and disorganized fibroblastic regions as well as areas of necrosis. The normal tendons presented distinct immunoreactivity for COMP and expression of COMP mRNA and type I collagen in the normal aligned fiber structures, but no immunolabeling of type III collagen. However, immunoreactivity for type III collagen was present in the endotenon surrounding the fiber bundles, where no expression of COMP could be seen. Immunostaining for type I and III collagens was present in all of the pathologic regions indicating repair tissue. Interestingly, the granulation tissues showed immunostaining for COMP and expression of COMP mRNA, indicating a role for COMP in repair and remodeling of the tendon after fiber degeneration and rupture. The present results suggest that not only type III collagen but also COMP is involved in the repair and remodeling processes of the tendon. PMID:23020676

Substance P up-regulates matrix metalloproteinase-1 and down-regulates collagen in human lung fibroblast.

PubMed

Ramos, Carlos; Montaño, Martha; Cisneros, Jose; Sommer, Bettina; Delgado, Javier; Gonzalez-Avila, Georgina

2007-01-01

Substance P is involved in inflammatory processes, but its effect on extracellular matrix metabolism has not been studied; therefore, the authors evaluated its effect on collagen synthesis and degradation, expression of pro-alpha1(I) collagen, matrix metalloproteinase-1 and -2, and tissue inhibitor of metalloproteinase-1 and -2 in normal human lung fibroblast strains. Substance P induced a decrease in collagen biosynthesis, concomitant to a down-regulation of pro-alpha1(I) collagen mRNA. In contrast, an increase in collagen degradation was observed, accompanied with an up-regulation of matrix metalloproteinase-1. Substance P did not influence tissue inhibitor of metalloproteinase-1 and -2 or matrix metalloproteinase-2 expression. The results suggest that substance P participates in extracellular matrix metabolism.

MMP-14 is necessary but not sufficient for invasion of three-dimensional collagen by human muscle satellite cells

PubMed Central

Lund, Dane K.; Mouly, Vincent

2014-01-01

The twenty-five known matrix metalloproteases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteases (TIMPs), mediate cell invasion through the extracellular matrix (ECM). In a comparative three-dimensional assay, we analyzed human and mouse satellite cells' competence to invade an artificial ECM (collagen I). We identified a single MMP that 1) is expressed by human muscle satellite cells; 2) is induced at the mRNA/protein level by adhesion to collagen I; and 3) is necessary for invasion into a collagen I matrix. Interestingly, murine satellite cells neither express this MMP, nor invade the collagen matrix. However, exogenous human MMP-14 is not sufficient to induce invasion of a collagen matrix by murine cells, emphasizing species differences. PMID:24898588

Targeting Discoidin Domain Receptors in Prostate Cancer

DTIC Science & Technology

2016-08-01

DDRs), a set of kinase receptors that signal in response to collagen . The project’s goal is to define the expression and therapeutic potential of...antibody, which blocks receptor activation by collagen I. Mice were inoculated with PC3 cells and anti-DDR1 or control antibody treatment. The study... collagen , the major organic component of the bone extracellular matrix. Purpose: To investigate the expression, therapeutic potential, and

The mesangial matrix in the normal and sclerotic glomerulus.

PubMed

Rosenblum, N D

1994-02-01

Mesangial sclerosis is a final common pathway to glomerular destruction in a variety of glomerular diseases. The expression of several classes of extracellular matrix (ECM) molecules has been defined in the normal and diseased mesangial matrix (MM). However, the manner in which these ECM components determine the three dimensional structure and function of the MM remains to be defined. Structural studies of the MM suggest that its constituent molecules are regionally organized into subcompartments with different three dimensional structures. The diversity of matrix molecules expressed within the MM as well as the organization of these components in nonrenal ECM's, such as the cornea, provides further support for this organizational model. The study of the cornea has also revealed that novel short chain collagenous proteins partially determine the three dimensional structure of the matrix. Recently, a novel collagen, type VIII collagen, has been described in mesangial cells and in the intact glomerulus. It is hypothesized that type VIII collagen is expressed both as a polymer and as a monomer within the glomerulus, and depending on its conformation, may serve unique functions. In the chronically diseased MM, normal MM components are overexpressed and fibrillar collagens are expressed de novo in a delayed fashion. Enhanced proteoglycan expression, observed early in disease, may determine increased volume of the mesangium. This, in turn, may stimulate the production of fibrillar collagens by mesangial cells resulting in a fibrillar noncompliant mesangial matrix.

Influence of cyclic hydrostatic pressure on fibrocartilaginous metaplasia of achilles tendon fibroblasts.

PubMed

Shim, J W; Elder, S H

2006-11-01

The goal of this study was to demonstrate whether cyclically imposed hydrostatic pressure, compressive in nature, could induce fibrocartilaginous metaplasia in a purely tendinous cell source in vitro. The effect of short-duration cyclic hydrostatic pressure on tendon fibroblasts (tenocytes) expanded from rat Achilles tendon was studied. Total RNA was isolated either immediately after loading or 24 h later. The mRNA expression of tendon and cartilage specific markers - Collagen types I and II, Sox9, and Aggrecan was quantified by real-time reverse transcription polymerase chain reaction over multiple biological samples (n=6). For immediately isolated RNA samples, there were statistically significant increases in mRNA expression of Aggrecan and Collagen type II, while Collagen type I significantly decreased. Noticeably, for RNA samples isolated 24 h later, there were further increases in mRNA expression of Aggrecan and Collagen type II, whereas Collagen type I increased roughly three-fold relative to the non-loaded control. These findings support the hypothesis that cyclic hydrostatic pressurization can induce fibrocartilaginous metaplasia in tenocytes by upregulation of cartilaginous gene expression. Also, it was demonstrated that changes in mRNA expression as a result of single 2 h pressurization persist even up to 24 h.

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

PubMed Central

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

Targeting the LRP5 pathway improves bone properties in a mouse model of Osteogenesis Imperfecta

PubMed Central

Jacobsen, Christina M.; Barber, Lauren A.; Ayturk, Ugur M.; Roberts, Heather J.; Deal, Lauren E.; Schwartz, Marissa A.; Weis, MaryAnn; Eyre, David; Zurakowski, David; Robling, Alexander G.; Warman, Matthew L.

2014-01-01

The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5p.A214V) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis Imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5+/p.A214V mice to Col1a2+/p.G610C mice, which model human type IV OI. We found that Col1a2+/p.G610C;Lrp5+/p.A214V offspring had significantly increased bone mass and strength compared to Col1a2+/p.G610C;Lrp5+/+ littermates. The improved bone properties were not due to altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2+/p.G610C mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody treated mice had significantly increased bone mass and strength compared to vehicle treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. PMID:24677211

Targeting the LRP5 pathway improves bone properties in a mouse model of osteogenesis imperfecta.

PubMed

Jacobsen, Christina M; Barber, Lauren A; Ayturk, Ugur M; Roberts, Heather J; Deal, Lauren E; Schwartz, Marissa A; Weis, MaryAnn; Eyre, David; Zurakowski, David; Robling, Alexander G; Warman, Matthew L

2014-10-01

The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5(p.A214V) ) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5(+/p.A214V) mice to Col1a2(+/p.G610C) mice, which model human type IV OI. We found that Col1a2(+/p.G610C) ;Lrp5(+/p.A214V) offspring had significantly increased bone mass and strength compared to Col1a2(+/p.G610C) ;Lrp5(+/+) littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2(+/p.G610C) mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody-treated mice had significantly increased bone mass and strength compared to vehicle-treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

PubMed Central

Yang, Xu; Vezeridis, Peter S; Nicholas, Brian; Crisco, Joseph J; Moore, Douglas C; Chen, Qian

2006-01-01

Objective Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. Results Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression. PMID:17150098

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

PubMed Central

Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

2016-01-01

Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

PubMed

Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

2016-03-21

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification

PubMed Central

2014-01-01

Background Extracellular matrix (ECM) disarray is found in calcific aortic valvular disease (CAVD), yet much remains to be learned about the role of individual ECM components in valvular interstitial cell (VIC) function and dysfunction. Previous clinical analyses have shown that calcification is associated with decreased collagen content, while previous in vitro work has suggested that the presence of collagen attenuates the responsiveness of VICs to pro-calcific stimuli. The current study uses whole leaflet cultures to examine the contributions of endogenous collagen in regulating the phenotype and calcification of VICs. Methods A “top-down” approach was used to characterize changes in VIC phenotype in response to collagen alterations in the native 3D environment. Collagen-deficient leaflets were created via enzymatic treatment and cultured statically for six days in vitro. After culture, leaflets were harvested for analysis of DNA, proliferation, apoptosis, ECM composition, calcification, and gene/protein expression. Results In general, disruption of collagen was associated with increased expression of disease markers by VICs in whole organ leaflet culture. Compared to intact control leaflets, collagen-deficient leaflets demonstrated increased VIC proliferation and apoptosis, increased expression of disease-related markers such as alpha-smooth muscle actin, alkaline phosphatase, and osteocalcin, and an increase in calcification as evidenced by positive von Kossa staining. Conclusions These results indicate that disruption of the endogenous collagen structure in aortic valves is sufficient to stimulate pathological consequences in valve leaflet cultures, thereby highlighting the importance of collagen and the valve extracellular matrix in general in maintaining homeostasis of the valve phenotype. PMID:24581344

Differential proteomics reveals S100-A11 as a key factor in aldosterone-induced collagen expression in human cardiac fibroblasts.

PubMed

Martínez-Martínez, Ernesto; Ibarrola, Jaime; Lachén-Montes, Mercedes; Fernández-Celis, Amaya; Jaisser, Frederic; Santamaría, Enrique; Fernández-Irigoyen, Joaquín; López-Andrés, Natalia

2017-08-23

Aldosterone (Aldo) could induce cardiac fibrosis, a hallmark of heart disease. Aldo direct effects on collagen production in cardiac fibroblasts remain controversial. Our aim is to characterize changes in the proteome of adult human cardiac fibroblasts treated with Aldo to identify new proteins altered that might be new therapeutic targets in cardiovascular diseases. Aldo increased collagens expressions in human cardiac fibroblasts. Complementary, using a quantitative proteomic approach, 30 proteins were found differentially expressed between control and Aldo-treated cardiac fibroblasts. Among these proteins, 7 were up-regulated and 23 were down-regulated by Aldo. From the up-regulated proteins, collagen type I, collagen type III, collagen type VI and S100-A11 were verified by Western blot. Moreover, protein interaction networks revealed a functional link between a third of Aldo-modulated proteome and specific survival routes. S100-A11 was identified as a possible link between Aldo and collagen. Interestingly, CRISPR/Cas9-mediated knock-down of S100-A11 blocked Aldo-induced collagen production in human cardiac fibroblasts. In adult human cardiac fibroblasts treated with Aldo, proteomic analyses revealed an increase in collagen production. S100-A11 was identified as a new regulator of Aldo-induced collagen production in human cardiac fibroblasts. These data could identify new candidate proteins for the treatment of cardiac fibrosis in cardiovascular diseases. S100-A11 is identified by a proteomic approach as a novel regulator of Aldosterone-induced collagen production in human cardiac fibroblasts. Our data could identify new candidate proteins of interest for the treatment of cardiac fibrosis in cardiovascular diseases. Copyright © 2017. Published by Elsevier B.V.

Collagen VI Ablation Retards Brain Tumor Progression Due to Deficits in Assembly of the Vascular Basal Lamina

PubMed Central

You, Weon-Kyoo; Bonaldo, Paolo; Stallcup, William B.

2012-01-01

To investigate the importance of the vascular basal lamina in tumor blood vessel morphogenesis and function, we compared vessel development, vessel function, and progression of B16F10 melanoma tumors in the brains of wild-type and collagen VI-null mice. In 7-day tumors in the absence of collagen VI, the width of the vascular basal lamina was reduced twofold. Although the ablation of collagen VI did not alter the abundance of blood vessels, a detailed analysis of the number of either pericytes or endothelial cells (or pericyte coverage of endothelial cells) showed that collagen VI-dependent defects during the assembly of the basal lamina have negative effects on both pericyte maturation and the sprouting and survival of endothelial cells. As a result of these deficits, vessel patency was reduced by 25%, and vessel leakiness was increased threefold, resulting in a 10-fold increase in tumor hypoxia along with a fourfold increase in hypoxia-inducible factor-1α expression. In 12-day collagen VI-null tumors, vascular endothelial growth factor expression was increased throughout the tumor stroma, in contrast to the predominantly vascular pattern of vascular endothelial growth factor expression in wild-type tumors. Vessel size was correspondingly reduced in 12-day collagen VI-null tumors. Overall, these vascular deficits produced a twofold decrease in tumor volume in collagen VI-null mice, confirming that collagen VI-dependent basal lamina assembly is a critical aspect of vessel development. PMID:22200614

Harnessing the Versatility of Bacterial Collagen to Improve the Chondrogenic Potential of Porous Collagen Scaffolds

PubMed Central

Parmar, Paresh A.; St-Pierre, Jean-Philippe; Chow, Lesley W.; Puetzer, Jennifer L.; Stoichevska, Violet; Peng, Yong Y.; Werkmeister, Jerome A.; Ramshaw, John A. M.; Stevens, Molly M.

2017-01-01

Collagen I foams are used in the clinic as scaffolds to promote articular cartilage repair as they provide a bioactive environment for cells with chondrogenic potential. However, collagen I as a base material does not allow for precise control over bioactivity. Alternatively, recombinant bacterial collagens can be used as “blank slate” collagen molecules to offer a versatile platform for incorporation of selected bioactive sequences and fabrication into 3D scaffolds. Here, we show the potential of Streptococcal collagen-like 2 (Scl2) protein foams modified with peptides designed to specifically and noncovalently bind hyaluronic acid and chondroitin sulfate to improve chondrogenesis of human mesenchymal stem cells (hMSCs) compared to collagen I foams. Specific compositions of functionalized Scl2 foams lead to improved chondrogenesis compared to both nonfunctionalized Scl2 and collagen I foams, as indicated by gene expression, extracellular matrix accumulation, and compression moduli. hMSCs cultured in functionalized Scl2 foams exhibit decreased collagens I and X gene and protein expression, suggesting an advantage over collagen I foams in promoting a chondrocytic phenotype. These highly modular foams can be further modified to improve specific aspects chondrogenesis. As such, these scaffolds also have the potential to be tailored for other regenerative medicine applications. PMID:27219220

The effect of collagen coating on titanium with nanotopography on in vitro osteogenesis.

PubMed

Costa, Daniel G; Ferraz, Emanuela P; Abuna, Rodrigo P F; de Oliveira, Paulo T; Morra, Marco; Beloti, Marcio M; Rosa, Adalberto L

2017-10-01

Several studies have shown the positive effects of Ti either with nanotopography or coated with collagen on osteoblast differentiation. Thus, we hypothesized that the association of nanotopography with collagen may increase the in vitro osteogenesis on Ti surface. Ti discs with nanotopography with or without collagen coating were characterized by scanning electron microscopy and atomic force microscopy. Rat calvaria-derived osteoblastic cells were cultured on both Ti surfaces for up to 14 days and the following parameters were evaluated: cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, protein expression of bone sialoprotein (BSP) and osteopontin (OPN), and gene expression of collagen type 1a (Coll1a), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OC), Ki67, Survivin, and Bcl2-associated X protein (BAX). Surface characterization evidenced that collagen coating did not alter the nanotopography. Collagen coating increased cell proliferation, ALP activity, extracellular matrix mineralization, and Coll1a, OSX, OC, and BAX gene expression. Also, OPN and BSP proteins were strongly detected in cultures grown on both Ti surfaces. In conclusion, our results showed that the combination of nanotopography with collagen coating stimulates the early, intermediate, and final events of the in vitro osteogenesis and may be considered a potential approach to promote osseointegration of Ti implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2783-2788, 2017. © 2017 Wiley Periodicals, Inc.

Platelets and Plasma Proteins Are Both Required to Stimulate Collagen Gene Expression by Anterior Cruciate Ligament Cells in Three-Dimensional Culture

PubMed Central

Cheng, Mingyu; Wang, Hao; Yoshida, Ryu

2010-01-01

Collagen–platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, little is known about the cellular mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two (PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elongation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP, did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis. PMID:19958169

Molecular, histologic, and trace mineral characterization of metacarpophalangeal and metatarsophalangeal joint hyperextension in juvenile llamas.

PubMed

Semevolos, Stacy A; Reed, Shannon K

2011-04-01

To evaluate molecular and histologic characteristics of the superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT), and suspensory ligament (SL) and assess trace-mineral concentrations in serum, liver, and hair of juvenile llamas with metacarpophalangeal and metatarsophalangeal joint hyperextension. 12 juvenile llamas (6 with bilateral hyperextension of metacarpophalangeal joints, metatarsophalangeal joints, or both and 6 clinically normal control llamas). Radiography and ultrasonography of metacarpophalangeal and metatarsophalangeal regions were performed. Llamas were euthanized, and SDFT, DDFT, and SL samples were collected for histologic evaluation of collagen and elastin content and orientation, proteoglycan content, and collagen type III immunohistochemistry. Total RNA was isolated from SL tissue, and gene expression of collagen types I and III, lysyl oxidase, and matrix metalloproteinase-13 was evaluated via real-time quantitative reverse transcriptase PCR assay. Liver, serum, and hair samples were evaluated for trace mineral content. Collagen type III gene expression and proteoglycan content were significantly increased in SL samples of affected juvenile llamas, compared with those of control llamas. No difference was detected in collagen and elastin content and orientation or in gene expression of collagen type I, lysyl oxidase, or matrix metalloproteinase-13 between groups. Affected llamas had significantly increased serum molybdenum and decreased liver cobalt concentrations, compared with values for control llamas. Increased collagen type III gene expression and proteoglycan content in SL samples of affected juvenile llamas provided evidence of ongoing SL matrix repair. Trace mineral differences may have been attributable to dietary imbalances in affected llamas.

Genetics Home Reference: Alport syndrome

MedlinePlus

... particularly the organ of Corti, that transform sound waves into nerve impulses for the brain. Alterations in type IV collagen often result in abnormal inner ear function, which can lead to hearing loss. In the ...

Mapping structural landmarks, ligand binding sites and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions and variation in phenotypes in inherited diseases affecting basement membranes

PubMed Central

Des Parkin, J.; San Antonio, James D.; Pedchenko, Vadim; Hudson, Billy; Jensen, Shane T.; Savige, Judy

2016-01-01

Collagen IV is the major protein found in basement membranes. It comprises 3 heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (‘interactomes’) that indicated major structural landmarks, known and predicted ligand-binding sites, and missense mutations, in order to identify functional and disease-associated domains, potential interactions between ligands, and genotype-phenotype relationships. The maps documented more than 30 known ligand-binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases and heparin. The maps indicated that mutations affecting major ligand-binding sites, for example for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms and muscle Cramps (HANAC) syndrome, and early onset Alport syndrome respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. PMID:21280145

[Expression of collagen and elastin fibers in the rectum of patients with obstructed defecation syndrome and its significance].

PubMed

Li, Juan; Lin, Hongcheng; Ren, Donglin

2015-12-01

To detect the expression of collagen and elastin fibers in the rectum of patients with obstructive defecation syndrome (ODS), and to explore the possible role of these fibers in the pathogenesis of ODS. The rectum specimens of 20 patients with ODS were collected. These patients had undergone stapled transanal rectal resection (STARR) surgery in our hospital since 2012. Full-thickness rectal specimens were stained with Masson and EVG staining to show collagen and elastin fibers. As the control, rectum specimens of 20 patients with severe prolapsed hemorrhoids also undergoing STARR surgery in our hospital during the same period were collected. Masson staining showed that the structure of collagen fibers in submucosa was slender, area decreased [(13.88±7.02)% vs. (30.98±3.46)%, P<0.01], and the expression level was significantly lower in ODS group compared with control group. EVG staining also showed that collagen fiber area of ODS patients was reduced compared with control group [(17.18±7.24)% vs. (27.04±9.65)%, P<0.01]. Meanwhile EVG staining revealed that elastic fibers were fragmented in ODS group and the expression level was lower compared with control group [(1.20±1.39)% vs. (1.47±1.06)%], whereas the difference was not statistically significant(P>0.05). The expression level of collagen fibers in patients with ODS is significantly reduced, which may play an important role in the pathogenesis of ODS.

Collagen and matrix metalloproteinase-2 and -9 in the ewe cervix during the estrous cycle.

PubMed

Rodríguez-Piñón, M; Tasende, C; Casuriaga, D; Bielli, A; Genovese, P; Garófalo, E G

2015-09-15

The cervical collagen remodeling during the estrous cycle of the ewe was examined. The collagen concentration determined by a hydroxyproline assay and the area occupied by collagen fibers (%C), determined by van Gieson staining, were assessed in the cranial and caudal cervix of Corriedale ewes on Days 1 (n = 6), 6 (n = 5), or 13 (n = 6) after estrous detection (defined as Day 0). In addition, the gelatinase activity by in situ and SDS-PAGE gelatin zymographies and matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9, respectively) expression by immunohistochemistry were determined. The collagen concentration and %C were lowest on Day 1 of the estrous cycle (P < 0.04), when MMP-2 activity was highest (P < 0.006) and the ratio of activated to latent MMP-2 trend to be highest (P = 0.0819). The MMP-2 activity was detected in 73% of the homogenized cervical samples, and its expression was mainly detected in active fibroblasts. By contrast, the MMP-9 activity was detected in 9% of the samples, and its scarce expression was associated with plasmocytes, macrophages, and lymphocytes. Matrix metalloproteinase-2 expression was maximal on Day 1 in the cranial cervix and on Day 13 in the caudal cervix and was lower in the cranial than in the caudal cervix (P < 0.0001). This time-dependent increase in MMP-2 expression that differed between the cranial and caudal cervix may reflect their different physiological roles. The decrease in the collagen content and increase in fibroblast MMP-2 activity in sheep cervix on Day 1 of the estrous cycle suggests that cervical dilation at estrus is due to the occurrence of collagen fiber degradation modulated by changes in periovulatory hormone levels. Copyright © 2015 Elsevier Inc. All rights reserved.

