Extracellular matrix regenerative graft attenuates the negative impact of polypropylene prolapse mesh on vagina in rhesus macaque

PubMed Central

Liang, Rui; Knight, Katrina; Barone, William; Powers, Robert W.; Nolfi, Alexis; Palcsey, Stacy; Abramowitch, Steven; Moalli, Pamela A.

2016-01-01

BACKGROUND The use of wide pore lightweight polypropylene mesh to improve anatomical outcomes in the surgical repair of prolapse has been hampered by mesh complications. One of the prototype prolapse meshes has been found to negatively impact the vagina by inducing a decrease in smooth muscle volume and contractility and the degradation of key structural proteins (collagen and elastin), resulting in vaginal degeneration. Recently, bioscaffolds derived from extracellular matrix have been used to mediate tissue regeneration and have been widely adopted in tissue engineering applications. OBJECTIVE Here we aimed to: (1) define whether augmentation of a polypropylene prolapse mesh with an extracellular matrix regenerative graft in a primate sacrocolpopexy model could mitigate the degenerative changes; and (2) determine the impact of the extracellular matrix graft on vagina when implanted alone. STUDY DESIGN A polypropylene-extracellular matrix composite graft (n = 9) and a 6-layered extracellular matrix graft alone (n = 8) were implanted in 17 middle-aged parous rhesus macaques via sacrocolpopexy and compared to historical data obtained from sham (n = 12) and the polypropylene mesh (n = 12) implanted by the same method. Vaginal function was measured in passive (ball-burst test) and active (smooth muscle contractility) mechanical tests. Vaginal histomorphologic/ biochemical assessments included hematoxylin-eosin and trichrome staining, immunofluorescent labeling of α-smooth muscle actin and apoptotic cells, measurement of total collagen, collagen subtypes (ratio III/ I), mature elastin, and sulfated glycosaminoglycans. Statistical analyses included 1-way analysis of variance, Kruskal-Wallis, and appropriate posthoc tests. RESULTS The host inflammatory response in the composite mesh-implanted vagina was reduced compared to that following implantation with the polypropylene mesh alone. The increase in apoptotic cells observed with the polypropylene mesh was blunted in the composite (overall P < .001). Passive mechanical testing showed inferior parameters for both polypropylene mesh alone and the composite compared to sham whereas the contractility and thickness of smooth muscle layer in the composite were improved with a value similar to sham, which was distinct from the decreases observed with polypropylene mesh alone. Biochemically, the composite had similar mature elastin content, sulfated glycosaminoglycan content, and collagen subtype III/I ratio but lower total collagen content when compared to sham (P = .011). Multilayered extracellular matrix graft alone showed overall comparable values to sham in aspects of the biomechanical, histomorphologic, or biochemical end-points of the vagina. The increased collagen subtype ratio III/I with the extracellular matrix graft alone (P = .033 compared to sham) is consistent with an ongoing active remodeling response. CONCLUSION Mesh augmentation with a regenerative extracellular matrix graft attenuated the negative impact of polypropylene mesh on the vagina. Application of the extracellular matrix graft alone had no measurable negative effects suggesting that the benefits of this extra-cellular matrix graft occur when used without a permanent material. Future studies will focus on understanding mechanisms. PMID:27615441

SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease.

PubMed

Li, Fan; Li, Lisha; Cheng, Meijuan; Wang, Xiumin; Hao, Jun; Liu, Shuxia; Duan, Huijun

2017-01-22

Tubular interstitial extracellular matrix accumulation, which plays a key role in the pathogenesis and progression of diabetic kidney disease (DKD), is believed to be mediated by activation of PI3K/Akt signal pathway. However, it is still not clear whether SH2 domain-containing inositol 5'-phosphatase (SHIP), known as a negative regulator of PI3K/Akt pathway is also involved in extracellular matrix metabolism of diabetic kidney. In the present study, decreased SHIP and increased phospho-Akt (Ser 473, Thr 308) were found in renal tubular cells of diabetic mice accompanied by overexpression of connective tissue growth factor (CTGF) and extracellular matrix deposition versus normal mice. Again, high glucose attenuated SHIP expression in a time-dependent manner, concomitant with activation of PI3K/Akt signaling and extracellular matrix production in human renal proximal tubular epithelial cells (HK2) cultured in vitro, which was significantly prevented by transfection of M90-SHIP vector. Furthermore, in vivo delivery of rAd-INPP5D vector (SHIP expression vector) via intraperitoneal injection in diabetic mice increased SHIP expression by 3.36 times followed by 65.26%, 70.38% and 46.71% decreases of phospho-Akt (Ser 473), phospho-Akt (Thr 308) and CTGF expression versus diabetic mice receiving rAd-EGFP vector. Meanwhile, increased renal extracellular matrix accumulation of diabetic mice was also inhibited with intraperitoneal injection of rAd-INPP5D vector. These above data suggested that overexpression of SHIP might be a potent method to lessen renal extracellular matrix accumulation via inactivation of PI3K/Akt pathway and suppression of CTGF expression in DKD. Copyright © 2016 Elsevier Inc. All rights reserved.

Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms.

PubMed

Klein, Marlise I; Hwang, Geelsu; Santos, Paulo H S; Campanella, Osvaldo H; Koo, Hyun

2015-01-01

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

PubMed Central

Klein, Marlise I.; Hwang, Geelsu; Santos, Paulo H. S.; Campanella, Osvaldo H.; Koo, Hyun

2015-01-01

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases. PMID:25763359

The extracellular matrix of Staphylococcus aureus biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH.

PubMed

Foulston, Lucy; Elsholz, Alexander K W; DeFrancesco, Alicia S; Losick, Richard

2014-09-02

Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix. Staphylococcus aureus is a leading cause of multiantibiotic-resistant nosocomial infections and is often found growing as a biofilm in catheters and chronic wounds. Biofilm formation is an important pathogenicity strategy that enhances resistance to antimicrobials, thereby limiting treatment options and ultimately contributing to increased morbidity and mortality. Cells in a biofilm are held together by an extracellular matrix that consists in whole or in part of protein, but the nature of the proteins in the S. aureus matrix is not well understood. Here we postulate that S. aureus recycles proteins from the cytoplasm to form the extracellular matrix. This strategy, of cytoplasmic proteins moonlighting as matrix proteins, could allow enhanced flexibility and adaptability for S. aureus in forming biofilms under infection conditions and could promote the formation of mixed-species biofilms in chronic wounds. Copyright © 2014 Foulston et al.

Teaching the Extracellular Matrix and Introducing Online Databases within a Multidisciplinary Course with i-Cell-MATRIX: A Student-Centered Approach

ERIC Educational Resources Information Center

Sousa, Joao Carlos; Costa, Manuel Joao; Palha, Joana Almeida

2010-01-01

The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure.…

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

PubMed

Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

2016-09-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

PubMed Central

Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

2016-01-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

Three-dimensional finite element modeling of pericellular matrix and cell mechanics in the nucleus pulposus of the intervertebral disk based on in situ morphology.

PubMed

Cao, Li; Guilak, Farshid; Setton, Lori A

2011-02-01

Nucleus pulposus (NP) cells of the intervertebral disk (IVD) have unique morphological characteristics and biologic responses to mechanical stimuli that may regulate maintenance and health of the IVD. NP cells reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties may significantly influence cell and extracellular matrix mechanics. In this study, a computational model was developed to predict the stress-strain, fluid pressure and flow fields for cells and their surrounding PCM in the NP using three-dimensional (3D) finite element models based on the in situ morphology of cell-PCM regions of the mature rat NP, measured using confocal microscopy. Three-dimensional geometries of the extracellular matrix and representative cell-matrix units were used to construct 3D finite element models of the structures as isotropic and biphasic materials. In response to compressive strain of the extracellular matrix, NP cells and PCM regions were predicted to experience volumetric strains that were 1.9-3.7 and 1.4-2.1 times greater than the extracellular matrix, respectively. Volumetric and deviatoric strain concentrations were generally found at the cell/PCM interface, while von Mises stress concentrations were associated with the PCM/extracellular matrix interface. Cell-matrix units containing greater cell numbers were associated with higher peak cell strains and lower rates of fluid pressurization upon loading. These studies provide new model predictions for micromechanics of NP cells that can contribute to an understanding of mechanotransduction in the IVD and its changes with aging and degeneration.

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

PubMed Central

Hansen, R K; Bissell, M J

2010-01-01

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function. PMID:10903527

Limitation of Cell Adhesion by the Elasticity of the Extracellular Matrix

PubMed Central

Nicolas, Alice; Safran, Samuel. A.

2006-01-01

Cell/matrix adhesions are modulated by cytoskeletal or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity arises from the activation of a mechanosensor located within the adhesion itself. We show that this mechanism accounts for the observed directional growth of focal adhesions and the reduction or even cessation of their growth when cells adhere to a soft extracellular matrix. We predict quantitatively that both the elasticity and the thickness of the matrix play a key role in the dynamics of focal adhesions. Two different types of dynamics are expected depending on whether the thickness of the matrix is of order of or much larger than the adhesion size. In the latter situation, we predict that the adhesion region reaches a saturation size that can be tuned by the mechanical properties of the matrix. PMID:16581840

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

PubMed

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2