[Effects of Fluoxetine on Nogo Expression and Collagen Production with Decrease of Pulmonary Artery Pressure in Rats with Right Ventricular Failure.

PubMed

Ran, Xun; Zhao, Jian-Xun; Nie, Hu; Chen, Yu-Cheng

2016-11-01

To investigate the effect of fluoxetine on neurite growth inhibitor (Nogo) expession and collagen production of cardiac tissue in rats with right heart failure and pulmonary hypertension. Thirty one male SD rats were randomly divided into the treatment group,right heart failure group and normal control group.The rats in the treatment group and right heart failure group received intrapertioneal injection of monocrotaline (MCT,60 mg/kg) to induce pulmonary hypertension and right heart failure.After 21 days,the rats in treatment group were given fluoxetine of 10 mg/(kg×d) by gavage per day for 21 days,the rats in the other two groups were given saline.HE staining was used to observe the pulmonary artery and right ventricular myocardial tissue in rats.The collagen formation in right ventricular myocardium was observed by Masson staining.The expressions of Nogo-A, Nogo-B ,type1collagen and type 3 collagen mRNA in myocardium were measured by real-time fluorescence quantitative PCR,while the semi quantitative measurement of Nogo protein level was detected by Western blot. After the intervention of fluoxetine,pulmonary artery stenosis was significantly reduced,myocardial tissue lesion decreased,collagen synthesis decreased in right ventricular myocardium.RT-PCR showed that mRNA of Nogo-A decreased,and mRNA of Nogo-B increased ( P <0.05).Western blot showed that the expression of Nogo-A protein decreased,while Nogo-B1 protein expression increased ( P <0.05),Nogo-B2 expression was not significantly changed ( P >0.05). Nogo may affect the collagen synthesis in right heart failure,and partly involved in myocardial fibrosis.

[Effects and mechanisms of ursodeoxycholic acid on isoprenaline-Induced myocardial fibrosis in mice].

PubMed

Li, X; Han, K Q; Shi, Y N; Men, S Z; Li, S; Sun, M H; Dong, H; Lu, J J; Ma, L J; Zhao, M; Li, D; Liu, W

2017-02-07

Objective: To investigate the effects and possible mechanisms of ursodeoxycholic acid (UDCA) on myocardial fibrosis in mice. Method: To observe the expression of transforming growth factor(TGF) -β1, CTGF, MMPs and the degree of myocardial fibrosis, 61 male Kunming mice were randomly divided into normal group, low dose UDCA group, high dose of UDCA group, spironolactone group, and the control group.Isoproterenol (ISO) injection was given subcutaneously (30 d) to make the model of myocardial fibrosis.Corresponding anti-fibrosis drugs (UDCA or spironolactone) were given by gavage.HE staining and Masson staining were performed to explore the inflammation and fibrosis in the myocardium.The expression of collagen Ⅰ and collagen Ⅲ protein was detected by immunohistochemistry to evaluate the degree of fibrosis among the groups.Western blot was used to detect the expression of transforming growth factor, (TGF)-β1, connective tissue growth factor (CTGF), matrix metalloproteinase (MMP)-2, -9, tissue inhibitor of metalloproteinase (TIMP)-4, -1 and anti-phospho-NFKBIA (p-IκB-α) inhibitor of NF-κB (IκB) protein in myocardium. Results: HE and Masson staining results showed that in the normal group, myocardial fibrosis is less, while the control group showed a large amount of fibrotic tissue ( P <0.05). Tissue fibrosis in the low/high dose UDCA group and spironolactone group was significantly reduced compared with the control group ( P <0.05), in which high dose of UDCA reduces fibrosis more significantly.Immunohistochemistry results showed that collagen Ⅰ and collagen Ⅲ protein expression was significantly increased ( P <0.05). Whereas in the low/high UDCA dose group and spironolactone group, collagen Ⅰ and collagen Ⅲ expression were significantly decreased ( P <0.05), the high UDCA dose group decreased more significantly.Western blot results suggest that TGFβ-1 expression in the myocardial tissue was significantly increased compared to the normal group ( P <0.05), whereas low/high UDCA dose group and spironolactone group, TGFβ-1 protein expression were significantly decreased [UDCA(1.52±0.16), (1.02±0.12), (1.01±0.21)vs (2.73±0.12), P <0.05], in which high UDCA dose group TGFβ-1 protein expression level decreased more significantly.However, there was no significant difference in the expression of CTGF, MMP2/9 and TIMP1/4 protein among the groups ( P >0.05). UDCA decrease p-IκB-α expression and increase IκB protein expression dose-dependently. Conclusions: UDCA can relieve isoproterenol induced myocardial fibrosis and reduce the myocardial collagen Ⅰ and collagen Ⅲ deposition in a dose dependent manner.Down-regulating of TGFβ-1 protein expression through the inhibition of TGR5-NF-κB signal transduction pathway might be a potential mechanism underlying UDCA's effects.

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

Efficient Production of Hydroxylated Human-Like Collagen Via the Co-Expression of Three Key Genes in Escherichia coli Origami (DE3).

PubMed

Tang, Yunping; Yang, Xiuliang; Hang, Baojian; Li, Jiangtao; Huang, Lei; Huang, Feng; Xu, Zhinan

2016-04-01

Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and D-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.

Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression.

PubMed

Eghbali, M; Weber, K T

1990-07-17

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.

Current status of synthetic epikeratoplasty.

PubMed

Thompson, K P; Hanna, K; Waring, G O; Gipson, I; Liu, Y; Gailitis, R P; Johnson-Wint, B; Green, K

1991-01-01

Many of the deficiencies with human tissue epikeratoplasty might be improved by the use of a suitable synthetic lenticule. Potential biomaterials for epikeratoplasty include collagen (types I, III, or IV), collagen-hydrogel copolymers, bioactive synthetics, and coated hydrogels. The biomaterial must be engineered to achieve strict specifications of optical clarity, support of epithelial migration and adhesion, permeability to solutes, and stability to corneal proteases. Attaching synthetic lenticules to the cornea without cutting Bowman's layer by adhesives, laser welding, or direct adhesion may also improve the efficacy of synthetic epikeratoplasty.

UV-Induced Triggering of a Biomechanical Initiation Switch Within Collagen Promotes Development of a Melanoma-Permissive Microenvironment in the Skin

DTIC Science & Technology

2012-09-01

addition, our new data suggest that pre-treating mice with anti-HU177 antibody inhibited UVB -induced accumulation of mast cells in the skin. Moreover, pre...work is provided below. Our previous studies have indicated that UVA and UVB irradiation can dose dependently trigger conformational changes in both...vitro following UV-irradiation was unlikely do to thermal denaturation. Interestingly, macrophage adhesion to UVB irradiated collagen type-IV but not

FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.

PubMed

Knüppel, Larissa; Heinzelmann, Katharina; Lindner, Michael; Hatz, Rudolf; Behr, Jürgen; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2018-04-19

In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

Histopathologic and Myogenic Gene Expression Changes Associated with Wooden Breast in Broiler Breast Muscles.

PubMed

Velleman, Sandra G; Clark, Daniel L

2015-09-01

The wooden breast condition is a myopathy affecting the pectoralis major (p. major) muscle in fast-growing commercial broiler lines. Currently, wooden breast-affected birds are phenotypically detected by palpation of the breast area, with affected birds having a very hard p. major muscle that is of lower value. The objective of this study was to compare the wooden breast myopathy in two fast-growing broiler lines (Lines A and B) with incidence of wooden breast to a slower growing broiler Line C with no phenotypically observable wooden breast. One of the characteristics of the wooden breast condition is fibrosis of the p. major muscle. Morphologic assessment of Lines A and B showed significant fibrosis in both lines, but the collagen distribution and arrangement of the collagen fibrils was different. In Line A, the collagen fibrils were tightly packed, whereas in Line B the collagen fibrils were diffuse. This difference in collagen organization may be due to the expression of the extracellular matrix proteoglycan decorin. Decorin is a regulator of collagen crosslinking and is expressed at significantly higher levels in Line A wooden breast-affected p. major muscle, which would lead to tightly packed collagen fibers due to high levels of collagen crosslinking. Furthermore, expression of the muscle-specific transcriptional regulatory factors for proliferation and differentiation of muscle cells leading to the regeneration of muscle in response to muscle damage was significantly elevated in Line A, and only the factor for differentiation, myogenin, was increased in Line B. The results from this study provide initial evidence that the etiology of the wooden breast myopathy may vary between fast-growing commercial broiler lines.

The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover

PubMed Central

Bay-Jensen, Anne-Christine; Tabassi, Nadine CB; Sondergaard, Lene V; Andersen, Thomas L; Dagnaes-Hansen, Frederik; Garnero, Patrick; Kassem, Moustapha; Delaissé, Jean-Marie

2009-01-01

Introduction The urinary level of the type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomised rats, suggesting that oestrogen deprivation induces cartilage breakdown. Here we investigate whether this response to oestrogen is also true for other type II collagen turnover markers known to be affected in osteoarthritis, and whether it relates to its presence in specific areas of cartilage tissue. Methods The type II collagen degradation markers CTX-II and Helix-II were measured in the body fluids of premenopausal and postmenopausal women and in those of ovariectomised rats receiving oestrogen or not. Levels of PIIANP, a marker of type II collagen synthesis, were also measured in rats. Rat knee cartilage was analysed for immunoreactivity of CTX-II and PIIANP and for type II collagen expression. Results As expected, urinary levels of CTX-II are significantly increased in postmenopausal women and also in oestrogen-deprived rats, although only transiently. However, in neither case were these elevations paralleled by a significant increase of Helix-II levels and PIIANP levels did not change at any time. CTX-II immunoreactivity and collagen expression were detected in different cartilage areas. The upper zone is the area where CTX-II immunoreactivity and collagen expression best reflected the differences in urinary levels of CTX-II measured in response to oestrogen. However, correlations between urinary levels of CTX-II and tissue immunostainings in individual rats were not statistically significant. Conclusions We found only a small effect of oestrogen deprivation on cartilage. It was detected by CTX-II, but not by other type II collagen turnover markers typically affected in osteoarthritis. PMID:20527083

Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation*

PubMed Central

Walker, Natalie M.; Belloli, Elizabeth A.; Stuckey, Linda; Chan, Kevin M.; Lin, Jules; Lynch, William; Chang, Andrew; Mazzoni, Serina M.; Fingar, Diane C.; Lama, Vibha N.

2016-01-01

Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states. PMID:26755732

Macrophage phenotype in the epigallocatechin-3-gallate (EGCG)-modified collagen determines foreign body reaction.

PubMed

Chu, Chenyu; Liu, Li; Wang, Yufei; Wei, Shimin; Wang, Yuanjing; Man, Yi; Qu, Yili

2018-04-28

Collagen has been widely used in guided bone regeneration, and the implantation of collagen membranes will elicit the foreign body reaction (FBR). The imbalance of FBR often leads to failure of dental implants. Therefore, modulation of the FBR after implantation of collagen membranes becomes increasingly important. Macrophages, pivotal in FBR, have been distinguished into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Epigallocatechin-3-gallate (EGCG)-modified collagen membranes have been previously shown to regulate secretion of inflammatory factors. In this study, immunohistochemistry of CD31 showed that areas of blood vessels were significantly enlarged after implantation of EGCG-modified collagen membranes compared with those treated with pure collagen membranes. Besides, haematoxylin-eosin staining and immunofluorescence showed an increased number of M2 macrophages after implantation of EGCG-modified collagen membranes. In addition, quantitative real-time polymerase chain reaction showed that after implantation of EGCG-modified collagen membranes, expression of CXCL1 (predominant chemoattractants to neutrophils and inflammation promotors) was significantly downregulated, whereas expressions of STAB1, CCR2, CCR3, CCL2, and CCL3 (related to M2 macrophages) were significantly upregulated. From these findings, we conclude that EGCG-modified collagen membranes were able to regulate the recruitment and polarization of macrophages, so that ameliorate FBR. Copyright © 2018 John Wiley & Sons, Ltd.

Loss of Interneuron-Derived Collagen XIX Leads to a Reduction in Perineuronal Nets in the Mammalian Telencephalon.

PubMed

Su, Jianmin; Cole, James; Fox, Michael A

2017-02-01

Perineuronal nets (PNNs) are lattice-like supramolecular assemblies of extracellular glycoproteins that surround subsets of neuronal cell bodies in the mammalian telencephalon. PNNs emerge at the end of the critical period of brain development, limit neuronal plasticity in the adult brain, and are lost in a variety of complex brain disorders diseases, including schizophrenia. The link between PNNs and schizophrenia led us to question whether neuronally expressed extracellular matrix (ECM) molecules associated with schizophrenia contribute to the assembly of these specialized supramolecular ECM assemblies. We focused on collagen XIX-a minor, nonfibrillar collagen expressed by subsets of telencephalic interneurons. Genetic alterations in the region encoding collagen XIX have been associated with familial schizophrenia, and loss of this collagen in mice results in altered inhibitory synapses, seizures, and the acquisition of schizophrenia-related behaviors. Here, we demonstrate that loss of collagen XIX also results in a reduction of telencephalic PNNs. Loss of PNNs was accompanied with reduced levels of aggrecan (Acan), a major component of PNNs. Despite reduced levels of PNN constituents in collagen XIX-deficient mice ( col19a1 - / - ), we failed to detect reduced expression of genes encoding these ECM molecules. Instead, we discovered a widespread upregulation of extracellular proteases capable of cleaving Acan and other PNN constituents in col19a1 - / - brains. Taken together, these results suggest a mechanism by which the loss of collagen XIX speeds PNN degradation and they identify a novel mechanism by which the loss of collagen XIX may contribute to complex brain disorders.

Immunogenicity moderation effect of interleukin-24 on myelogenous leukemia cells.

PubMed

Yu, Xin; Miao, Jingcheng; Xia, Wei; Gu, Zong-Jiang

2018-04-01

Previous studies have shown that interleukin-24 (IL-24) has tumor-suppressing activity by multiple pathways. However, the immunogenicity moderation effect of IL-24 on malignant cells has not been explored extensively. In this study, we investigated the role of IL-24 in immunogenicity modulation of the myelogenous leukemia cells. Data show that myelogenous leukemia cells express low levels of immunogenicity molecules. Treatment with IL-24 could enhance leukemia cell immunogenicity, predominantly regulate leukemia cells to produce immune-associated cytokines, and improve the cytotoxic sensitivity of these cells to immune effector cells. IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. Moreover, IL-24 had marked effects on downregulating the expression of angiogenesis-related proteins vascular endothelial growth factor, cluster of differentiation (CD) 31, CD34, collagen IV and metastasis-related factors CD147, membrane type-1 matrix metalloproteinase (MMP), and MMP-2 and MMP-9 in transplanted tumors. These findings indicated novel functions of this antitumor gene and characterized IL-24 as a promising agent for further clinical trial for hematologic malignancy immunotherapy.

Transforming Growth Factor β1 Induces the Expression of Collagen Type I by DNA Methylation in Cardiac Fibroblasts

PubMed Central

Pan, Xiaodong; Chen, Zhongpu; Huang, Rong; Yao, Yuyu; Ma, Genshan

2013-01-01

Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression. PMID:23560091

Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re-Activation

PubMed Central

Stellavato, Antonietta; Corsuto, Luisana; D’Agostino, Antonella; La Gatta, Annalisa; Diana, Paola; Bernini, Patrizia; De Rosa, Mario

2016-01-01

Hyaluronic Acid (HA)-based dermal formulations have rapidly gained a large consensus in aesthetic medicine and dermatology. HA, highly expressed in the Extracellular Matrix (ECM), acts as an activator of biological cascades, stimulating cell migration and proliferation, and operating as a regulator of the skin immune surveillance, through specific interactions with its receptors. HA may be used in topical formulations, as dermal inducer, for wound healing. Moreover, intradermal HA formulations (injectable HA) provide an attractive tool to counteract skin aging (e.g., facial wrinkles, dryness, and loss of elasticity) and restore normal dermal functions, through simple and minimally invasive procedures. Biological activity of a commercially available hyaluronic acid, Profhilo®, based on NAHYCO™ technology, was compared to H-HA or L-HA alone. The formation of hybrid cooperative complexes was confirmed by the sudden drop in η0 values in the rheological measurements. Besides, hybrid cooperative complexes proved stable to hyaluronidase (BTH) digestion. Using in vitro assays, based on keratinocytes, fibroblasts cells and on the Phenion® Full Thickness Skin Model 3D, hybrid cooperative complexes were compared to H-HA, widely used in biorevitalization procedures, and to L-HA, recently proposed as the most active fraction modulating the inflammatory response. Quantitative real-time PCR analyses were accomplished for the transcript quantification of collagens and elastin. Finally immunofluorescence staining permitted to evaluate the complete biosynthesis of all the molecules investigated. An increase in the expression levels of type I and type III collagen in fibroblasts and type IV and VII collagen in keratinocytes were found with the hybrid cooperative complexes, compared to untreated cells (CTR) and to the H-HA and L-HA treatments. The increase in elastin expression found in both cellular model and in the Phenion® Full Thickness Skin Model 3D also at longer time (up to 7 days), supports the clinically observed improvement of skin elasticity. The biomarkers analyzed suggest an increase of tissue remodeling in the presence of Profhilo®, probably due to the long lasting release and the concurrent action of the two HA components. PMID:27723763

Impaired anti-fibrotic effect of bone marrow-derived mesenchymal stem cell in a mouse model of pulmonary paracoccidioidomycosis

PubMed Central

Arango, Julián Camilo; Puerta-Arias, Juan David; Pino-Tamayo, Paula Andrea; Salazar-Peláez, Lina María; Rojas, Mauricio

2017-01-01

Bone marrow-derived mesenchymal stem cells (BMMSCs) have been consider as a promising therapy in fibrotic diseases. Experimental models suggest that BMMSCs may be used as an alternative therapy to treat chemical- or physical-induced pulmonary fibrosis. We investigated the anti-fibrotic potential of BMMSCs in an experimental model of lung fibrosis by infection with Paracoccidioides brasiliensis. BMMSCs were isolated and purified from BALB/c mice using standardized methods. BALB/c male mice were inoculated by intranasal infection of 1.5x106 P. brasiliensis yeasts. Then, 1x106 BMMSCs were administered intra venous at 8th week post-infection (p.i.). An additional group of mice was treated with itraconazole (ITC) two weeks before BMMSCs administration. Animals were sacrificed at 12th week p.i. Histopathological examination, fibrocytes counts, soluble collagen and fibrosis-related genes expression in lungs were evaluated. Additionally, human fibroblasts were treated with homogenized lung supernatants (HLS) to determine induction of collagen expression. Histological analysis showed an increase of granulomatous inflammatory areas in BMMSCs-treated mice. A significant increase of fibrocytes count, soluble collagen and collagen-3α1, TGF-β3, MMP-8 and MMP-15 genes expression were also observed in those mice. Interestingly, when combined therapy BMMSCs/ITC was used there is a decrease of TIMP-1 and MMP-13 gene expression in infected mice. Finally, human fibroblasts stimulated with HLS from infected and BMMSCs-transplanted mice showed a higher expression of collagen I. In conclusion, our findings indicate that late infusion of BMMSCs into mice infected with P. brasiliensis does not have any anti-fibrotic effect; possibly because their interaction with the fungus promotes collagen expression and tissue remodeling. PMID:29040281

Effect of single- and double-row rotator cuff repair at the tendon-to-bone interface: preliminary results using an in vivo sheep model.

PubMed

Baums, M H; Schminke, B; Posmyk, A; Miosge, N; Klinger, H-M; Lakemeier, S

2015-01-01

The clinical superiority of the double-row technique is still a subject of controversial debate in rotator cuff repair. We hypothesised that the expression of different collagen types will differ between double-row and single-row rotator cuff repair indicating a faster healing response by the double-row technique. Twenty-four mature female sheep were randomly assembled to two different groups in which a surgically created acute infraspinatus tendon tear was fixed using either a modified single- or double-row repair technique. Shoulder joints from female sheep cadavers of identical age, bone maturity, and weight served as untreated control cluster. Expression of type I, II, and III collagen was observed in the tendon-to-bone junction along with recovering changes in the fibrocartilage zone after immunohistological tissue staining at 1, 2, 3, 6, 12, and 26 weeks postoperatively. Expression of type III collagen remained positive until 6 weeks after surgery in the double-row group, whereas it was detectable for 12 weeks in the single-row group. In both groups, type I collagen expression increased after 12 weeks. Type II collagen expression was increased after 12 weeks in the double-row versus single-row group. Clusters of chondrocytes were only visible between week 6 and 12 in the double-row group. The study demonstrates differences regarding the expression of type I and type III collagen in the tendon-to-bone junction following double-row rotator cuff repair compared to single-row repair. The healing response in this acute repair model is faster in the double-row group during the investigated healing period.

The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma.

PubMed

Makino, Katsunari; Jinnin, Masatoshi; Hirano, Ayaka; Yamane, Keitaro; Eto, Mitsuhiko; Kusano, Takamitsu; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Sakai, Keisuke; Masuguchi, Shinichi; Fukushima, Satoshi; Ihn, Hironobu

2013-04-15

Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3'-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression

PubMed Central

Saby, Charles; Buache, Emilie; Brassart-Pasco, Sylvie; El Btaouri, Hassan; Courageot, Marie-Pierre; Van Gulick, Laurence; Garnotel, Roselyne; Jeannesson, Pierre; Morjani, Hamid

2016-01-01

Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D). We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen. In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2. PMID:27121132

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

PubMed

Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

2008-06-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiacmore » fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen formation. ► Aspirin decreases the transcription of angiotensin II type 1 receptor by inhibiting NADPH oxidase–NF-κB pathway. ► The inhibition of angiotensin II type 1 receptor expression may be the basis for reduction in fibroblast growth and collagen formation. ► The effects of aspirin appear to be mediated via its salicylate moiety.« less

In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats.

PubMed

Fujiwara, Ken; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2010-12-01

Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.

Peripheral Blood Mononuclear Cells Enhance the Anabolic Effects of Platelet-Rich Plasma on Anterior Cruciate Ligament Fibroblasts

PubMed Central

Yoshida, Ryu; Murray, Martha M.

2012-01-01

Use of platelet-rich plasma (PRP) has shown promise in various orthopaedic applications, including treatment of anterior cruciate ligament (ACL) injuries. However, various components of blood, including peripheral blood mononuclear cells (PBMCs), are removed in the process of making PRP. It is yet unknown whether these PBMCs have a positive or negative effect on fibroblast behavior. To begin to define the effect of PBMCs on ACL fibroblasts, ACL fibroblasts were cultured on three-dimensional collagen scaffolds for 14 days with and without PBMCs. ACL fibroblasts exposed to PBMCs showed increased type I and type III procollagen gene expression, collagen protein expression, and cell proliferation when the cells were cultured in the presence of platelets and plasma. However, addition of PBMCs to cells cultured without the presence of platelets had no effect. The increase in collagen gene and protein expression was accompanied by an increase in IL-6 expression by the PBMCs with exposure to the platelets. Our results suggest that the interaction between platelets and PBMCs leads to an IL-6 mediated increase in collagen expression by ACL fibroblasts. PMID:22767425

Alport syndrome and thin glomerular basement membrane nephropathy: a practical approach to diagnosis.

PubMed

Haas, Mark

2009-02-01

Alport syndrome and thin glomerular basement membrane nephropathy (TBMN) are genetically heterogeneous conditions characterized by structural abnormalities in the glomerular basement membrane and an initial presentation that usually involves hematuria. Approximately 40% of patients with TBMN are heterozygous carriers for autosomal recessive Alport syndrome, with mutations at the genetic locus encoding type IV collagen alpha(3) [alpha(3)(IV)] and alpha(4) chains. However, although the clinical course of TBMN is usually benign, Alport syndrome, particularly the X-linked form with mutations in the locus encoding the alpha(5) chain of type IV collagen [alpha(5)(IV)], typically results in end-stage renal disease. Electron microscopy is essential to diagnosis of TBMN and Alport syndrome on renal biopsy, although electron microscopy alone is of limited value in distinguishing between TBMN, the heterozygous carrier state of X-linked Alport syndrome, autosomal recessive Alport syndrome, and even early stages of X-linked Alport syndrome. To review diagnostic pathologic features of each of the above conditions, emphasizing the need for immunohistology for alpha(3)(IV) and alpha(5)(IV) in addition to electron microscopy to resolve this differential diagnosis on a renal biopsy. The diagnostic value of immunofluorescence studies for alpha(5)(IV) on a skin biopsy in family members of patients with Alport syndrome also is reviewed. Original and comprehensive review articles on the diagnosis of Alport syndrome and TBMN from the past 35 years, primarily the past 2 decades, and experience in our own renal pathology laboratory. Although Alport syndrome variants and TBMN do not show characteristic light microscopic findings and can be difficult to differentiate from each other even by electron microscopy, using a combination of electron microscopy and immunohistology for alpha(3)(IV) and alpha(5)(IV) enables pathologists to definitively diagnose these disorders on renal biopsy in most cases.

Decreased expression of microRNA-29 family in leiomyoma contributes to increased major fibrillar collagen production.

PubMed

Marsh, Erica E; Steinberg, Marissa L; Parker, J Brandon; Wu, Ju; Chakravarti, Debabrata; Bulun, Serdar E

2016-09-01

To determine the expression and function of the microRNA-29 family (miRNA-29a, miRNA-29b, miRNA-29c) in human leiomyoma and myometrium. Basic science experimental design. Academic medical center. Women undergoing surgery for symptomatic uterine fibroids. Overexpression and knockdown of miRNA-29a, miRNA-29b, and miRNA-29c in primary leiomyoma and myometrial cells. [1] Expression of the miRNA-29 family members in vivo in leiomyoma versus myometrium; [2] Major fibrillar collagen (I, II, III) expression in leiomyoma and myometrial cells with manipulation of miRNA-29 species. Members of the miRNA-29 family (29a, 29b, 29c) are all down-regulated in leiomyoma versus myometrium in vivo. The expression of the miRNA-29 family can be successfully modulated in primary leiomyoma and myometrial cells. Overexpression of the miRNA-29 family in leiomyoma cells results in down-regulation of the major fibrillar collagens. Down-regulation of the miRNA-29 species in myometrium results in an increase in collagen type III deposition. The miRNA-29 family is consistently down-regulated in leiomyoma compared to matched myometrial tissue. This down-regulation contributes to the increased collagen seen in leiomyomas versus myometrium. When miRNA-29 members are overexpressed in leiomyoma cells, protein levels of all of the major fibrillar collagens decrease. The miRNA-29 members are potential therapeutic targets in this highly prevalent condition. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

In vivo topical application of acetyl aspartic acid increases fibrillin-1 and collagen IV deposition leading to a significant improvement of skin firmness.

PubMed

Gillbro, J M; Merinville, E; Cattley, K; Al-Bader, T; Hagforsen, E; Nilsson, M; Mavon, A

2015-10-01

Acetyl aspartic acid (A-A-A) was discovered through gene array analysis with corresponding Cmap analysis. We found that A-A-A increased keratinocyte regeneration, inhibited dermal matrix metalloprotease (MMP) expression and relieved fibroblast stiffness through reduction of the fibroblast stiffness marker F-actin. Dermal absorption studies showed successful delivery to both the epidermal and dermal regions, and in-use trial demonstrated that 1% A-A-A was well tolerated. In this study, the aim was to investigate whether A-A-A could stimulate the synthesis of extracellular matrix supporting proteins in vivo and thereby improving the viscoelastic properties of human skin by conducting a dual histological and biophysical clinical study. Two separate double-blind vehicle-controlled in vivo studies were conducted using a 1% A-A-A containing oil-in-water (o/w) emulsion. In the histological study, 16 female volunteers (>55 years of age) exhibiting photodamaged skin on their forearm were included, investigating the effect of a 12-day treatment of A-A-A on collagen IV (COLIV) and fibrillin-1. In a subsequent pilot study, 0.1% retinol was used for comparison to A-A-A (1%). The biomechanical properties of the skin were assessed in a panel of 16 women (>45 years of age) using the standard Cutometer MPA580 after topical application of the test products for 28 days. The use of multiple suction enabled the assessment of F4, an area parameter specifically representing skin firmness. Twelve-day topical application of 1% A-A-A significantly increased COLIV and fibrillin with 13% and 6%, respectively, compared to vehicle. 1% A-A-A and 0.1% retinol were found to significantly reduce F4 after 28 days of treatment by 15.8% and 14.7%, respectively, in the pilot Cutometer study. No significant difference was found between retinol and A-A-A. However, only A-A-A exhibited a significant effect vs. vehicle on skin firmness which indicated the incremental benefit of A-A-A as a skin-firming active ingredient. In this study, we showed the in vivo efficacy of 1% A-A-A both on a protein level (fibrillin and collagen IV) and on a clinical end point, specifically skin firmness, providing proof that, acetyl aspartic acid has a strong potential as an anti-ageing 'cosmeceutical' ingredient answering the needs of our key consumer base. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

PubMed Central

1994-01-01

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect. PMID:7964480

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy.

PubMed

Shah, Anu; Xia, Ling; Masson, Elodie A Y; Gui, Chloe; Momen, Abdul; Shikatani, Eric A; Husain, Mansoor; Quaggin, Susan; John, Rohan; Fantus, I G

2015-12-01

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP(-/-), and TxNIP(+/-) mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP(-/-) mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP(-/-) mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms' tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP(-/-) mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O2 (-) generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target. Copyright © 2015 by the American Society of Nephrology.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

PubMed

Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara Dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro Dos Santos; Behrens, Maria Dutra; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

2016-08-01

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

PubMed Central

Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro dos Santos; Behrens, Maria Dutra

2016-01-01

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography–mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment. PMID:27579922

A Drug-Induced Hybrid Electrospun Poly-Capro-Lactone: Cell-Derived Extracellular Matrix Scaffold for Liver Tissue Engineering.

PubMed

Grant, Rhiannon; Hay, David C; Callanan, Anthony

2017-07-01

Liver transplant is the only treatment option for patients with end-stage liver failure, however, there are too few donor livers available for transplant. Whole organ tissue engineering presents a potential solution to the problem of rapidly escalating donor liver shortages worldwide. A major challenge for liver tissue engineers is the creation of a hepatocyte microenvironment; a niche in which liver cells can survive and function optimally. While polymers and decellularized tissues pose an attractive option for scaffold manufacturing, neither alone has thus far proved sufficient. This study exploited cell's native extracellular matrix (ECM) producing capabilities using two different histone deacetylase inhibitors, and combined these with the customizability and reproducibility of electrospun polymer scaffolds to produce a "best of both worlds" niche microenvironment for hepatocytes. The resulting hybrid poly-capro-lactone (PCL)-ECM scaffolds were validated using HepG2 hepatocytes. The hybrid PCL-ECM scaffolds maintained hepatocyte growth and function, as evidenced by metabolic activity and DNA quantitation. Mechanical testing revealed little significant difference between scaffolds, indicating that cells were responding to a biochemical and topographical profile rather than mechanical changes. Immunohistochemistry showed that the biochemical profile of the drug-derived and nondrug-derived ECMs differed in ratio of Collagen I, Laminin, and Fibronectin. Furthermore, the hybrid PCL-ECM scaffolds influence the gene expression profile of the HepG2s drastically; with expression of Albumin, Cytochrome P450 Family 1 Subfamily A Polypeptide 1, Cytochrome P450 Family 1 Subfamily A Polypeptide 2, Cytochrome P450 Family 3 Subfamily A Polypeptide 4, Fibronectin, Collagen I, and Collagen IV undergoing significant changes. Our results demonstrate that drug-induced hybrid PCL-ECM scaffolds provide a viable, translatable platform for creating a niche microenvironment for hepatocytes, supporting in vivo phenotype and function. These scaffolds offer great potential for tissue engineering and regenerative medicine strategies for whole organ tissue engineering.

Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid

PubMed Central

Jamieson, Hamish A; Hilmer, Sarah N; Cogger, Victoria C; Warren, Alessandra; Cheluvappa, Rajkumar; Abernethy, Darrell R; Everitt, Arthur V; Fraser, Robin; de Cabo, Rafael; Le Couteur, David G

2007-01-01

Age-related changes in the hepatic sinusoid, called pseudocapillarization, may contribute to the pathogenesis of dyslipidaemia. Caloric restriction (CR) is a powerful model for the study of aging because it extends lifespan. We assessed the effects of CR on the hepatic sinusoid to determine whether pseudocapillarization is preventable and hence a target for the prevention of age-related dyslipidemia. Livers from young (6 months) and old (24 months) CR and ad libitum fed (AL) F344 rats were examined using electron microscopy and immunohistochemistry. In old age, there was increased thickness of the liver sinusoidal endothelium and reduced endothelial fenestration porosity. In old CR rats, endothelial thickness was less and fenestration porosity was greater than in old AL rats. Immunohistochemistry showed that CR prevented age-related decrease in caveolin-1 expression and increase in peri-sinusoidal collagen IV staining, but did not alter the age-related increase of von Willebrand’s factor. CR reduces age-related pseudocapillarization of the hepatic sinusoid and correlates with changes in caveolin-1 expression. PMID:17204388

Mesh-based Monte Carlo code for fluorescence modeling in complex tissues with irregular boundaries

NASA Astrophysics Data System (ADS)

Wilson, Robert H.; Chen, Leng-Chun; Lloyd, William; Kuo, Shiuhyang; Marcelo, Cynthia; Feinberg, Stephen E.; Mycek, Mary-Ann

2011-07-01

There is a growing need for the development of computational models that can account for complex tissue morphology in simulations of photon propagation. We describe the development and validation of a user-friendly, MATLAB-based Monte Carlo code that uses analytically-defined surface meshes to model heterogeneous tissue geometry. The code can use information from non-linear optical microscopy images to discriminate the fluorescence photons (from endogenous or exogenous fluorophores) detected from different layers of complex turbid media. We present a specific application of modeling a layered human tissue-engineered construct (Ex Vivo Produced Oral Mucosa Equivalent, EVPOME) designed for use in repair of oral tissue following surgery. Second-harmonic generation microscopic imaging of an EVPOME construct (oral keratinocytes atop a scaffold coated with human type IV collagen) was employed to determine an approximate analytical expression for the complex shape of the interface between the two layers. This expression can then be inserted into the code to correct the simulated fluorescence for the effect of the irregular tissue geometry.

Prohibitin is associated with antioxidative protection in hypoxia/reoxygenation-induced renal tubular epithelial cell injury

NASA Astrophysics Data System (ADS)

Zhou, Tian-Biao; Qin, Yuan-Han; Lei, Feng-Ying; Huang, Wei-Fang; Drummen, Gregor P. C.

2013-11-01

Prohibitin is an evolutionary conserved and pleiotropic protein that has been implicated in various cellular functions, including proliferation, tumour suppression, apoptosis, transcription, and mitochondrial protein folding. We recently demonstrated that prohibitin downregulation results in increased renal interstitial fibrosis. Here we investigated the role of oxidative stress and prohibitin expression in a hypoxia/reoxygenation injury system in renal tubular epithelial cells with lentivirus-based delivery vectors to knockdown or overexpress prohibitin. Our results show that increased prohibitin expression was negatively correlated with reactive oxygen species, malon dialdehyde, transforming-growth-factor-β1, collagen-IV, fibronectin, and apoptosis (r = -0.895, -0.764, -0.798, -0.826, -0.817, -0.735 each P < 0.01), but positively correlated with superoxide dismutase, glutathione and mitochondrial membrane potential (r = 0.807, 0.815, 0.739; each P < 0.01). We postulate that prohibitin acts as a positive regulator of mechanisms that counteract oxidative stress and extracellular matrix accumulation and therefore has an antioxidative effect.

Altered procollagen gene expression in mid-gestational mouse excisional wounds.

PubMed

Goldberg, Stephanie R; Quirk, Gerald L; Sykes, Virginia W; Kordula, Tomasz; Lanning, David A

2007-11-01

Many pathologic conditions are characterized by excessive tissue contraction and scar formation. Previously, we developed a murine model of excisional wound healing in which mid-gestational wounds heal scarlessly compared with late-gestational wounds. We theorized that variations in procollagen gene expression may contribute to the scarless and rapid closure. Time-dated pregnant FVB strain mice underwent laparotomy and hysterotomy on embryonic days 15 (E15) and 18 (E18). Full-thickness, excisional wounds (3 mm) were made on each of 4 fetuses per doe and then harvested at 32, 48, or 72 h. Control tissue consisted of age-matched normal fetal skin. Procollagen types 1alpha1, 1alpha2, and 3 gene expressions were measured by real-time polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase. Trichrome staining was also performed. Procollagen 1alpha1 expression was decreased in E15 wounds at 32 h compared with their normal skin groups. Procollagen types 1alpha2 and 3 expressions were both increased in the E15 groups compared with the E18 groups at 48 h. At 72 h, the E15 wounds had a collagen density similar to the surrounding normal skin while E18 wounds exhibited increased collagen deposition in a disorganized pattern. This study demonstrates that the pattern of gene expression for types 1 and 3 collagen varies between mid- and late-gestational mouse excisional wounds. These alterations in procollagen expression may contribute to a pattern of collagen deposition in the mid-gestational fetuses that is more favorable for scarless healing with less type 1 and more type 3 collagen.

Optic nerve head and intraocular pressure in the guinea pig eye.

PubMed

Ostrin, Lisa A; Wildsoet, Christine F

2016-05-01

The guinea pig is becoming an increasingly popular model for studying human myopia, which carries an increased risk of glaucoma. As a step towards understanding this association, this study sought to characterize the normal, developmental intraocular pressure (IOP) profiles, as well as the anatomy of the optic nerve head (ONH) and adjacent sclera of young guinea pigs. IOP was tracked in pigmented guinea pigs up to 3 months of age. One guinea pig was imaged in vivo with OCT and one with a fundus camera. The eyes of pigmented and albino guinea pigs (ages 2 months) were enucleated and sections from the posterior segment, including the ONH and surrounding sclera, processed for histological analyses - either hematoxylin and eosin (H&E) staining of paraffin embedded, sectioned tissue (n = 1), or cryostat sectioned tissue, processed for immunohistochemistry (n = 3), using primary antibodies against collagen types I-V, elastin, fibronectin and glial fibrillary acidic protein (GFAP). Transmission and scanning electron microscopy (TEM, SEM) studies of ONHs were also undertaken (n = 2 & 5 respectively). Mean IOPs ranged from 17.33 to 22.7 mmHg, increasing slightly across the age range studied, and the IOPs of individual animals also exhibited diurnal variations, peaking in the early morning (mean of 25.8, mmHg, ∼9 am), and decreasing across the day. H&E-stained sections showed retinal ganglion cell axons organized into fascicles in the prelaminar and laminar region of the ONHs, with immunostained sections revealing collagen types I, III, IV and V, as well as elastin, GFAP and fibronectin in the ONHs. SEM revealed a well-defined lamina cribrosa (LC), with radially-oriented collagen beams. TEM revealed collagen fibrils surrounding non-myelinated nerve fiber bundles in the LC region, with myelination and decreased collagen posterior to the LC. The adjacent sclera comprised mainly crimped collagen fibers in a crisscross arrangement. Both the sclera and LC were qualitatively similar in structure in pigmented and albino guinea pigs. The well-organized, collagen-based LC of the guinea pig ONH is similar to that described for tree shrews and more similar to the human LC than that of other rodents that lack collagen. Based on these latter structural similarities the guinea pig would seem a promising model for investigating the relationship between myopia and glaucoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inflammatory PAF Receptor Signaling Initiates Hedgehog Signaling and Kidney Fibrogenesis During Ethanol Consumption

PubMed Central

Latchoumycandane, Calivarathan; Hanouneh, Mohamad; Nagy, Laura E.; McIntyre, Thomas M.

2015-01-01

Acute inflammation either resolves or proceeds to fibrotic repair that replaces functional tissue. Pro-fibrotic hedgehog signaling and induction of its Gli transcription factor in pericytes induces fibrosis in kidney, but molecular instructions connecting inflammation to fibrosis are opaque. We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis. Ethanol ingestion stimulated transcription of TGF-ß, collagens I and IV, and alpha-smooth muscle actin with accumulation of these proteins. This was accompanied by deposition of extracellular fibrils. Ethanol catabolism by CYP2E1 in kidney generates local reactive oxygen species that oxidize cellular phospholipids to phospholipid products that activate the Platelet-activating Factor receptor (PTAFR) for inflammatory phospholipids. Genetically deleting this ptafr locus abolished accumulation of mRNA for TGF-ß, collagen IV, and α-smooth muscle actin. Loss of PTAFR also abolished ethanol-stimulated Sonic (Shh) and Indian hedgehog (Ihh) expression, and abolished transcription and accumulation of Gli1. Shh induced in pericytes and Ihh in tubules escaped to urine of ethanol-fed mice. Neutrophil myeloperoxidase (MPO) is required for ethanol-induced kidney inflammation, and Shh was not present in kidney or urine of mpo -/- mice. Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney. We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly biopsied. PMID:26720402

Ascorbic acid: Chemistry, biology and the treatment of cancer☆

PubMed Central

Du, Juan; Cullen, Joseph J.; Buettner, Garry R.

2013-01-01

Since the discovery of vitamin C, the number of its known biological functions is continually expanding. Both the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its role in the synthesis of collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes, for example via the HIF system, as well as via the epigenetic landscape of cells and tissues. In fact, all known physiological and biochemical functions of ascorbate are due to its action as an electron donor. The ability to donate one or two electrons makes AscH− an excellent reducing agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H2O2). In the presence of catalytic metals this oxidation is accelerated. In this review, we show that the chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant properties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as prodrug to deliver a significant flux of H2O2 to tumors. This new knowledge has rekindled interest and spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especially the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer. PMID:22728050

PEP-1-SIRT2-induced matrix metalloproteinase-1 and -13 modulates type II collagen expression via ERK signaling in rabbit articular chondrocytes.

PubMed

Eo, Seong-Hui; Choi, Soo Young; Kim, Song Ja

2016-11-01

Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1-SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1-SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1-SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1-SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

Collagen type XI alpha1 may be involved in the structural plasticity of the vertebral column in Atlantic salmon (Salmo salar L.).

PubMed

Wargelius, A; Fjelldal, P G; Nordgarden, U; Grini, A; Krossøy, C; Grotmol, S; Totland, G K; Hansen, T

2010-04-01

Atlantic salmon (Salmo salar L.) vertebral bone displays plasticity in structure, osteoid secretion and mineralization in response to photoperiod. Other properties of the vertebral bone, such as mineral content and mechanical strength, are also associated with common malformations in farmed Atlantic salmon. The biological mechanisms that underlie these changes in bone physiology are unknown, and in order to elucidate which factors might be involved in this process, microarray assays were performed on vertebral bone of Atlantic salmon reared under natural or continuous light. Eight genes were upregulated in response to continuous light treatment, whereas only one of them was upregulated in a duplicate experiment. The transcriptionally regulated gene was predicted to code for collagen type XI alpha1, a protein known to be involved in controlling the diameter of fibrillar collagens in mammals. Furthermore, the gene was highly expressed in the vertebrae, where spatial expression was found in trabecular and compact bone osteoblasts and in the chordoblasts of the notochordal sheath. When we measured the expression level of the gene in the tissue compartments of the vertebrae, the collagen turned out to be 150 and 25 times more highly expressed in the notochord and compact bone respectively, relative to the expression in the trabecular bone. Gene expression was induced in response to continuous light, and reduced in compressed vertebrae. The downregulation in compressed vertebrae was due to reduced expression in the compact bone, while expression in the trabecular bone and the notochord was unaffected. These data support the hypothesis that this gene codes for a presumptive collagen type XI alpha1, which may be involved in the regulatory pathway leading to structural adaptation of the vertebral architecture.

SENP1 attenuates the liver fibrosis through down-regulating the expression of SMAD2.

PubMed

Wu, Linshi; Qiu, Weiqing; Sun, Jianhua; Wang, Jian

2018-01-01

To investigate whether SENP1 could play a regulating role in the liver fibrosis process, the Sprague-Dawley (SD) rats were used to establish the liver fibrosis rat models by intraperitoneally injecting with 1 ml/kg of 10% CCl 4 , while the control normal rats were injected with olive oil. Then confirmation experiments to verify the successful establishment of these models were conducted by detecting the cellular and lobular architecture, and liver function indexes using hematoxylin-eosin staining, Masson's trichrome staining and microplate method, respectively. In addition, the expression levels of fibrosis markers including collagen I, collagen III, α-SMA and TGF-β1 were inspected using quantitative real-time PCR (qRT-PCR), as well as SMAD2. Subsequently, the relative mRNA and protein level of SENP1 was also determined via qRT-PCR and western blot analysis. Next, the HSC-T6 cells of SENP1 knock-down were constructed and used to test the relative protein expression levels of α-SMA and SMAD2 in these cells. The results of hematoxylin-eosin staining, Masson's trichrome staining and microplate method turned out that the rat liver fibrosis models were constructed successfully, which was further confirmed by the increased expression of collagen I, collagen III, α-SMA and TGF-β1 in mRNA and protein level, as well as SMAD2. Then the expression of SENP1 was overexpressed in the rat liver fibrosis models induced by CCl 4 and the TGF-β1 treatment could increase the protein expression level of collagen I, collagen III and α-SMA. Lastly, the SENP1 knockdown HSC-T6 cells were successfully constructed, while the silence of SENP1 down-regulated the protein expression of α-SMA and SMAD2. In conclusion, this study provided a new regulation mechanism about the liver fibrosis process. Copyright © 2017 Elsevier Inc. All rights reserved.

Pancreatic Cancer Cells Enhance the Ability of Collagen Internalization during Epithelial–Mesenchymal Transition

PubMed Central

Ikenaga, Naoki; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Akagawa, Shin; Fujiwara, Kenji; Eguchi, Daiki; Kozono, Shingo; Ohtsuka, Takao; Takahata, Shunichi; Tanaka, Masao

2012-01-01

Background Extracellular matrix (ECM) remodeling is predominantly mediated by fibroblasts using intracellular and extracellular pathways. Although it is well known that extracellular degradation of the ECM by proteases derived from cancer cells facilitates cellular invasion, the intracellular degradation of ECM components by cancer cells has not been clarified. The aim of this study was to characterize collagen internalization, which is the initial step of the intracellular degradation pathway in pancreatic cancer cells, in light of epithelial–mesenchymal transition (EMT). Methodology/Principal Findings We analyzed the function of collagen internalization in two pancreatic cancer cell lines, SUIT-2 and KP-2, and pancreatic stellate cells (PSCs) using Oregon Green 488-gelatin. PSCs had a strong ability for collagen uptake, and the pancreatic cancer cells also internalized collagen although less efficiently. The collagen internalization abilities of SUIT-2 and KP-2 cells were promoted by EMT induced by human recombinant transforming growth factor β1 (P<0.05). Expression of Endo180, a collagen uptake receptor, was high in mesenchymal pancreatic cancer cell lines, as determined by EMT marker expression (P<0.01). Quantitative RT-PCR and western blot analyses showed that Endo180 expression was also increased by EMT induction in SUIT-2 and KP-2 cells. Endo180 knockdown by RNA interference attenuated the collagen uptake (P<0.01) and invasive abilities (P<0.05) of SUIT-2 and KP-2 cells. Conclusions/Significance Pancreatic cancer cells are capable of collagen internalization, which is enhanced by EMT. This ECM clearance system may be a novel mechanism for cellular invasion and a potential therapeutic target in pancreatic cancer. PMID:22792318

Global DNA hypomethylation has no impact on lung function or serum inflammatory and fibrosis cytokines in asbestos-exposed population.

PubMed

Yu, Min; Lou, Jianlin; Xia, Hailing; Zhang, Min; Zhang, Yixiao; Chen, Junqiang; Zhang, Xing; Ying, Shibo; Zhu, Lijin; Liu, Lihong; Jia, Guang

2017-04-01

To examine the effect of asbestos exposure on global DNA methylation and determine whether lung function and inflammatory and fibrosis biomarkers are correlated with the methylation state. A total of 26 healthy subjects without asbestos exposure (Group 1), 47 healthy subjects with exposure (Group 2), and 52 subjects with benign asbestos-related disorders (ARDs) (Group 3) participated in this cross-sectional study. Blood global 5-methylcytosine (5mC) and serum TNF-α, collagen IV, CCL5 and CC16 concentrations were analyzed using enzyme-linked immunosorbent assay-like assays. Spirometric maneuvers were performed to assess lung function. Decreased 5mC levels were observed in Groups 2 and 3 compared to Group 1, irrespective of lung function (p < 0.01). There was no significant change in 5mC between Groups 2 and 3. Overall, 5mC was negatively correlated with CCL5 and collagen IV (p < 0.05), but no significant inverse relationship was found between 5mC and CCL5 or collagen IV in each group. Additionally, both 5mC and CC16 were inversely associated with FEV1/FVC% (p = 0.001, adjusted R 2  = 0.145) for non-smokers, and consistently significant inverse relationships were found between CC16 and FEV1/FVC%, independent of asbestos exposure. Asbestos exposure causes global DNA hypomethylation. DNA hypomethylation has no influence on serum biomarkers and lung function in asbestos-exposed population with or without pleural and pulmonary parenchymal abnormalities.

Albumin contributes to kidney disease progression in Alport syndrome

PubMed Central

Knutsen, Russell H.; Mecham, Robert P.

2016-01-01

Alport syndrome is a familial kidney disease caused by defects in the collagen type IV network of the glomerular basement membrane. Lack of collagen-α3α4α5(IV) changes the glomerular basement membrane morphologically and functionally, rendering it leaky to albumin and other plasma proteins. Filtered albumin has been suggested to be a cause of the glomerular and tubular injuries observed at advanced stages of Alport syndrome. To directly investigate the role that albumin plays in the progression of disease in Alport syndrome, we generated albumin knockout (Alb−/−) mice to use as a tool for removing albuminuria as a component of kidney disease. Mice lacking albumin were healthy and indistinguishable from control littermates, although they developed hypertriglyceridemia. Dyslipidemia was observed in Alb+/− mice, which displayed half the normal plasma albumin concentration. Alb mutant mice were bred to collagen-α3(IV) knockout (Col4a3−/−) mice, which are a model for human Alport syndrome. Lack of circulating and filtered albumin in Col4a3−/−;Alb−/− mice resulted in dramatically improved kidney disease outcomes, as these mice lived 64% longer than did Col4a3−/−;Alb+/+ and Col4a3−/−;Alb+/− mice, despite similar blood pressures and serum triglyceride levels. Further investigations showed that the absence of albumin correlated with reduced transforming growth factor-β1 signaling as well as reduced tubulointerstitial, glomerular, and podocyte pathology. We conclude that filtered albumin is injurious to kidney cells in Alport syndrome and perhaps in other proteinuric kidney diseases, including diabetic nephropathy. PMID:27147675

Albumin contributes to kidney disease progression in Alport syndrome.

PubMed

Jarad, George; Knutsen, Russell H; Mecham, Robert P; Miner, Jeffrey H

2016-07-01

Alport syndrome is a familial kidney disease caused by defects in the collagen type IV network of the glomerular basement membrane. Lack of collagen-α3α4α5(IV) changes the glomerular basement membrane morphologically and functionally, rendering it leaky to albumin and other plasma proteins. Filtered albumin has been suggested to be a cause of the glomerular and tubular injuries observed at advanced stages of Alport syndrome. To directly investigate the role that albumin plays in the progression of disease in Alport syndrome, we generated albumin knockout (Alb(-/-)) mice to use as a tool for removing albuminuria as a component of kidney disease. Mice lacking albumin were healthy and indistinguishable from control littermates, although they developed hypertriglyceridemia. Dyslipidemia was observed in Alb(+/-) mice, which displayed half the normal plasma albumin concentration. Alb mutant mice were bred to collagen-α3(IV) knockout (Col4a3(-/-)) mice, which are a model for human Alport syndrome. Lack of circulating and filtered albumin in Col4a3(-/-);Alb(-/-) mice resulted in dramatically improved kidney disease outcomes, as these mice lived 64% longer than did Col4a3(-/-);Alb(+/+) and Col4a3(-/-);Alb(+/-) mice, despite similar blood pressures and serum triglyceride levels. Further investigations showed that the absence of albumin correlated with reduced transforming growth factor-β1 signaling as well as reduced tubulointerstitial, glomerular, and podocyte pathology. We conclude that filtered albumin is injurious to kidney cells in Alport syndrome and perhaps in other proteinuric kidney diseases, including diabetic nephropathy. Copyright © 2016 the American Physiological Society.

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.

PubMed Central

Gil, M L; Peñalver, M C; Lopez-Ribot, J L; O'Connor, J E; Martinez, J P

1996-01-01

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease. PMID:8945572

A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling.

PubMed

Monaghan, Michael; Browne, Shane; Schenke-Layland, Katja; Pandit, Abhay

2014-04-01

Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.

PubMed

Kreikemeyer, Bernd; Nakata, Masanobu; Oehmcke, Sonja; Gschwendtner, Caroline; Normann, Jana; Podbielski, Andreas

2005-09-30

The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.

Low-level laser therapy induces an upregulation of collagen gene expression during the initial process of bone healing: a microarray analysis

NASA Astrophysics Data System (ADS)

Tim, Carla Roberta; Bossini, Paulo Sérgio; Kido, Hueliton Wilian; Malavazi, Iran; von Zeska Kress, Marcia Regina; Carazzolle, Marcelo Falsarella; Rennó, Ana Cláudia; Parizotto, Nivaldo Antonio

2016-08-01

This study investigates the histological modifications produced by low level laser therapy (LLLT) on the first day of bone repair, as well as evaluates the LLLT effects on collagen expression on the site of a fracture. Twenty Wistar rats were distributed into a control group (CG) and a laser group (LG). Laser irradiation of Ga-Al-As laser 830 nm, 30 mW, 94 s, 2.8 J was performed in five sessions. Animals were euthanized on day 5 postsurgery. Histopathological analysis showed that LLLT was able to increase deposition of granulation tissue and newly formed bone at the site of the injury. In addition, picrosirius analysis showed that collagen fiber organization in the LG was enhanced compared to CG. Microarray analysis demonstrated that LLLT produced an upregulation type I collagen (COL-I). Immunohistochemical analysis revealed that the subjects that were treated presented a higher immunoexpression of COL-I. Our findings indicated that LLLT improves bone healing by producing a significant increase in the expression of collagen genes.

[EXPERIMENTAL RESEARCH OF DIFFERENTIATION OF HUMAN AMNIOTIC MESENCHYMAL STEM CELLS INTO LIGAMENT CELLS IN VITRO].

PubMed

Jin, Ying; Li, Yuwan; Zhang, Chenghao; Wu, Shuhong; Cheng, Daixiong; Liu, Yi

2016-02-01

To discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction. The hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor beta1 (TGF-beta1) + basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-beta1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining. The flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001). The hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.

Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Kairui; Zhang, Sheng; Li, Qianqian

Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aimsmore » to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.« less

Development of a conjunctival tissue substitute on the basis of plastic compressed collagen.

PubMed

Drechsler, C C; Kunze, A; Kureshi, A; Grobe, G; Reichl, S; Geerling, G; Daniels, J T; Schrader, S

2017-03-01

Ocular surface disorders, such as pterygium, cicatricial pemphigoid and external disruptions, can cause severe inflammation, scarring, fornix shortening as well as ankyloblepharon. Current treatments do not resolve these conditions sufficiently. The aim of this study was to evaluate clinical applicability and suitability of plastic compressed collagen to serve as a substrate for the expansion of human conjunctival epithelial cells in order to develop an epithelialized conjunctival substitute for fornix reconstruction. Human conjunctival epithelial cells were expanded on plastic compressed collagen gels. Epithelial cell characteristics were evaluated by haematoxylin and eosin staining, electron microscopy and cytokeratin expression. The expression of putative epithelial progenitor cell markers p63α, ABCG2 and CK15 was assessed by immunostaining. The proliferative capacity and clonal growth of the cells was evaluated before (P0) and after expansion (P1) on the plastic compressed collagen gels by colony forming efficiency assay. The potential clinical applicability of this gel substitutes was evaluated by assessment of their biomechanical properties as well as their surgical handling. Human conjunctival epithelial cells cultured on plastic and plastic compressed collagen gels formed a confluent cell layer and expressed CK19. The cells showed expression of the putative epithelial progenitor cell markers p63α, ABCG2 and CK15 and sustained colony forming ability. The compressed collagen gels showed a high ultimate tensile strength and elasticity and the surgical handling of gels was comparable to amniotic membrane. An epithelialized conjunctival tissue construct on the basis of compressed collagen might therefore be a promising alternative bioartificial tissue substitute for conjunctival reconstruction. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Fibrin gels exhibit improved biological, structural, and mechanical properties compared with collagen gels in cell-based tendon tissue-engineered constructs.

PubMed

Breidenbach, Andrew P; Dyment, Nathaniel A; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T; Rowe, David W; Kadler, Karl E; Butler, David L

2015-02-01

The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair.

Fibrin Gels Exhibit Improved Biological, Structural, and Mechanical Properties Compared with Collagen Gels in Cell-Based Tendon Tissue-Engineered Constructs

PubMed Central

Dyment, Nathaniel A.; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T.; Rowe, David W.; Kadler, Karl E.; Butler, David L.

2015-01-01

The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair. PMID:25266738

Cathepsin S is associated with degradation of collagen I in abdominal aortic aneurysm.

PubMed

Klaus, Veronika; Schmies, Fadwa; Reeps, Christian; Trenner, Matthias; Geisbüsch, Sarah; Lohoefer, Fabian; Eckstein, Hans-Henning; Pelisek, Jaroslav

2018-06-01

Cathepsins have been described in the pathogenesis of abdominal aortic aneurysm (AAA), their exact role, especially in collagen degradation, is still unclear. The aim of the present study was therefore to analyse relevant cathepsins in human AAA tissue samples in relation to collagen I, III, and their degradation products. Samples from 37 AAA patients obtained from elective open surgical repair and eight healthy non-aneurysmatic aortas from kidney donors were included. Expression of cathepsins B, D, K, L, S, cystatin C, collagen I and III, their degraded products C-Telopeptide of type 1 and 3 collagen (CTX-I, CTX-III), cellular markers for leukocytes (CD45), T cells (CD3), macrophage scavenger receptor-1 (MSR-1), synthetic, and contractile smooth muscle cells (SMCs) (smoothelin: SMTH, collagen I and III, myosin heavy chain: MHC, embryonic smooth muscle myosin heavy chain: SMemb) were determined at messenger RNA (mRNA) level, using SYBRGreen-based quantitative PCR and at protein level using enzyme-linked immunosorbent assay (ELISA). Expression of cathepsins B, D, L, and S at mRNA level was significantly elevated in AAA compared to control aorta (1.7-fold, p = 0.025; 2.5-fold, p = 0.002; 2.6-fold, p = 0.034; and 7.0-fold, p = 0.003). Expression of cathepsin S correlated significantly with leukocytes and macrophages (ρ = 0.398, p = 0.033 and ρ = 0.422, p = 0.020), synthetic SMCs were significantly associated with cathepsins B, D, and L (ρ = 0.522, p = 0.003; ρ = 0.431, p = 0.015 and ρ = 0.467, p = 0.008). At protein level, cathepsins B and S were elevated in AAA compared to controls (5.4-fold, p = 0.001 and 7.3-fold, p < 0.001). Significant correlations were observed between collagen I, its degraded product, and cathepsin S (r = -0.350, p = 0.034 and r = +0.504, p < 0.001). Expression of cathepsin B was associated with SMCs, expression of cathepsin S with inflammatory cells. Particularly cathepsin S was associated with the degradation product of collagen I and thus might be involved in the progression of AAA. Furthermore, cathepsin S correlated with inflammatory cells.

Suramin, Genistein and Collagen Matrix (DuraGen) for Delayed Adjustment after Strabismus Surgery: Which One is Best?

PubMed

Oktem, Caglar; Oto, Sibel; Toru, Serap; Bakar, Coskun; Ozdemir, Handan; Akova, Yonca Aydin

2016-01-01

To evaluate the efficacy and safety of suramin, genistein and collagen matrix for the prevention of inflammation, the reduction of fibrosis and the delay in adjustment after strabismus surgery on a rabbit model. By using an adjustable suture technique, a recession of the superior rectus muscle (SRM) was made in 36 eyes of 18 rabbits. Three study groups were created using genistein, suramin and collagen matrix (n = 6 per group). Two control groups utilized dimethyl sulphoxide (DMSO) (n = 6) and balanced salt solution (n = 12). The adjustments and measurements were made on days 2, 7, 14. After enucleation was done on day 21, the degree of inflammation was evaluated quantitatively in histopathological sections and immunohistochemical investigations were performed for tissue expression of cytoplasmic vascular endothelial growth factor (VEGF), MAC 387, TGF-β and bFGF. The adhesions between conjunctiva and SRM were significantly less in the collagen matrix and suramin groups (p = 0.002) and adhesions between the sclera and SRM were considerably reduced in the genistein and DMSO groups (p = 0.006) on day 7. Force exerted for adjustment was significantly less in the collagen matrix and suramin groups on day 14 (p = 0.006). Expression of b-FGF was significantly lower in the conjunctival epithelium in the suramin and genistein groups (p = 0.0001 for both). TGF-β was significantly lower (p = 0.001) in the suramin group and VEGF expression was totally absent. MAC 387 expression was lower in the genistein and suramin groups (p = 0.0001). Suramin, genistein and collagen matrix successfully reduce adhesions, and facilitate adjustment following recession surgery. Both suramin and genistein effectively suppress growth factor expression, while collagen matrix offers the longest time interval for adjustability after strabismus surgery.

Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration.

PubMed

Kaufman, Gili; Whitescarver, Ryan A; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

2018-01-24

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by ImageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within three days of incubation, fibroblast spheroids interacted with the fibers, and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment.

Effect of testosterone on the proliferation and collagen synthesis of cardiac fibroblasts induced by angiotensin II in neonatal rat

PubMed Central

Yang, Xiaocun; Wang, Ying; Yan, Shuxun; Sun, Lina; Yang, Guojie; Li, Yuan; Yu, Chaonan

2017-01-01

ABSTRACT The objective is to explore the effect of testosterone on the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts (CF) induced by Angiotensin II (Ang II) and the underlying mechanisms. Derived from neonatal rats, the CFs were divided into 4 groups: the control group, Ang II group, testosterone group, and testosterone + Ang II group in vitro. Cell cycle distribution, collagen counts, and phosphorylated extracellular signal-regulated kinase (ERK1/2) (p - ERK1/2) expression were assessed by flow cytometry, VG staining, and immunocytochemistry, respectively. The Ang II group had a much higher proportion of cells in the S-phase, higher collagen contents, and a higher p - ERK1/2 expression level than either the control or testosterone group. However, these factors were significantly reduced in the testosterone + Ang II group as compared to the Ang II group. In terms of cells in the S-phase and the collagen contents, there was not a significant difference between the testosterone group and the control. However, the protein expression of p-ERK1/2 was significantly increased in the testosterone group as compared to the control. Testosterone inhibits the proliferation and collagen synthesis of CF induced by Ang II. The underlying mechanism may involve the ERK1/2 signaling pathway. PMID:27791460

[Effect of Combined Intervention of Electroacupuncture and Astragaloside IV on Myocardial Hypertrophy and TGF-β 1/Smad Signaling in Rats with Myocardial Fibrosis].

PubMed

Li, Jia-Shen; Zhu, Xiao-Yu; Lu, Mei-Li; Gao, Jun-Hong; Wang, Hong-Xin; Yu, Xiao-Chun

2017-12-25

To observe the effect of combined intervention of electroacupuncture (EA) and astragaloside IV(ASIV) on cardiac hypertrophy and transforming growth factor β 1 (TGF-β 1)/Smad signaling in isoproterenol (ISO) induced cardiac hypertrophy rats, so as to investigate its underlying mechanisms in improving myocardial fibrosis. A total of 50 SD rats were randomly divided into 5 groups: normal control, model (ISO), Propranolol (PRO)，ASIV and EA＋ASIV groups ( n ＝10 in each group). The myocardial fibrosis model was established by intraperitoneal injection (i.p.) of ISO (10 mg·kg －1 ·d －1 ), once daily for 30 days. Rats of the control group were given normal saline (i.p.), those of the PRO group given with PRO (40 mg·kg －1 ·d －1 , gavage), and those of the ASIV and EA＋ASIV groups were treated by gavage of ASIV (40 mg·kg －1 ·d －1 ), once daily for 30 days. EA (20 Hz, 6 V) was applied to bilateral "Neiguan" (PC 6) for 10 min, once every day for 30 d. The heart mass index (HMI, whole heart weight/body weight) and left ventricular (LV) mass index (LVMI, weight of the LV/body weight) were calculated to assess the state of cardiac hypertrophy. The enzyme linked immunosorbent assay (ELISA) was used to determine the levels of procollagen I carboxy-terminal propeptide (PICP，a marker of extracellular matrix remodeling) and carboxyterminal telopeptide of type I collagen (ICTP, a metabolite of type I collagen) in serum, and Western blot was used to test protein contents of TGF- β 1, Smad 2 / 3, Smad 4, Smad 7 in the left ventricle tissue of the heart. After modeling, the HMI and LVMI, serum PICP and ICTP contents and the expression levels of myocardial TGF-β 1, Smad 2/3 and Smad 4 proteins were significantly increased in the model (ISO) group ( P <0.05), suggesting a deposition of collagen and cardiac hypertrophy, and were considerably decreased in PRO, ASIV and EA＋ASIV groups after the intervention ( P <0.05). The expression level of myocardial Smad 7 protein was significantly lower in the model group than in the normal control group ( P <0.05), and significantly up-regulated in PRO, ASIV and EA＋ASIV groups ( P <0.05). Sirius Red staining of the left ventricular myocardium showed a dense deposition of collagen and a severer myocardial fibrosis in the model group, and a relatively lighter fibrosis in the PRO, ASIV and EA＋ASIV groups. The therapeutic effects of EA＋ASIV were comparable to those of PRO, and were significantly superior to those of ASIV in down-regulating HMI, serum ICTP, and myocardial Smad 2/3 and Smad 4 expression and up-regulating Smad 7 protein ( P <0.05). There were no significant differences among the PRO, ASIV and EA＋ASIV groups in LVMI, PICP and TGF-β 1 levels, and between the PRO and EA＋ ASIV groups in HMI, ICTP, Smad 2/3, Smad 4 and Smad 7 levels ( P > 0.05). EA stimulation of PC 6 combined with ASIV can relieve cardiac hypertrophy and myocardial fibrosis in rats, which may be associated with its effects in regulating myocardial TGF-β 1/Smad signaling pathway.

The triple helix of collagens - an ancient protein structure that enabled animal multicellularity and tissue evolution.

PubMed

Fidler, Aaron L; Boudko, Sergei P; Rokas, Antonis; Hudson, Billy G

2018-04-09

The cellular microenvironment, characterized by an extracellular matrix (ECM), played an essential role in the transition from unicellularity to multicellularity in animals (metazoans), and in the subsequent evolution of diverse animal tissues and organs. A major ECM component are members of the collagen superfamily -comprising 28 types in vertebrates - that exist in diverse supramolecular assemblies ranging from networks to fibrils. Each assembly is characterized by a hallmark feature, a protein structure called a triple helix. A current gap in knowledge is understanding the mechanisms of how the triple helix encodes and utilizes information in building scaffolds on the outside of cells. Type IV collagen, recently revealed as the evolutionarily most ancient member of the collagen superfamily, serves as an archetype for a fresh view of fundamental structural features of a triple helix that underlie the diversity of biological activities of collagens. In this Opinion, we argue that the triple helix is a protein structure of fundamental importance in building the extracellular matrix, which enabled animal multicellularity and tissue evolution. © 2018. Published by The Company of Biologists Ltd.

PDL Progenitor-Mediated PDL Recovery Contributes to Orthodontic Relapse.

PubMed

Feng, L; Yang, R; Liu, D; Wang, X; Song, Y; Cao, H; He, D; Gan, Y; Kou, X; Zhou, Y

2016-08-01

Periodontal ligament (PDL) is subjected to mechanical force during physiologic activities. PDL stem /: progenitor cells are the main mesenchymal stem cells in PDL. However, how PDL progenitors participate in PDL homeostasis upon and after mechanical force is largely unknown. In this study, force-triggered orthodontic tooth movement and the following relapse were used as models to demonstrate the response of PDL progenitors and their role in PDL remodeling upon and after mechanical force. Upon orthodontic force, PDL collagen on the compression side significantly degraded, showing a broken and disorganized pattern. After force withdrawal, the degraded PDL collagen recovered during the early stage of relapse. Correspondingly, increased CD90(+) PDL progenitors with suppressed expression of type I collagen (Col-I) were observed upon orthodontic force, whereas these cells accumulated at the degradation regions and regained Col-I expression after force withdrawal during early relapse. Our results further showed that compressive force altered cell morphology and repressed collagen expression in cultured PDL progenitors, which both recovered after force withdrawal. Force withdrawal-induced recovery of collagen expression in cultured PDL progenitors could be regulated by transforming growth factor-β (TGF-β), a key molecule for tissue homeostasis and extracellular matrix remodeling. More interesting, inhibiting the regained Col-I expression in CD90(+) PDL progenitors by blocking TGF-β interrupted PDL collagen recovery and partially inhibited the early relapse. These data suggest that PDL progenitors can respond to mechanical force and may process intrinsic stability to recover to original status after force withdrawal. PDL progenitors with intrinsic stability are required for PDL recovery and consequently contribute to early orthodontic relapse, which can be regulated by TGF-β signaling. © International & American Associations for Dental Research 2016.

Transforming growth factor-β-mediated CD44/STAT3 signaling contributes to the development of atrial fibrosis and fibrillation.

PubMed

Chang, Shang-Hung; Yeh, Yung-Hsin; Lee, Jia-Lin; Hsu, Yu-Juei; Kuo, Chi-Tai; Chen, Wei-Jan

2017-09-04

Atrial fibrillation (AF) is associated with atrial fibrosis. Inhibition of atrial fibrosis might be a plausible approach for AF prevention and therapy. This study is designed to evaluate the potential role of CD44, a membrane receptor known to regulate fibrosis, and its related signaling in the pathogenesis of atrial fibrosis and AF. Treatment of cultured rat atrial fibroblasts with transforming growth factor-β (TGF-β, a key mediator of atrial fibrosis) led to a higher expression of hyaluronan (HA), CD44, STAT3, and collagen (a principal marker of fibrosis) than that of ventricular fibroblasts. In vivo, TGF-β transgenic mice and AF patients exhibited a greater expression of HA, CD44, STAT3, and collagen in their atria than wild-type mice and sinus rhythm subjects, respectively. Treating TGF-β transgenic mice with an anti-CD44 blocking antibody resulted in a lower expression of STAT3 and collagen in their atria than those with control IgG antibody. Programmed stimulation triggered less AF episodes in TGF-β transgenic mice treated with anti-CD44 blocking antibody than in those with control IgG. Blocking CD44 signaling with anti-CD44 antibody and mutated CD44 plasmids attenuated TGF-β-induced STAT3 activation and collagen expression in cultured atrial fibroblasts. Deletion and mutational analysis of the collagen promoter along with chromatin immunoprecipitation demonstrated that STAT3 served as a vital transcription factor in collagen expression. TGF-β-mediated HA/CD44/STAT3 pathway plays a crucial role in the development of atrial fibrosis and AF. Blocking CD44-dependent signaling may be a feasible way for AF management.

Integrin distributions in renal cell carcinomas of various grades of malignancy.

PubMed Central

Korhonen, M.; Laitinen, L.; Ylänne, J.; Koukoulis, G. K.; Quaranta, V.; Juusela, H.; Gould, V. E.; Virtanen, I.

1992-01-01

We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1443050

Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

PubMed

Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

2000-01-01

The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

PubMed Central

1996-01-01

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

Cordyceps cicadae extracts ameliorate renal malfunction in a remnant kidney model*

PubMed Central

Zhu, Rong; Chen, Yi-ping; Deng, Yue-yi; Zheng, Rong; Zhong, Yi-fei; Wang, Lin; Du, Lan-ping

2011-01-01

Background and Objectives: Chronic kidney disease (CKD) is a growing public health problem with an urgent need for new pharmacological agents. Cordyceps cicadae is widely used in traditional Chinese medicine (TCM) and has potential renoprotective benefits. The current study aimed to determine any scientific evidence to support its clinical use. Methods: We analyzed the potential of two kinds of C. cicadae extract, total extract (TE) and acetic ether extract (AE), in treating kidney disease simulated by a subtotal nephrectomy (SNx) model. Sprague-Dawley rats were divided randomly into seven groups: sham-operated group, vehicle-treated SNx, Cozaar, 2 g/(kg∙d) TE SNx, 1 g/(kg∙d) TE SNx, 92 mg/(kg∙d) AE SNx, and 46 mg/(kg∙d) AE SNx. Renal injury was monitored using urine and serum analyses, and hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) stainings were used to analyze the level of fibrosis. The expression of type IV collagen (Col IV), fibronectin (FN), transforming growth factor-β1 (TGF-β1), and connective tissue growth factor (CTGF) was detected by immunohistochemistry. Results: Renal injury, reflected in urine and serum analyses, and pathological changes induced by SNx were attenuated by TE and AE intervention. The depositions of Col IV and FN were also decreased by the treatments and were accompanied by reduced expression of TGF-β1 and CTGF. In some respects, 2 g/(kg∙d) of TE produced better effects than Cozaar. Conclusions: For the first time, we have shown that C. cicadae may inhibit renal fibrosis in vivo through the TGF-β1/CTGF pathway. Therefore, we conclude that the use of C. cicadae could provide a rational strategy for combating renal fibrosis. PMID:22135152

Glomerular pathology in Alport syndrome: a molecular perspective

PubMed Central

Cosgrove, Dominic

2012-01-01

We have known for some time that mutations in the genes encoding 3 of the 6 type IV collagen chains are the underlying defect responsible for both X-linked (where the COL4A5 gene is involved) and autosomal (where either COL4A3 or COL4A4 genes are involved) Alport syndrome. The result of these mutations is the absence of the sub-epithelial network of all three chains in the glomerular basement membrane (GBM) resulting, at maturity, in a type IV collagen GBM network comprised of only α1(IV) and α2(IV) chains. The altered GBM functions adequately in early life. Eventually there is onset of proteinuria associated with the classic and progressive irregular thickening, thinning, and splitting of the GBM, which culminates in end stage renal failure. We have learned much about the molecular events associated with disease onset and progression through the study of animal models for Alport syndrome, and have identified some potential therapeutic approaches that may serve to delay the onset or slow the progression of the disease. This review focuses on where we are in our understanding of the disease, where we need to go to understand the molecular triggers that set the process in motion, and what emergent therapeutic approaches show promise for ameliorating disease progression in the clinic. PMID:21455721

A new method for meniscus repair using type I collagen scaffold and infrapatellar fat pad.

PubMed

Oda, Shuhei; Otsuki, Shuhei; Kurokawa, Yoshitaka; Hoshiyama, Yoshiaki; Nakajima, Mikio; Neo, Masashi

2015-05-01

The aim of this study was to investigate a new method for meniscal repair by combinative transplantation with type I collagen scaffold and infrapatellar fat pad. Two-mm cylindrical defects at the anterior part of bilateral medial menisci were prepared in nine Japanese white rabbits. The 18 knees were equally divided into three groups: I, no treatment; II, collagen scaffold transplantation; and III, collagen scaffold and infrapatellar fat pad transplantation. Another three rabbits (six knees) underwent sham surgery and served as controls. Rabbits were sacrificed at eight weeks after transplantation. Surface area of the medial meniscus was evaluated using macrophotographs. Ishida score for meniscal regeneration was used for assessment. To evaluate the composition of regenerated tissue, immunohistochemistry was analyzed with anti-type I and anti-type II collagen antibodies, and anti-Ki67 antibody. To investigate the effects of collagen scaffold on human meniscus, cells were isolated from human meniscus and infrapatellar fat pad, and cultured with collagen scaffold for three weeks. After that, gene expression was evaluated by using quantitative real-time polymerase chain reaction. In group I, the meniscus shrank anterior to posterior, and the surface area was significantly less than that of normal meniscus. However, the surface area was maintained in group III. Ishida score and Ki67-positive cell ratio in group III were significantly higher than that in any other group, and staining with type I and type II collagen was similar to that of the control. Expression of matrix metalloproteinase was significantly lower in cocultures of collagen scaffold, meniscus cell, and infrapatellar fat pad cell than in monocultured meniscus cell, and expression of interleukin-1β was not increased. This new method for meniscal repair by combinative transplantation with type I collagen scaffold and infrapatellar fat pad showed meniscal regeneration and potential for suppressing inflammation. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

[The effect of calcitonin gene-related peptide on collagen accumulation in pulmonary arteries of rats with hypoxic pulmonary arterial hypertension].

PubMed

Li, Xian-Wei; Du, Jie; Li, Yuan-Jian

2013-03-01

To observe the effect of calcitonin gene-related peptide (CGRP) on pulmonary vascular collagen accumulation in hypoxia rats in order to study the effect of CGRP on hypoxic pulmonary vascular structural remodeling and its possible mechanism. Rats were acclimated for 1 week, and then were randomly divided into three groups: normoxia group, hypoxia group, and hypoxia plus capsaicin group. Pulmonary arterial hypertension was induced by hypoxia in rats. Hypoxia plus capsaicin group, rats were given capsaicin (50 mg/(kg x d), s.c) 4 days before hypoxia to deplete endogenous CGRP. Hypoxia (3% O2) stimulated proliferation of pulmonary arterial smooth muscle cells (PASMCs) and proliferation was measured by BrdU marking. The expression levels of CGRP, phosphorylated ERK1/2 (p-ERK1/ 2), collagen I and collagen III were detected by real-time PCR or Western blot. Right ventricle systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) of pulmonary arterial hypertension (PAH) rats induced by hypoxia were higher than those of normoxia rats. By HE and Masson staining, it was demonstrated that hypoxia also significantly induced hypertrophy of pulmonary arteries and increased level of collagen accumulation. Hypoxia dramatically decreased the CGRP level and increased the expression of p-ERK1/2, collagen I, collagen III in pulmonary arteries. All these effects of hypoxia were further aggravated by pre-treatment of rats with capsaicin. CGRP concentration-dependently inhibited hypoxia-induced proliferation of PASMCs, markedly decreased the expression of p-ERK1/2, collagen I and collagen III. All these effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that CGRP might inhibit hypoxia-induced PAH and pulmonary vascular remodeling, through inhibiting phosphorylation of ERK1/2 and alleviating the collagen accumulation of pulmonary arteries.

Hemodynamic and renal implications of sodium-glucose cotransporter- 2 inhibitors in type 2 diabetes mellitus.

PubMed

Tejedor Jorge, Alberto

2016-11-01

In DM2, there is increased expression of the proximal glucose transporter SGLT2. The increased glucose reabsorption from the urine to the proximal tubule and subsequently to the bloodstream, has three direct effects on the prognosis of patients with DM2: a) it increases the daily glucose load by raising the renal threshold for glucose, thus augmenting requirements for oral antidiabetics and insulin. This progressive increase occurs throughout the course of the disease and in parallel with the increase in renal mass (renal hypertrophy); b) because of the greater glucose reabsorption, glycosuria is lower than the level corresponding to glycaemia, decreasing the stimulus on the tubuloglomerular feedback system of the distal nephron. As a result, the glomerular vasodilation caused by hyperglycaemia is not arrested, maintaining glomerular hyperfiltration, and c) the excess glucose transported to the proximal tubular cells modifies their redox status, increasing local production of glycosylating products and activating local production of proinflammatory and profibrotic proliferative mediators. These mediators are responsible for the direct free radical damage to proximal tubular cells, for increased SGLT2 expression, increased production of collagen IV and extracellular matrix, and activation of monocyte/macrophages able to cause endothelial injury. The use of SGLT2 inhibitors not only reduces the reabsorption of glucose from the glomerular filtrate back into the circulationthus improving metabolic control in diabetesbut also restores tubuloglomerular feedback by increasing glycosuria and distal urinary flow. However, the most notable effect is due to inhibition of glucose entry to the proximal tubular cells. Glycosuria is toxic to the kidney: it harms glucosetransporting cells, that is, the proximal cells, which contain SGLT2. In animal models, SGLT2 inhibition reduces local production of oxygen-free radicals, the formation of mesangial matrix and collagen IV, glomerular infiltration by inflammatory cells and monocyte/macrophage-dependent arteriosclerosis. In humans, SGLT2 have a demonstrated ability to reduce renal injury and cardiovascular risk in patients with type 2 diabetes mellitus. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion

PubMed Central

Dong, Shengzhang; Balaraman, Velmurugan; Kantor, Asher M.; Lin, Jingyi; Grant, DeAna G.; Held, Nicole L.

2017-01-01

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24–32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus. PMID:28961239

2-(2-Benzofuranyl)-2-Imidazoline Mediates Neuroprotection by Regulating the Neurovascular Unit Integrity in a Rat Model of Focal Cerebral Ischemia.

PubMed

Zhang, Zheng; Zhang, Linlei; Chen, Jiaou; Cao, Yungang; Qu, Man; Lin, Xinda; Han, Zhao; Ji, Xunming

2018-06-01

We showed previously that 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a ligand to type 2 imidazoline receptor (I2R) exerts neuroprotective effects in ischemia stroke via an unknown mechanism. The present study was to investigate whether 2-BFI can protect the neurovascular unit (NVU) using a rat model of 90 min focal cerebral ischemia. Rats were randomly divided into three groups: thesham-operated group; the vehicle control group and the 2-BFI group which received 2-BFI (3 mg/kg) immediately after the start of middle cerebralartery occlusion (MCAO). Neurological deficit score, infarct size, apoptosis level, brain water content and Evans Blue extravasation were assessed at 24 h after stroke. Expressions of occludin and zonula occludens 1 (ZO-1), collagen IV, aquaporin-4 (AQP-4), matrix metalloproteinase-9 (MMP-9) and MMP-2 were assessed by Western blotting. 2-BFI treatment was associated with significant improvement of neurological performance and decreased infarct volume at 24 h after stroke. Apoptosis level reduced significantly by 2-BFI compared to the vehicle group (34.3 ± 5.4% vs 56.1 ± 7.9%, p < 0.05). Significant decreased of brain water content (79.5 ± 2.6% vs 84.62 ± 2%, p < 0.05) and Evans Blue extravasation (1.2 ± 0.5 vs 2.5 ± 0.41 µg/g, p < 0.05) of ipsilateral hemisphere was observed in 2-BFI group compared to vehicle group. Expressions of occludin, ZO-1 and collagen IV were significantly higher while MMP-9 level significantly lower in 2-BFI group. AQP-4 and MMP-2 showed no difference between 2-BFI and the vehicle groups. These results suggest that the neuroprotective effects of 2-BFI in acute ischemic brain damage are at least partly due to the drug's ability to improve the functions of NVU. Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Splicing defect in FKBP10 gene causes autosomal recessive osteogenesis imperfecta disease: a case report.

PubMed

Maghami, Fatemeh; Tabei, Seyed Mohammad Bagher; Moravej, Hossein; Dastsooz, Hassan; Modarresi, Farzaneh; Silawi, Mohammad; Faghihi, Mohammad Ali

2018-05-25

Osteogenesis imperfecta (OI) is a group of connective tissue disorder caused by mutations of genes involved in the production of collagen and its supporting proteins. Although the majority of reported OI variants are in COL1A1 and COL1A2 genes, recent reports have shown problems in other non-collagenous genes involved in the post translational modifications, folding and transport, transcription and proliferation of osteoblasts, bone mineralization, and cell signaling. Up to now, 17 types of OI have been reported in which types I to IV are the most frequent cases with autosomal dominant pattern of inheritance. Here we report an 8- year- old boy with OI who has had multiple fractures since birth and now he is wheelchair-dependent. To identify genetic cause of OI in our patient, whole exome sequencing (WES) was carried out and it revealed a novel deleterious homozygote splice acceptor site mutation (c.1257-2A > G, IVS7-2A > G) in FKBP10 gene in the patient. Then, the identified mutation was confirmed using Sanger sequencing in the proband as homozygous and in his parents as heterozygous, indicating its autosomal recessive pattern of inheritance. In addition, we performed RT-PCR on RNA transcripts originated from skin fibroblast of the proband to analyze the functional effect of the mutation on splicing pattern of FKBP10 gene and it showed skipping of the exon 8 of this gene. Moreover, Real-Time PCR was carried out to quantify the expression level of FKBP10 in the proband and his family members in which it revealed nearly the full decrease in the level of FKBP10 expression in the proband and around 75% decrease in its level in the carriers of the mutation, strongly suggesting the pathogenicity of the mutation. Our study identified, for the first time, a private pathogenic splice site mutation in FKBP10 gene and further prove the involvement of this gene in the rare cases of autosomal recessive OI type XI with distinguished clinical manifestations.

Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

PubMed

Mori, Yu; Adams, Douglas; Hagiwara, Yusuke; Yoshida, Ryu; Kamimura, Masayuki; Itoi, Eiji; Rowe, David W

2016-11-01

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

Glycosaminoglycans of abdominal skin after massive weight loss in post-bariatric female patients.

PubMed

Veiga, Daniela Francescato; Bussolaro, Rodolpho A; Kobayashi, Elsa Y; Medeiros, Valquíria P; Martins, João R M; Garcia, Elvio B; Novo, Neil F; Nader, Helena B; Ferreira, Lydia M

2011-06-01

The number of post-bariatric patients had a significant increase over the last years, and a better understanding of the consequences of massive weight loss on skin is imperative. Despite weight-loss-related changes in collagen and elastin have been reported, less is known about changes in another of the matrix components of the skin, the glycosaminoglycans. The objective of this study is to evaluate abdominal skin glycosaminoglycans concentrations and perlecan and collagen III expression in post-bariatric female patients. Skin tissue samples from the abdomen of lean (n = 19) and post-bariatric (n = 24) female patients were compared. Sulfated glycosaminoglycans and hyaluronic acid were extracted, characterized and quantified. Perlecan and collagen III expression was assessed by immunofluorescence. The major glycosaminoglycans found were dermatan sultafe and hyaluronic acid; the others were found in smaller amounts. The skin of the post-bariatric patients had lower concentrations of heparan sulfate (p = 0.002) while hyaluronic acid, dermatan sulfate, and chondroitin sulfate concentrations were similar to the lean women's skin. Post-bariatric skin showed decreased expression of perlecan and increased expression of collagen III. No correlation was found among glycosaminoglycans concentrations and age, body mass index, frequency of pregnancies, or skin types, but it was observed in higher skin heparan sulfate concentrations in post-bariatric patients who had their weights stabilized for over than 24 months (p = 0.000). Abdominal skin of post-bariatric women presented decreased heparan sulfate concentrations and perlecan expression and increased expression of collagen III.

Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

PubMed

Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

2016-07-01

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. © 2015 Wiley Periodicals, Inc.

Periostin in Mature Stage Localized Scleroderma.

PubMed

Kim, Min-Woo; Park, Jung Tae; Kim, Jung Ho; Koh, Seong-Joon; Yoon, Hyun-Sun; Cho, Soyun; Park, Hyun-Sun

2017-06-01

Periostin is a novel matricellular protein expressed in many tissues, including bone, periodontal ligament, and skin. Although its expression is prominent in various fibrotic conditions, studies of periostin in localized scleroderma are rare. To investigate the expression of periostin and other molecules in localized scleroderma. A retrospective study of 14 patients with confirmed mature stage localized scleroderma was undertaken. Fourteen age-matched and biopsy site-matched subjects with normal skin were included as controls. Collagen fiber deposition, periostin, procollagen, transforming growth factor-β, and matrix metalloproteinase (MMP)-1 expression were assessed and compared between the two groups. Co-localization of α-smooth muscle actin and periostin was evaluated using confocal microscopy. Periostin was predominantly expressed along the dermo-epidermal junction in the controls. Conversely, patients with localized scleroderma demonstrated increased collagen fiber deposition and periostin expression that was more widely distributed along the entire dermis. MMP-1 staining showed increased expression in the epidermis and dermis of patients compared to scanty expression in the controls. A semi-quantitative evaluation showed a higher proportion of excessive collagen bundle deposition (57.1% vs. 7.1%, p =0.013), diffuse periostin positivity (42.9% vs. 0%, p =0.016), and moderate MMP-1 positivity (71.4% vs. 7.1%, p =0.001) in patients than in the controls. Compared to the controls, patients with localized scleroderma had enhanced periostin expression corresponding to increased collagen fiber deposition and unexpected overexpression of MMP-1. The results of this human in vivo study may implicate the pathogenesis of localized scleroderma.

Defective Expression and Function of the Leukocyte Associated Ig-like Receptor 1 in B Lymphocytes from Systemic Lupus Erythematosus Patients

PubMed Central

Colombo, Barbara M.; Canevali, Paolo; Magnani, Ottavia; Rossi, Edoardo; Puppo, Francesco; Zocchi, Maria Raffaella; Poggi, Alessandro

2012-01-01

Systemic lupus erythematosus (SLE) is characterized by the production of a wide array of autoantibodies and dysregulation of B cell function. The leukocyte associated Immunoglobulin (Ig)-like receptor (LAIR)1 is a transmembrane molecule belonging to Ig superfamily which binds to different types of collagen. Herein, we have determined the expression and function of LAIR1 on B lymphocyte from SLE patients. LAIR1 expression in peripheral blood B lymphocytes from 54 SLE, 24 mixed connective tissue disease (MCTD), 20 systemic sclerosis (SSc) patients, 14 rheumatoid arthritis (RA) and 40 sex and age matched healthy donors (HD) have been analyzed by immunofluorescence. The effect of LAIR1 ligation by specific monoclonal antibodies, collagen or collagen producing mesenchymal stromal cells from reactive lymph nodes or bone marrow on Ig production by pokeweed mitogen and B cell receptor (BCR)-mediated NF-kB activation was assessed by ELISA and TransAM assay. The percentage of CD20+ B lymphocytes lacking or showing reduced expression of LAIR1 was markedly increased in SLE and MCTD but not in SSc or RA patients compared to HD. The downregulation of LAIR1 expression was not dependent on corticosteroid therapy. Interestingly, LAIR1 engagement by collagen or collagen-producing mesenchymal stromal cells in SLE patients with low LAIR1 expression on B cells delivered a lower inhibiting signal on Ig production. In addition, NF-kB p65 subunit activation upon BCR and LAIR1 co-engagement was less inhibited in SLE patients than in HD. Our findings indicate defective LAIR1 expression and function in SLE B lymphocytes, possible contributing to an altered control of B lymphocytes behavior. PMID:22355402

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

PubMed

Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

2004-11-01

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

Juvenile porcine temporomandibular joint: Three different cartilaginous structures?

PubMed

Tabeian, Hessam; Bakker, Astrid D; de Vries, Teun J; Zandieh-Doulabi, Behrouz; Lobbezoo, Frank; Everts, Vincent

2016-12-01

The temporomandibular joint (TMJ) consists of three cartilaginous structures: the fossa, disc, and condyle. In juvenile idiopathic arthritis (JIA), inflammation of the TMJ leads to destruction of the condyle, but not of the fossa or the disc. Such a different effect of inflammation might be related to differences in matrix composition of the cartilaginous structures. The matrix composition of the three TMJ structures was analyzed in juvenile porcine samples and in an in vitro system of cells isolated from each anatomical structure embedded in 3% agarose gels. The matrix of all three anatomical structures of the TMJ contained collagen type I and its gene expression was maintained after isolation. The condyle and the fossa stained positive for collagen type II and proteoglycans, but the condyle contained considerably more collagen type II and proteoglycans than the fossa. The disc contained neither collagen type II protein nor expression of its gene, and the disc did not stain positive for proteoglycans. Aggrecan gene expression was lower in the disc compared to condyle and fossa cell-isolates. In general, the cell-isolates in vitro closely mimicked the characteristic features found in the tissue. The collagen type II content of the condyle clearly distinguished this cartilaginous structure from the disc and fossa. Since autoimmunity against collagen type II is associated with JIA, the relatively abundant presence of this type of collagen in the condyle might provide an explanation why primarily this cartilaginous structure of the TMJ is affected in JIA patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

A collagen α2(I) mutation impairs healing after experimental myocardial infarction.

PubMed

Hofmann, Ulrich; Bonz, Andreas; Frantz, Stefan; Hu, Kai; Waller, Christiane; Roemer, Katrin; Wolf, Jürgen; Gattenlöhner, Stefan; Bauersachs, Johann; Ertl, Georg

2012-01-01

Collagen breakdown and de novo synthesis are important processes during early wound healing after myocardial infarction (MI). We tested the hypothesis that collagen I, the main constituent of the extracellular matrix, affects wound healing after MI. The osteogenesis imperfecta mouse (OIM), lacking procollagen-α2(I) expression, represents a model of the type III form of the disease in humans. Homozygous (OIM/OIM), heterozygous (OIM/WT), and wild-type (WT/WT) mice were subjected to a permanent myocardial infarction protocol or sham surgery. Baseline functional and geometrical parameters determined by echocardiography did not differ between genotypes. After MI but not after sham surgery, OIM/OIM animals exhibited significantly increased mortality, due to early ventricular rupture between day 3 and 7. Echocardiography at day 1 demonstrated increased left ventricular dilation in OIM/OIM animals. Less collagen I mRNA within the infarct area was found in OIM/OIM animals. At 2 days after MI, MMP-9 expression in the infarct border zone was higher in OIM/OIM than in WT/WT animals. Increased granulocyte infiltration into the infarct border zone occurred in OIM/OIM animals. Neither granulocyte depletion nor MMP inhibition reduced mortality in OIM/OIM animals. In this murine model, deficiency of collagen I leads to a myocardial wound-healing defect. Both structural alterations within pre-existing collagen matrix and impaired collagen de novo expression contribute to a high rate of early myocardial rupture after MI. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Spatiotemporal variations in gene expression, histology and biomechanics in an ovine model of tendinopathy

PubMed Central

Blaker, Carina; Clarke, Elizabeth; Jeffcott, Leo; Little, Christopher

2017-01-01

Flexor tendinopathy is a common problem affecting humans and animals. Tendon healing is poorly understood and the outcomes of conservative and surgical management are often suboptimal. While often considered a localized injury, recent evidence indicates that in the short term, tendinopathic changes are distributed widely throughout the tendon, remote from the lesion itself. Whether these changes persist throughout healing is unknown. The aim of this study was to document gene expression, histopathological and biomechanical changes that occur throughout the superficial digital flexor tendon (SDFT) up to 16 weeks post-injury, using an ovine surgical model of tendinopathy. Partial tendon transection was associated with decreased gene expression for aggrecan, decorin, fibromodulin, tissue inhibitors of metalloproteinases (TIMPS 1, 2 and 3), collagen I and collagen II. Gene expression for collagen III, lumican and matrix metalloproteinase 13 (MMP13) increased locally around the lesion site. Expression of collagen III and MMP13 decreased with time, but compared to controls, collagen III, MMP13 and lumican expression remained regionally high throughout the study. An increase in TIMP3 was observed over time. Histologically, operated tendons had higher pathology scores than controls, especially around the injured region. A chondroid phenotype was observed with increased cellular rounding and marked proteoglycan accumulation which only partially improved with time. Biomechanically, partial tendon transection resulted in a localized decrease in elastic modulus (in compression) but only at 8 weeks postoperatively. This study improves our understanding of tendon healing, demonstrating an early ‘peak’ in pathology characterized by altered gene expression and notable histopathological changes. Many of these pathological changes become more localized to the region of injury during healing. Collagen III and MMP13 expression levels remained high close to the lesion throughout the study and may reflect the production of tendon tissue with suboptimal biomechanical properties. Further studies evaluating the long-term response of tendon to injury (6–12 months) are warranted to provide additional information on tendon healing and provide further understanding of the mechanisms underlying the pathology observed in this study. PMID:29023489

Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes.

PubMed

Lin, Guiting; Qiu, Xuefeng; Fandel, Thomas M; Albersen, Maarten; Wang, Zhong; Lue, Tom F; Lin, Ching-Shwun

2011-10-01

To investigate whether fluorochrome-conjugated phalloidin can delineate cavernous smooth muscle (CSM) cells and whether it can be combined with immunofluorescence (IF) staining to quantify erectile dysfunction (ED)-associated changes. ED was induced by cavernous nerve crush in rats. Penile tissues of control and ED rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen (RECA), CD31, neuronal nitric oxide synthase (nNOS), and collagen-IV (Col-IV). Phalloidin was able to delineate CSM as composed of a circular and a longitudinal compartment. When combined with IF stain for CD31 or RECA, it helped the identification of the helicine arteries as covered by endothelial cells on both sides of the smooth muscle layer. When combined with IF stain for nNOS, it helped the identification that nNOS-positive nerves were primarily localized within the dorsal nerves and in the adventitia of dorsal arteries. When combined with IF stain for Col-IV, it helped identify that Col-IV was localized around smooth muscles and beneath the endothelium. Phalloidin also facilitated the quantitative analysis of ED-related changes in the penis. In rats with cavernous nerve injury, RECA or Col-IV expression did not change significantly, but CSM and nNOS nerve contents decreased significantly. Phalloidin stain improved penile histology, enabling the visualization of the circular and longitudinal compartments in the CSM. It also worked synergistically with IF stain, permitting the visualization of the dual endothelial covering in helicine arteries, and facilitating the quantification of ED-related histologic changes. Copyright © 2011 Elsevier Inc. All rights reserved.

The Mice Drawer System (MDS) Tissue Sharing Programme: effect of space conditions on skin metabolic activity and vascularization and potential impact of radiations in mice.

NASA Astrophysics Data System (ADS)

Nusgens, Betty; Lambert, Charles; Liu, Yi; Cancedda, Ranieri; Tavella, Sara; Ruggiu, Alessandra; Colige, Alain

Our aim was to investigate the effect of prolonged microgravity on skin physiology in mice and to identify potentially altered metabolic and vascular parameters. Osteoporosis-induced microgravity is a well documented space flight problem in Man and animals.We have previously demonstrated that fibroblasts from the dermis also sense and react to mechanical forces issued from the surrounding extracellular matrix. Conversely, fibroblasts are able to remodel their environment by a dynamic process of synthesis and degradation. Fibroblasts and cells of the vascular network also react to soluble mediators secreted by the keratinocytes such as IL-1, IL-6, VEGF, .... Skin fibroblasts upon relaxation of mechanical tension adopt a catabolic phenotype and produce significant amount of IL-1 and IL-6, of various matrix metalloproteinases (MMP) while the collagen synthesis is reduced. In microgravity, we have shown that the expression of MMP-1 and IL-6 by dermal fibroblasts is increased indicating that cell might interpret micro-gravity as a mechanical relaxation. This concept is supported by a reduction of the actin stress fibers and focal adhesions in fibroblasts in microgravity. Altogether, microgravity might alter metabolic equilibrium of the skin and perhaps of other soft connective tissues such as tendons or interstitial matrix of internal organs. The Mice Drawer System (MDS) is an Italian Space Agency facility which is able to support mice onboard the International Space Station during long-duration exploration missions by providing living space, food, water, ventilation and light-ing. Details on the mission that took place from August 28, 2009 to November 27, 2009, can be found at the URL indicated below. A similar ground control mission has been performed using the same wild type and transgenic mice overexpressing OSF-1. Upon receiving mice at KSC, the team has collected pieces of skin that were handled for histological, transcriptomic and biochemical analyses. The morphological and immunochemical analysis aim at investigating dermal and epidermal thickness, number and surface of blood vessels (CD31, VW factor, type IV collagen) and lymphatics (D2-40, Lyve), score of acanthosis and papillomatosis, proliferation index (Ki67), epidermal markers of differentiation and FXIIIa positive dendritic cells. Another fragment of skin is used for measuring water content, total collagen content (hydroxyproline) and total proteoglycans content (uronic acid). A differential extraction procedure allows to quantify the newly synthetized collagen and the progressively more heavily cross-linked colla-gen, the pattern of extraction reflecting the turn-over rate of collagen. Quantitative RT-PCR procedure is used to evaluate the expression of extracellular matrix components (fibrillar and FACIT collagens), the enzymes involved in their postranslational modifications (ADAMTS-2, -3, -14, BMP1, prolylhydroxylase, lysyloxydase), the MMPs and their physiologic activators and inhibitors, proteoglycans of the hyalectans family and the SLRP, hyaluronansynthase, ag-grecanases of the ADAMTS family, and vascular markers (CD31, VEGF-A, -C, -D, VEGF-R1, -R2, -R3, PlGF, NRP1 2, SEMA3). A special attention will be paid to the splice variants of VEGF-A. We recently discovered a new splice variant, VEGF111, lacking exons 5, 6 and 7, bio-logically active and resistant to proteolysis that is specifically induced by genotoxic agents such as radiations. Its expression might reflect an impact of radiations during the long stay of the animals in space environment. Additionally, a genome-wide analysis of gene expression will be performed using DNA microarrays (Affimetrix). Partial results of the analyses under way will be presented. http://www.nasa.gov/missionp ages/station/science/experiments/M DS.html

p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase.

PubMed

Teodoro, Jose G; Parker, Albert E; Zhu, Xiaochun; Green, Michael R

2006-08-18

Recent evidence suggests that antiangiogenic therapy is sensitive to p53 status in tumors, implicating a role for p53 in the regulation of angiogenesis. Here we show that p53 transcriptionally activates the alpha(II) collagen prolyl-4-hydroxylase [alpha(II)PH] gene, resulting in the extracellular release of antiangiogenic fragments of collagen type 4 and 18. Conditioned media from cells ectopically expressing either p53 or alpha(II)PH selectively inhibited growth of primary human endothelial cells. When expressed intracellularly or exogenously delivered, alpha(II)PH significantly inhibited tumor growth in mice. Our results reveal a genetic and biochemical linkage between the p53 tumor suppressor pathway and the synthesis of antiangiogenic collagen fragments.

A new class of animal collagen masquerading as an insect silk

PubMed Central

Sutherland, Tara D.; Peng, Yong Y.; Trueman, Holly E.; Weisman, Sarah; Okada, Shoko; Walker, Andrew A.; Sriskantha, Alagacone; White, Jacinta F.; Huson, Mickey G.; Werkmeister, Jerome A.; Glattauer, Veronica; Stoichevska, Violet; Mudie, Stephen T.; Haritos, Victoria S.; Ramshaw, John A. M.

2013-01-01

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials. PMID:24091725

Tissue distribution and developmental expression of type XVI collagen in the mouse.

PubMed

Lai, C H; Chu, M L

1996-04-01

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

PubMed

Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

2012-06-29

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

PubMed Central

Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

2012-01-01

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

Polymerized-type I collagen downregulates inflammation and improves clinical outcomes in patients with symptomatic knee osteoarthritis following arthroscopic lavage: a randomized, double-blind, and placebo-controlled clinical trial.

PubMed

Furuzawa-Carballeda, Janette; Lima, Guadalupe; Llorente, Luis; Nuñez-Álvarez, Carlos; Ruiz-Ordaz, Blanca H; Echevarría-Zuno, Santiago; Hernández-Cuevas, Virgilio

2012-01-01

Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA.

Histochemical and Immunohistochemical Study of α-SMA, Collagen, and PCNA in Epithelial Ovarian Neoplasm

PubMed

Anggorowati, Nungki; Ratna Kurniasari, Chatarina; Damayanti, Karina; Cahyanti, Titik; Widodo, Irianiwati; Ghozali, Ahmad; Romi, Muhammad Mansyur; Sari, Dwi Cahyani Ratna; Arfian, Nur

2017-03-01

Background: Alpha-smooth muscle actin (α-SMA) is an isoform of actin, positive in myofibroblasts and is an epithelial to mesenchymal transition (EMT) marker. EMT is a process by which tumor cells develop to be more hostile and able to metastasize. Progression of tumor cells is always followed by cell composition and extracellular matrix component alteration. Increased α-SMA expression and collagen alteration may predict the progressivity of ovarian neoplasms. Objective: The aim of this research was to analyse the characteristic of α-SMA and collagen in tumor cells and stroma of ovarian neoplasms. In this study, PCNA (proliferating cell nuclear antigen) expression was also investigated. Methods: Thirty samples were collected including serous, mucinous, endometrioid, and clear cell subtypes. The expression of α-SMA and PCNA were calculated in cells and stroma of ovarian tumors. Collagen was detected using Sirius Red staining and presented as area fraction. Results: The overexpressions of α-SMA in tumor cells were only detected in serous and clear cell ovarian carcinoma. The histoscore of α-SMA was higher in malignant than in benign or borderline ovarian epithelial neoplasms (105.3±129.9 vs. 17.3±17.1, P=0.011; mean±SD). Oppositely, stromal α-SMA and collagen area fractions were higher in benign than in malignant tumors (27.2±6.6 vs 20.5±8.4, P=0.028; 31.0±5.6 vs. 23.7±6.4, P=0.04). The percentages of epithelial and stromal PCNA expressions were not significantly different between benign and malignant tumors. Conclusion: Tumor cells of serous and clear cell ovarian carcinoma exhibit mesenchymal characteristic as shown by α-SMA positive expression. This expression might indicate that these subtypes were more aggressive. This research showed that collagen and α-SMA area fractions in stroma were higher in benign than in malignant neoplasms. 10.22034/APJCP.2017.18.3.667

Composition, Architecture, and Functional Implications of the Connective Tissue Network of the Extraocular Muscles

PubMed Central

McLoon, Linda K.; Vicente, André; Fitzpatrick, Krysta R.; Lindström, Mona

2018-01-01

Purpose We examined the pattern and extent of connective tissue distribution in the extraocular muscles (EOMs) and determined the ability of the interconnected connective tissues to disseminate force laterally. Methods Human EOMs were examined for collagens I, III, IV, and VI; fibronectin; laminin; and elastin using immunohistochemistry. Connective tissue distribution was examined with scanning electron microscopy. Rabbit EOMs were examined for levels of force transmission longitudinally and transversely using in vitro force assessment. Results Collagens I, III, and VI localized to the endomysium, perimysium, and epimysium. Collagen IV, fibronectin, and laminin localized to the basal lamina surrounding all myofibers. All collagens localized similarly in the orbital and global layers throughout the muscle length. Elastin had the most irregular pattern and ran longitudinally and circumferentially throughout the length of all EOMs. Scanning electron microscopy showed these elements to be extensively interconnected, from endomysium through the perimysium to the epimysium surrounding the whole muscle. In vitro physiology demonstrated force generation in the lateral dimension, presumably through myofascial transmission, which was always proportional to the force generated in the longitudinally oriented muscles. Conclusions A striking connective tissue matrix interconnects all the myofibers and extends, via perimysial connections, to the epimysium. These interconnections are significant and allow measurable force transmission laterally as well as longitudinally, suggesting that they may contribute to the nonlinear force summation seen in motor unit recording studies. This provides strong evidence that separate compartmental movements are unlikely as no region is independent of the rest of the muscle. PMID:29346490

Attachment of Actinobacillus suis H91-0380 and Its Isogenic Adhesin Mutants to Extracellular Matrix Components of the Tonsils of the Soft Palate of Swine

PubMed Central

Bujold, Adina R.

2016-01-01

Tonsils conduct immune surveillance of antigens entering the upper respiratory tract. Despite their immunological function, they are also sites of persistence and invasion of bacterial pathogens. Actinobacillus suis is a common resident of the tonsils of the soft palate in pigs, but under certain circumstances it can invade, causing septicemia and related sequelae. Twenty-four putative adhesins are predicted in the A. suis genome, but to date, little is known about how they might participate in colonization or invasion. To better understand these processes, swine tonsil lysates were characterized by mass spectrometry. Fifty-nine extracellular matrix (ECM) proteins were identified, including small leucine-rich proteoglycans, integrins, and other cell surface receptors. Additionally, attachment of the wild type and 3 adhesin mutants to 5 ECM components was evaluated. Exponential cultures of wild-type A. suis adhered significantly more than stationary cultures to all ECM components studied except collagen I. During exponential growth, the A. suis Δflp1 mutant attached less to collagen IV while the ΔompA mutant attached less to all ECMs. The ΔcomE1 strain attached less to collagen IV, fibronectin, and vitronectin during exponential growth and exhibited differential attachment to collagen I over short adherence time points. These results suggest that Flp1, OmpA, and ComE1 are important during early stages of attachment to ECM components found in tonsils, which supports the notion that other adhesins have compensatory effects during later stages of attachment. PMID:27481253

Molecular and Genetic Analyses of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention.

PubMed

Jeanne, Marion; Jorgensen, Jeff; Gould, Douglas B

2015-05-05

Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases, including cerebral microbleeds, porencephaly, and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown, and there is a lack of effective treatment. Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small-vessel disease, recurrent hemorrhagic strokes, and age-related macroangiopathy. We showed that allelic heterogeneity, genetic context, and environmental factors such as intense exercise or anticoagulant medication modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes was a key triggering factor of ICH. Finally, we showed that treatment of mutant mice with a US Food and Drug Administration-approved chemical chaperone resulted in a decreased collagen intracellular accumulation and a significant reduction in ICH severity. Our data are the first to show therapeutic prevention in vivo of ICH resulting from Col4a1 mutation and imply that a mechanism-based therapy promoting protein folding might also prevent ICH in patients with COL4A1 and COL4A2 mutations. © 2015 American Heart Association, Inc.

Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation.

PubMed

Sarras, Michael P

2012-01-01

The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.

ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen.

PubMed

Urello, Morgan A; Kiick, Kristi L; Sullivan, Millicent O

2017-10-15

Gene therapies have great potential in regenerative medicine; however, clinical translation has been inhibited by low stability and limited transfection efficiencies. Herein, we incorporate collagen-mimetic peptide (CMP)-linked polyplexes in collagen scaffolds to increase DNA stability by up to 400% and enable tailorable in vivo transgene expression at 100-fold higher levels and 10-fold longer time periods. These improvements were directly linked to a sustained interaction between collagen and polyplexes that persisted during cellular remodeling, polyplex uptake, and intracellular trafficking. Specifically, incorporation of CMPs into polyethylenimine (PEI) polyplexes preserved serum-exposed polyplex-collagen activity over a period of 14days, with 4 orders-of-magnitude more intact DNA present in CMP-modified polyplex-collagen relative to unmodified polyplex-collagen after a 10day incubation under cell culture conditions. CMP-modification also altered endocytic uptake, as indicated by gene silencing studies showing a nearly 50% decrease in transgene expression in response to caveolin-1 silencing in modified samples versus only 30% in unmodified samples. Furthermore, cellular internalization studies demonstrated that polyplex-collagen association persisted within cells in CMP polyplexes, but not in unmodified polyplexes, suggesting that CMP linkage to collagen regulates intracellular transport. Moreover, experiments in an in vivo repair model showed that CMP modification enabled tailoring of transgene expression from 4 to 25days over a range of concentrations. Overall, these findings demonstrate that CMP decoration provides substantial improvements in gene retention, altered release kinetics, improved serum-stability, and improved gene activity in vivo. This versatile technique has great potential for multiple applications in regenerative medicine. In this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural process of collagen remodelling for high efficiency non-viral gene delivery. The incorporation of CMPs into DNA polyplexes, coupled with the innate affinity between CMPs and collagen, not only permitted improved control over polyplex retention and release, but also provided a series of substantial and highly unique benefits via the stable and persistent linkage between CMP-polyplexes and collagen fragments. Specifically, CMP-modification of polyplexes was demonstrated to (i) control release for nearly a month, (ii) improve vector stability under physiological-like conditions, and (iii) provide ligands able to efficiently transfer genes via endocytic collagen pathways. These unique properties overcome key barriers inhibiting non-viral gene therapy. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Differential expression of miR-31 between inflammatory bowel disease and microscopic colitis.

PubMed

Zhang, Chen; Zhao, Zijin; Osman, Hany; Watson, Rao; Nalbantoglu, Ilke; Lin, Jingmei

2014-01-01

Idiopathic inflammatory bowel disease (IBD) and microscopic colitis (MC) are distinct entities. However, patients with intermittent episodes of IBD and MC that are encountered in a clinical setting puzzle clinicians and pathologists. This study examined whether microRNA assisted in the classification of IBD and MC. Small RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) colon tissue and qRT-PCR was performed from cohorts of normal control (n=38), ulcerative colitis (n=36), Crohns disease (n=26), collagenous colitis (n=36), lymphocytic colitis (n=30), and patients with intermittent features of IBD and MC (n=6). Differential expression of miR-31 distinguished IBD (ulcerative colitis and Crohns disease) from MC (collagenous colitis and lymphocytic colitis), confirming the specificity of miR-31 expression in IBD (P=0.00001). In addition, expression of miR-31 was increased in collagenous colitis compared to that of lymphocytic colitis (P=0.010). Among 6 patients with alternating episodes of IBD and MC, one patient had matching miR-31 expression in different phases (lymphocytic colitis to ulcerative colitis, and then back to collagenous colitis). The other 5 patients had MC-like expression patterns in both MC and IBD episodes. In summary, IBD and MC have distinct miR-31 expression pattern. Therefore, miR-31 might be used as a biomarker to distinguish between IBD and MC in FFPE colonic tissue. In addition, miR-31 is differentially expressed in colonic tissue between lymphocytic colitis and collagenous colitis, suggesting them of separate disease processes. Finally, patients with alternating IBD and MC episodes represent a diverse group. Among them, the majority demonstrates MC-like miR-31 expression pattern in MC phases, which seems unlikely to support the speculation of MC as an inactive form of IBD. Although the mechanisms deserve further investigation, microRNA is a potentially useful biomarker to differentiate IBD and MC.

Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease.

PubMed

Liu, Bin; Li, Chenghai; Liu, Zijuan; Dai, Zonghan; Tao, Yunxia

2012-09-11

Polycystic Kidney Disease (PKD) kidneys exhibit increased extracellular matrix (ECM) collagen expression and metalloproteinases (MMPs) activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. We examined the role of type I collagen (collagen I) and membrane bound type 1 MMP (MT1-MMP) on cyst development using both in vitro 3 dimensional (3D) collagen gel culture and in vivo PCK rat model of PKD. We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

Supplementating with dietary astaxanthin combined with collagen hydrolysate improves facial elasticity and decreases matrix metalloproteinase-1 and -12 expression: a comparative study with placebo.

PubMed

Yoon, Hyun-Sun; Cho, Hyun Hee; Cho, Soyun; Lee, Se-Rah; Shin, Mi-Hee; Chung, Jin Ho

2014-07-01

Photoaging accounts for most age-related changes in skin appearance. It has been suggested that both astaxanthin, a potent antioxidant, and collagen hydrolysate can be used as antiaging modalities in photoaged skin. However, there is no clinical study using astaxanthin combined with collagen hydrolysate. We investigated the effects of using a combination of dietary astaxanthin and collagen hydrolysate supplementation on moderately photoaged skin in humans. A total of 44 healthy subjects were recruited and treated with astaxanthin (2 mg/day) combined with collagen hydrolysate (3 g/day) or placebos, which were identical in appearance and taste to the active supplementation for 12 weeks. The elasticity and hydration properties of facial skin were evaluated using noninvasive objective devices. In addition, we also evaluated the expression of procollagen type I, fibrillin-1, matrix metalloproteinase-1 (MMP-1) and -12, and ultraviolet (UV)-induced DNA damage in artificially UV-irradiated buttock skin before and after treatment. The supplement group showed significant improvements in skin elasticity and transepidermal water loss in photoaged facial skin after 12 weeks compared with the placebo group. In the supplement group, expression of procollagen type I mRNA increased and expression of MMP-1 and -12 mRNA decreased compared with those in the placebo group. In contrast, there was no significant difference in UV-induced DNA damage between groups. These results demonstrate that dietary astaxanthin combined with collagen hydrolysate can improve elasticity and barrier integrity in photoaged human facial skin, and such treatment is well tolerated.

Particulate wear debris activates protein tyrosine kinases and nuclear factor kappaB, which down-regulates type I collagen synthesis in human osteoblasts.

PubMed

Vermes, C; Roebuck, K A; Chandrasekaran, R; Dobai, J G; Jacobs, J J; Glant, T T

2000-09-01

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma.

PubMed

Etoh, Mitsuhiko; Jinnin, Masatoshi; Makino, Katsunari; Yamane, Keitaro; Nakayama, Wakana; Aoi, Jun; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Ihn, Hironobu

2013-01-01

Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

PubMed

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

2006-05-01

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes.

PubMed

Wong, Marcy; Siegrist, Mark; Goodwin, Kelly

2003-10-01

Endochondral ossification is regulated by many factors, including mechanical stimuli, which can suppress or accelerate chondrocyte maturation. Mathematical models of endochondral ossification have suggested that tension (or shear stress) can accelerate the formation of endochondral bone, while hydrostatic stress preserves the cartilage phenotype. The goal of this study was to test this hypothesis by examining the expression of hypertrophic chondrocyte markers (transcription factor Cbfa1, MMP-13, type X collagen, VEGF, CTGF) and cartilage matrix proteins under cyclic tension and cyclic hydrostatic pressure. Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers. Mammalian chondrocytes appear to have evolved complex mechanoresponsive mechanisms, the effects of which can be observed in the histomorphologic establishment of the cartilaginous skeleton during development and maturation.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium

PubMed Central

Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Prakash, Vittal P.; Qin, Xiang; Hook, Magnus; Weinstock, George M.; Murray, Barbara E.

2009-01-01

SUMMARY Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about pathogenic determinants of this organism. We have previously identified a cell wall anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad spectrum binding to extracellular matrix proteins. Here, we analyzed the draft genome of strain TX0016 for potential MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Genome-based bioinformatics identified 22 predicted cell wall anchored E. faeciumsurface proteins (Fms) of which 15 (including Acm) have typical characteristics of MSCRAMMs including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one (Fms10, redesignated Scm for second collagen adhesin of E. faeciu m) revealed that recombinant Scm65 (A- and B-domains) and Scm36 (A-domain) bound efficiently to collagen type V in a concentration dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism of recombinant Scm36 and of Acm37 indicated that these proteins are rich in β-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; 9 of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated as EbpCfm), detected a “ladder” pattern of high-molecular weight protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpCfm is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpAfm), fms5 (ebpBfm) and ebpCfm are co-transcribed, consistent with pilus-encoding gene clusters of other gram-positive bacteria. All 15 genes occurred frequently in 30 clinically-derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen. PMID:18832325

Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition

PubMed Central

Li, Xuelian; Zhao, Dandan; Guo, Zhenfeng; Li, Tianshi; Qili, Muge; Xu, Bozhi; Qian, Ming; Liang, Haihai; E, Xiaoqiang; Chege Gitau, Samuel; Wang, Lu; Huangfu, Longtao; Wu, Qiuxia; Xu, Chaoqian; Shan, Hongli

2016-01-01

Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis. PMID:27876880

The transmembrane collagen COL-99 guides longitudinally extending axons in C. elegans.

PubMed

Taylor, Jesse; Unsoeld, Thomas; Hutter, Harald

2018-06-01

We have identified the transmembrane collagen, COL-99, in a genetic screen for novel genes involved in axon guidance in the nematode C. elegans. COL-99 is similar to transmembrane collagens type XIII, XXIII and XXV in vertebrates. col-99 mutants exhibit guidance defects in axons extending along the major longitudinal axon tracts, most prominently the left ventral nerve cord (VNC). COL-99 is expressed in the hypodermis during the time of axon outgrowth. We provide evidence that a furin cleavage site in COL-99 is essential for function, suggesting that COL-99 is released from the cells producing it. Vertebrate homologs of COL-99 have been shown to be expressed in mammalian nervous systems and linked to various neurological disease but have not been associated with guidance of extending neurons. col-99 acts genetically with the discoidin domain receptors ddr-1 and ddr-2, which are expressed by neurons affected in col-99 mutants. Discoidin domain receptors are activated by collagens in vertebrates. DDR-1 and DDR-2 may function as receptors for COL-99. Our results establish a novel role for a transmembrane collagen in axonal guidance and asymmetry establishment of the VNC. Copyright © 2018 Elsevier Inc. All rights reserved.

Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

PubMed

Mizuno, Kazunori; Boudko, Sergei; Engel, Jürgen; Bächinger, Hans Peter

2013-06-28

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

PubMed

Schendel, Stephen A

2017-10-01

Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. 5. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

Accelerated collagen turnover in women with angina pectoris without obstructive coronary artery disease: An iPOWER substudy.

PubMed

Nielsen, Signe H; Mygind, Naja D; Michelsen, Marie M; Bechsgaard, Daria F; Suhrs, Hannah E; Genovese, Federica; Nielsen, Henning B; Brix, Susanne; Karsdal, Morten; Prescott, Eva; Kastrup, Jens

2018-05-01

Aim Collagens are major cardiac extracellular matrix components, known to be actively remodelled and accumulated during diffuse myocardial fibrosis. We evaluated whether accelerated collagen turnover described by neo-epitope biomarkers reflecting collagen formation and degradation separates patients with diffuse myocardial fibrosis from asymptomatic controls. Methods and results Seventy-one women with angina pectoris without significant coronary artery disease assessed by invasive coronary angiogram were included. Competitive enzyme-linked immunosorbent assays (ELISAs) measuring circulating protein fragments in serum assessed the formation and degradation of collagen type III (Pro-C3, C3M and C3C), IV (P4NP7S and C4M), V (Pro-C5 and C5M) and VI (Pro-C6 and C6M), and degradation of collagen type I (C1M). Serum samples from 32 age-matched asymptomatic women were included as controls. Symptomatic women presented significantly elevated levels of Pro-C6, C3C, C3M, C4M and C8-C ( p < 0.0001-0.0058) and significantly decreased levels of Pro-C3, C5M and C6M ( p < 0.0001-0.041), reflecting accelerated collagen turnover and an imbalanced collagen formation and degradation compared to controls. Cardiac magnetic resonance T1 mapping was performed to determine extracellular volume fraction and thus diffuse myocardial fibrosis. A significant association was identified between C5M and extracellular volume fraction by cardiac magnetic resonance ( p = 0.01). Conclusion Women with angina pectoris, but without significant obstructive coronary artery disease, showed an imbalanced collagen turnover compared to asymptomatic controls. The examined biomarkers are tools to monitor active collagen remodelling in patients with angina pectoris, in risk of developing myocardial fibrosis.

β2-Adrenoceptor is involved in connective tissue remodeling in regenerating muscles by decreasing the activity of MMP-9.

PubMed

Silva, Meiricris T; Nascimento, Tábata L; Pereira, Marcelo G; Siqueira, Adriane S; Brum, Patrícia C; Jaeger, Ruy G; Miyabara, Elen H

2016-07-01

We investigated the role of β2-adrenoceptors in the connective tissue remodeling of regenerating muscles from β2-adrenoceptor knockout (β2KO) mice. Tibialis anterior muscles from β2KO mice were cryolesioned and analyzed after 3, 10, and 21 days. Regenerating muscles from β2KO mice showed a significant increase in the area density of the connective tissue and in the amount of collagen at 10 days compared with wild-type (WT) mice. A greater increase occurred in the expression levels of collagen I, III, and IV in regenerating muscles from β2KO mice evaluated at 10 days compared with WT mice; this increase continued at 21 days, except for collagen III. Matrix metalloproteinase (MMP-2) activity increased to a similar extent in regenerating muscles from both β2KO and WT mice at 3 and 10 days. This was also the case for MMP-9 activity in regenerating muscles from both β2KO and WT mice at 3 days; however, at 10 days post-cryolesion, this activity returned to baseline levels only in WT mice. MMP-3 activity was unaltered in regenerating muscles at 10 days. mRNA levels of tumor necrosis factor-α increased in regenerating muscles from WT and β2KO mice at 3 days and, at 10 days post-cryolesion, returned to baseline only in WT mice. mRNA levels of interleukin-6 increased in muscles from WT mice at 3 days post-cryolesion and returned to baseline at 10 days post-cryolesion but were unchanged in β2KO mice. Our results suggest that the β2-adrenoceptor contributes to collagen remodeling during muscle regeneration by decreasing MMP-9 activity.

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

PubMed

Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

2018-02-01

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

PubMed

Clavarino, Giovanna; Gauthier, Arnaud; Hellmark, Thomas; Carron, Pierre-Louis; Giovannini, Diane; Colliard, Sophie; Dragon-Durey, Marie-Agnès; Segelmark, Mårten; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal

2018-04-12

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Research of Acellular pancreatic bioscaffoldas a natural 3D platform In Vitro

NASA Astrophysics Data System (ADS)

Wang, Xin; Li, Zhao

2018-03-01

AIM: To investigate the biochemical and functional properties of a rat acellular pancreatic bioscaffold (APB). METHODS: Fresh pancreata were soaked and perfused. The histological structure, the extracellular matrix (ECM) composition, and the DNA content of the APBs were evaluated. After biocompatibility studies, the proliferation, apoptosis and differentiation of AR42J pancreatic acinar cells cultured on APBs were assessed. RESULTS: The pancreatic tissues became translucent after decellularization. The native macroscopic 3D architecture and the ECM ultrastructure were preserved, with large ductal structures and vascular tissue branching from the greater pancreatic artery, but there were no visible vascular endothelial cells, cellular components or cracked cellular debris. The ECM components, including collagen I, collagen IV, fibronectin, laminin and sGAG, were not decreased after decellularization of the APB (P>0.05) however, the DNA content was decreased significantly (P<0.05). The subcutaneous implantation sites showed low immunological response and low cytotoxicity around the APB. The proliferation rate was higher and the apoptosis rate was lower when AR42J cells were cultured on APB than when they were cultured in media alone, on artificial scaffold or ECM (P<0.05). The gene expression of pancreatic duodenal homeodomain containing transcription factor (PDX-1) and pancreatic exocrine transcription factor (PTF-1) and the protein expression of α-Amy, cytokeratin 7 (CK7) and fetal liver kinase-1 (Flk-1) were higher for the APB group than for the other groups (P<0.001). CONCLUSION: Our findings support the biological utility of whole pancreas APBs as biomaterial scaffolds, which provides an improved approach for regenerative medicine.

Clonal population of adult stem cells: life span and differentiation potential.

PubMed

Seruya, Mitchel; Shah, Anup; Pedrotty, Dawn; du Laney, Tracey; Melgiri, Ryan; McKee, J Andrew; Young, Henry E; Niklason, Laura E

2004-01-01

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-beta1 (TGF-beta1) differentiated into a homogeneous population expressing alpha-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

PubMed

Wagner, Diane R; Lindsey, Derek P; Li, Kelvin W; Tummala, Padmaja; Chandran, Sheena E; Smith, R Lane; Longaker, Michael T; Carter, Dennis R; Beaupre, Gary S

2008-05-01

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

In microfluidico: Recreating in vivo hemodynamics using miniaturized devices

PubMed Central

Zhu, Shu; Herbig, Bradley A.; Li, Ruizhi; Colace, Thomas V.; Muthard, Ryan W.; Neeves, Keith B.; Diamond, Scott L.

2016-01-01

Microfluidic devices create precisely controlled reactive blood flows and typically involve: (i) validated anticoagulation/pharmacology protocols, (ii) defined reactive surfaces, (iii) defined flow-transport regimes, and (iv) optical imaging. An 8-channel device can be run at constant flow rate or constant pressure drop for blood perfusion over a patterned collagen, collagen/kaolin, or collagen/tissue factor (TF) to measure platelet, thrombin, and fibrin dynamics during clot growth. A membrane-flow device delivers a constant flux of platelet agonists or coagulation enzymes into flowing blood. A trifurcated device sheaths a central blood flow on both sides with buffer, an ideal approach for on-chip recalcification of citrated blood or drug delivery. A side-view device allows clotting on a porous collagen/TF plug at constant pressure differential across the developing clot. The core-shell architecture of clots made in mouse models can be replicated in this device using human blood. For pathological flows, a stenosis device achieves shear rates of >100,000 s−1 to drive plasma von Willebrand factor (VWF) to form thick long fibers on collagen. Similarly, a micropost-impingement device creates extreme elongational and shear flows for VWF fiber formation without collagen. Overall, microfluidics are ideal for studies of clotting, bleeding, fibrin polymerization/fibrinolysis, cell/clot mechanics, adhesion, mechanobiology, and reaction-transport dynamics. PMID:26600269

[Comparison of various noninvasive serum markers of liver fibrosis in chronic viral liver disease].

PubMed

Kim, Sun Min; Sohn, Joo Hyun; Kim, Tae Yeob; Roh, Young Wook; Eun, Chang Soo; Jeon, Yong Cheol; Han, Dong Soo; Oh, Young Ha

2009-12-01

The aim of this study was to determine the clinical performances of noninvasive serum markers for the prediction of liver fibrosis in chronic viral liver diseases. We analyzed a total of 225 patients with chronic viral liver diseases (180 with hepatitis B virus, 43 with hepatitis C virus, and 2 with hepatitis B+C virus) who underwent a liver biopsy procedure at the Hanyang University Guri Hospital between March 2002 and February 2007. Serum was also obtained at the time of liver biopsy. Liver fibrosis was staged according to the scoring system proposed by the Korean Study Group for the Pathology of Digestive Diseases. Various noninvasive serum markers were evaluated, including the aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (AAR), age-platelet (AP) index, AST/platelet ratio index (APRI), cirrhosis discriminant score (CDS), platelet count, hyaluronic acid (HA), and type IV collagen. There were 17, 40, 61, 74, and 33 patients at stages F0, F1, F2, F3, and F4, respectively. The overall diagnostic accuracies of each marker, as determined by the area under receiver operating characteristics curves, were APRI=0.822, CDS=0.776, platelet count=0.773, AP index=0.756, HA=0.749, type IV collagen=0.718, and AAR=0.642 for predicting significant fibrosis (> or =F2); and CDS=0.835, platelet count=0.795, AP index=0.794, HA=0.766, AAR=0.711, type IV collagen=0.697, and APRI=0.691 for predicting extensive fibrosis (> or =F3). All noninvasive serum markers evaluated in this study were useful for predicting significant or extensive liver fibrosis in chronic viral liver diseases. In particular, APRI was most useful for the prediction of significant fibrosis, and CDS was most useful for the prediction of extensive fibrosis.

Supramolecular Organization of the α121-α565 Collagen IV Network*

PubMed Central

Robertson, Wesley E.; Rose, Kristie L.; Hudson, Billy G.; Vanacore, Roberto M.

2014-01-01

Collagen IV is a family of 6 chains (α1-α6), that form triple-helical protomers that assemble into supramolecular networks. Two distinct networks with chain compositions of α121 and α345 have been established. These oligomerize into separate α121 and α345 networks by a homotypic interaction through their trimeric noncollagenous (NC1) domains, forming α121 and α345 NC1 hexamers, respectively. These are stabilized by novel sulfilimine (SN) cross-links, a covalent cross-link that forms between Met93 and Hyl211 at the trimer-trimer interface. A third network with a composition of α1256 has been proposed, but its supramolecular organization has not been established. In this study we investigated the supramolecular organization of this network by determining the chain identity of sulfilimine-cross-linked NC1 domains derived from the α1256 NC1 hexamer. High resolution mass spectrometry analyses of peptides revealed that sulfilimine bonds specifically cross-link α1 to α5 and α2 to α6 NC1 domains, thus providing the spatial orientation between interacting α121 and α565 trimers. Using this information, we constructed a three-dimensional homology model in which the α565 trimer shows a good chemical and structural complementarity to the α121 trimer. Our studies provide the first chemical evidence for an α565 protomer and its heterotypic interaction with the α121 protomer. Moreover, our findings, in conjunction with our previous studies, establish that the six collagen IV chains are organized into three canonical protomers α121, α345, and α565 forming three distinct networks: α121, α345, and α121-α565, each of which is stabilized by sulfilimine bonds between their C-terminal NC1 domains. PMID:25006246

Effect of Tissucol on connective tissue matrix during wound healing: an immunohistochemical study in rat skin.

PubMed

Romanos, G E; Strub, J R

1998-03-05

Fibrin sealants are very useful in different surgical fields. Fixation of free gingival grafts, root coverage procedures, and other techniques increasing connective tissue attachment may be associated with the application of Tissucol in periodontology. The aim of this study was to evaluate the influence of the fibrin sealant in the extracellular matrix, as well as alterations of the connective tissue matrix during wound-healing processes. In the back dermis of 15 Net male rats, Tissucol was implanted after intraperitoneal anesthesia. The implant material was placed in subcutaneous pockets (2 cm in length) which were sutured with interproximal sutures (test and control pockets). At 4, 7, 14, 21, and 28 days after surgery, biopsies of the healed and surrounding tissues were taken, frozen in liquid nitrogen, and examined histologically and immunohistochemically with antibodies against collagen types I, III, IV, V, VI, and VII. The findings showed thick and thin collagen type I and III fibers, respectively, with different orientations localized around the implant material. An increased amount of blood vessels and capillaries (their basement membranes containing collagen type IV) was observed during wound healing which may be associated with the implantation of the sealant. Collagen type V fibers were localized from the first days to the 4th postoperative week and, without any inflammatory reaction (according to histologic staining), formed a fibrillar extracellular matrix with high collagenase resistance. Collagen type VI showed a microfibrillar pattern of distribution, and collagen type VII was localized in the dermo epidermo junction and very deep in the connective tissue in the form of anchoring fibers (only in the test group) during the 4 postoperative weeks of healing. The data showed that Tissucol is a biocompatible component which cannot produce any extensive inflammatory reaction in the matrix. New blood vessel formation, an epithelial-connective tissue interface with high stability, as well as matrix alterations with high resistance in the proteolytic enzymes (i.e., collagenases) can be induced in the connective tissue after use of a fibrin sealant. All of these characteristics may be of great importance in connective tissue healing in periodontal surgical procedures.

Effect of doxycycline on transforming growth factor-beta-1-induced matrix metalloproteinase 2 expression, migration, and collagen contraction in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Kang, Byungjin; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2016-11-01

It is well known that doxycycline has antibacterial and anti-inflammatory effects. In this study, we aimed to investigate the effects of doxycycline on the transforming growth factor (TGF) beta 1-induced matrix metalloproteinase (MMP) 2 expression, migration, and collagen contraction, and to determine its molecular mechanism on nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from the nasal polyps of six patients. Doxycycline was used to pretreat TGF-beta-1-induced NPDFs and ex vivo organ cultures of nasal polyps. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Smad2/3 is one of the major transcription factors of TGF-beta signaling. The expression levels of MMP2 and Smad2/3 were measured by using Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The enzymic activity of MMP2 was analyzed by using gelatin zymography. Fibroblast migration was evaluated by using transwell migration assays. Contractile activity was measured by a collagen gel contraction assay. The expression level of MMP2 in nasal polyp tissues increased in comparison with inferior turbinate tissues. TGF-beta-1-induced NPDFs were not affected by doxycycline (0-40 μg/mL). The expression levels of MMP2 and activation of Smad2/3 in TGF-beta-1-induced NPDFs and in organ cultures of nasal polyps were significantly downregulated with doxycycline pretreatment. Doxycycline also reduced TGF-beta-1-induced fibroblast migration and collagen contraction in NPDFs. Doxycycline inhibited TGF-beta-1-induced MMP2 expression, migration, and collagen contraction via the Smad2/3 signal pathways in NPDFs.

Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

PubMed Central

Huh, Jeong-Eun; Park, Yeon-Cheol; Seo, Byung-Kwan; Lee, Jae-Dong; Baek, Yong-Hyeon; Choi, Do-Young; Park, Dong-Suk

2013-01-01

We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105) cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1β-treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone. PMID:23983790

Stage-Specific Action of Matrix Metalloproteinases Influences Progressive Hereditary Kidney Disease

PubMed Central

Zeisberg, Michael; Khurana, Mona; Rao, Velidi H; Cosgrove, Dominic; Rougier, Jean-Philippe; Werner, Michelle C; Shield, Charles F; Werb, Zena; Kalluri, Raghu

2006-01-01

Background Glomerular basement membrane (GBM), a key component of the blood-filtration apparatus in the in the kidney, is formed through assembly of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations or deletions involving α3(IV), α4(IV), or α5(IV) chains of type IV collagen in the GBM have been identified as the cause for Alport syndrome in humans, a progressive hereditary kidney disease associated with deafness. The pathological mechanisms by which such mutations lead to eventual kidney failure are not completely understood. Methods and Findings We showed that increased susceptibility of defective human Alport GBM to proteolytic degradation is mediated by three different matrix metalloproteinases (MMPs)—MMP-2, MMP-3, and MMP-9—which influence the progression of renal dysfunction in α3(IV) −/− mice, a model for human Alport syndrome. Genetic ablation of either MMP-2 or MMP-9, or both MMP-2 and MMP-9, led to compensatory up-regulation of other MMPs in the kidney glomerulus. Pharmacological ablation of enzymatic activity associated with multiple GBM-degrading MMPs, before the onset of proteinuria or GBM structural defects in the α3(IV) −/− mice, led to significant attenuation in disease progression associated with delayed proteinuria and marked extension in survival. In contrast, inhibition of MMPs after induction of proteinuria led to acceleration of disease associated with extensive interstitial fibrosis and early death of α3(IV) −/− mice. Conclusions These results suggest that preserving GBM/extracellular matrix integrity before the onset of proteinuria leads to significant disease protection, but if this window of opportunity is lost, MMP-inhibition at the later stages of Alport disease leads to accelerated glomerular and interstitial fibrosis. Our findings identify a crucial dual role for MMPs in the progression of Alport disease in α3(IV) −/− mice, with an early pathogenic function and a later protective action. Hence, we propose possible use of MMP-inhibitors as disease-preventive drugs for patients with Alport syndrome with identified genetic defects, before the onset of proteinuria. PMID:16509766

Stent-induced coronary artery stenosis characterized by multimodal nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Wang, Han-Wei; Simianu, Vlad; Locker, Mattew J.; Cheng, Ji-Xin; Sturek, Michael

2011-02-01

We demonstrate for the first time the applicability of multimodal nonlinear optical (NLO) microscopy to the interrogation of stented coronary arteries under different diet and stent deployment conditions. Bare metal stents and Taxus drug-eluting stents (DES) were placed in coronary arteries of Ossabaw pigs of control and atherogenic diet groups. Multimodal NLO imaging was performed to inspect changes in arterial structures and compositions after stenting. Sum frequency generation, one of the multimodalities, was used for the quantitative analysis of collagen content in the peristent and in-stent artery segments of both pig groups. Atherogenic diet increased lipid and collagen in peristent segments. In-stent segments showed decreased collagen expression in neointima compared to media. Deployment of DES in atheromatous arteries inhibited collagen expression in the arterial media.

Collagenolytic protease expression in cranial cruciate ligament and stifle synovial fluid in dogs with cranial cruciate ligament rupture.

PubMed

Muir, Peter; Danova, Nichole A; Argyle, David J; Manley, Paul A; Hao, Zhengling

2005-01-01

To determine expression of collagenolytic genes and collagen degradation in stifle tissues of dogs with ruptured cranial cruciate ligament (CCL). Six dogs with CCL rupture and 11 dogs with intact CCL. Gene expression in CCL tissue and synovial fluid cells was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). Collagen degradation was studied using CCL explant cultures and a synovial fluid bioassay. Expression of matrix metalloproteases (MMP) was not found in young Beagles with intact CCL; however, increased expression of MMP-3 was found in CCL tissue from older hounds with intact CCL, when compared with young Beagles. In dogs with ruptured CCL, expression of MMP-2 and -9 was increased in stifle tissues, when compared with dogs with intact CCL. Similar to MMP-9, expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin S was only found in stifle tissues from dogs with ruptured CCL; in contrast, expression of cathepsin K was found in all ruptured and intact CCL. Collagen degradation was increased in ruptured CCL, when compared with intact CCL. Rupture of the CCL is associated with up-regulation of expression of MMP-2 and -9 (gelatinase A and B), TRAP, and cathepsin S, and increased degradation of collagen. These findings suggest that MMP-2, -9, cathepsin S, and TRAP may be important mediators of progressive joint destruction in dogs with CCL rupture. These genes are markers for macrophages and dendritic cells. MMP and cathepsin S pathways may offer novel targets for anti-inflammatory medical therapy aimed at ameliorating joint degradation associated with inflammatory arthritis.

Periostin in Mature Stage Localized Scleroderma

PubMed Central

Kim, Min-Woo; Park, Jung Tae; Kim, Jung Ho; Koh, Seong-Joon; Yoon, Hyun-Sun; Cho, Soyun

2017-01-01

Background Periostin is a novel matricellular protein expressed in many tissues, including bone, periodontal ligament, and skin. Although its expression is prominent in various fibrotic conditions, studies of periostin in localized scleroderma are rare. Objective To investigate the expression of periostin and other molecules in localized scleroderma. Methods A retrospective study of 14 patients with confirmed mature stage localized scleroderma was undertaken. Fourteen age-matched and biopsy site-matched subjects with normal skin were included as controls. Collagen fiber deposition, periostin, procollagen, transforming growth factor-β, and matrix metalloproteinase (MMP)-1 expression were assessed and compared between the two groups. Co-localization of α-smooth muscle actin and periostin was evaluated using confocal microscopy. Results Periostin was predominantly expressed along the dermo-epidermal junction in the controls. Conversely, patients with localized scleroderma demonstrated increased collagen fiber deposition and periostin expression that was more widely distributed along the entire dermis. MMP-1 staining showed increased expression in the epidermis and dermis of patients compared to scanty expression in the controls. A semi-quantitative evaluation showed a higher proportion of excessive collagen bundle deposition (57.1% vs. 7.1%, p=0.013), diffuse periostin positivity (42.9% vs. 0%, p=0.016), and moderate MMP-1 positivity (71.4% vs. 7.1%, p=0.001) in patients than in the controls. Conclusion Compared to the controls, patients with localized scleroderma had enhanced periostin expression corresponding to increased collagen fiber deposition and unexpected overexpression of MMP-1. The results of this human in vivo study may implicate the pathogenesis of localized scleroderma. PMID:28566901

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

PubMed Central

Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Büchler, M W

1997-01-01

BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the pancreatic parenchyma. Images PMID:9155579

Quantification and Comparison of Anti-Fibrotic Therapies by Polarized SRM and SHG-Based Morphometry in Rat UUO Model

PubMed Central

Weldon, Steve M.; Matera, Damian; Lee, ChungWein; Yang, Haichun; Fryer, Ryan M.; Fogo, Agnes B.; Reinhart, Glenn A.

2016-01-01

Renal interstitial fibrosis (IF) is an important pathologic manifestation of disease progression in a variety of chronic kidney diseases (CKD). However, the quantitative and reproducible analysis of IF remains a challenge, especially in experimental animal models of progressive IF. In this study, we compare traditional polarized Sirius Red morphometry (SRM) to novel Second Harmonic Generation (SHG)-based morphometry of unstained tissues for quantitative analysis of IF in the rat 5 day unilateral ureteral obstruction (UUO) model. To validate the specificity of SHG for detecting fibrillar collagen components in IF, co-localization studies for collagens type I, III, and IV were performed using IHC. In addition, we examined the correlation, dynamic range, sensitivity, and ability of polarized SRM and SHG-based morphometry to detect an anti-fibrotic effect of three different treatment regimens. Comparisons were made across three separate studies in which animals were treated with three mechanistically distinct pharmacologic agents: enalapril (ENA, 15, 30, 60 mg/kg), mycophenolate mofetil (MMF, 2, 20 mg/kg) or the connective tissue growth factor (CTGF) neutralizing antibody, EX75606 (1, 3, 10 mg/kg). Our results demonstrate a strong co-localization of the SHG signal with fibrillar collagens I and III but not non-fibrillar collagen IV. Quantitative IF, calculated as percent cortical area of fibrosis, demonstrated similar response profile for both polarized SRM and SHG-based morphometry. The two methodologies exhibited a strong correlation across all three pharmacology studies (r2 = 0.89–0.96). However, compared with polarized SRM, SHG-based morphometry delivered a greater dynamic range and absolute magnitude of reduction of IF after treatment. In summary, we demonstrate that SHG-based morphometry in unstained kidney tissues is comparable to polarized SRM for quantitation of fibrillar collagens, but with an enhanced sensitivity to detect treatment-induced reductions in IF. Thus, performing SHG-based morphometry on unstained kidney tissue is a reliable alternative to traditional polarized SRM for quantitative analysis of IF. PMID:27257917

Coenzyme Q(10) enhances dermal elastin expression, inhibits IL-1α production and melanin synthesis in vitro.

PubMed

Zhang, M; Dang, L; Guo, F; Wang, X; Zhao, W; Zhao, R

2012-06-01

Coenzyme Q(10) (CoQ(10) ) is a well-known antioxidant and has been used in many skincare products for anti-ageing purpose. However, the molecular mechanisms of CoQ(10) function in skin cells are not fully understood. In this paper, we compared the effects of CoQ(10) on primary human dermal fibroblasts from three individuals, including adult. We demonstrated that CoQ(10) treatment promoted proliferation of fibroblasts, increased type IV collagen expression and reduced UVR-induced matrix metalloproteinases-1 (MMP-1) level in embryonic and adult cells. In addition, CoQ(10) treatment increased elastin gene expression in cultured fibroblasts and significantly decreased UVR-induced IL-1α production in HaCat cells. Taken together, CoQ(10) presented anti-ageing benefits against intrinsic ageing as well as photo damage. Interestingly, CoQ(10) was able to inhibit tyrosinase activity, resulting in reduced melanin content in B16 cells. Thus, CoQ(10) may have potential depigmentation effects for skincare. © 2012 Space Biology Research & Technology Center, CASC. ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Microbial BOD sensors based on Zr (IV)-loaded collagen fiber.

PubMed

Zhao, Lei; He, Li; Chen, Shujuan; Zou, Likou; Zhou, Kang; Ao, Xiaolin; Liu, Shuliang; Hu, Xinjie; Han, Guoquan

2017-03-01

Biochemical oxygen demand (BOD) sensors based on Zr (IV)-loaded collagen fiber (ZrCF), a novel material with great porous structure, were developed. This novel material shows adsorbability by microorganisms. Saccharomyces cerevisiae and Escherichia coli were used for the construction of BOD sensors. Factors affecting BOD sensor performance were examined. The ZrCF-based BOD sensor showed different sensitivities and linear response ranges with different biofilm densities. The amount of microorganisms strongly affected the performance of the BOD sensor. Poor permeability of previously reported immobilization carriers were greatly circumvented by ZrCF. The service life of the ZrCF-based BOD sensor was more than 42 days. The immobilized microorganisms can be stored for more than 6 months under 4°C in PB solution. There was good correlation between the results of the sensor method and the standard 5-day BOD method in the determination of pure organic substrates and real water samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Combined Alport syndrome and Klinefelter syndrome.

PubMed

Nishida, Masashi; Hashimoto, Fusako; Kaito, Hiroshi; Nozu, Kandai; Iijima, Kazumoto; Asada, Dai; Hamaoka, Kenji

2016-02-01

To date, there have been a very limited number of case reports on combined Alport syndrome (AS) and Klinefelter syndrome (KS). We herein describe the case of a 9-month-old boy diagnosed with concomitant AS and KS. KS was detected on chromosomal analysis of the amniotic fluid, and hematuria/proteinuria was identified in urinary screening at 6 months of age. Renal biopsy indicated AS, with complete deficit of the α5 chain of type IV collagen in the glomerular basement membranes. On genetic analysis for AS, de novo homozygote mutation (c.3605-2a > c) was seen in the gene encoding α5 chain of type IV collagen (COL4A5) on the X chromosomes of maternal origin. This is the first case report of combined AS and KS diagnosed during infancy, and it indicates the need to consider the concurrent existence of these two disorders in infants with urine abnormalities, even in the absence of a family history. © 2015 Japan Pediatric Society.

Constructing bio-layer of heparin and type IV collagen on titanium surface for improving its endothelialization and blood compatibility.

PubMed

Zhang, Kun; Chen, Jun-ying; Qin, Wei; Li, Jing-an; Guan, Fang-xia; Huang, Nan

2016-04-01

The modification of cardiovascular stent surface for a better micro-environment has gradually changed to multi-molecule, multi-functional designation. In this study, heparin (Hep) and type IV collagen (IVCol) were used as the functional molecule to construct a bifunctional micro-environment of anticoagulation and promoting endothelialization on titanium (Ti). The surface characterization results (AFM, Alcian Blue 8GX Staining and fluorescence staining of IVCol) indicated that the bio-layer of Hep and IVCol were successfully fabricated on the Ti surface through electrostatic self-assembly. The APTT and platelet adhesion test demonstrated that the bionic layer possessed better blood compatibility compared with Ti surface. The adhesion, proliferation, migration and apoptosis tests of endothelial cells proved that the Hep/IVCol layer was able to enhance the endothelialization of the Ti surface. The in vivo animal implantation results manifested that the bionic surface could encourage new endothelialization. This work provides an important reference for the construction of multifunction micro-environment on the cardiovascular scaffold surface.

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB.

PubMed

Wang, Hong-Ju; Li, Meng-Qi; Liu, Wei-Wei; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2017-09-01

Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Polymerized-Type I Collagen Downregulates Inflammation and Improves Clinical Outcomes in Patients with Symptomatic Knee Osteoarthritis Following Arthroscopic Lavage: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial

PubMed Central

Furuzawa-Carballeda, Janette; Lima, Guadalupe; Llorente, Luis; Nuñez-Álvarez, Carlos; Ruiz-Ordaz, Blanca H.; Echevarría-Zuno, Santiago; Hernández-Cuevas, Virgilio

2012-01-01

Objectives. Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. Aim. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). Methods. Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. Results. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Conclusion. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA. PMID:22545014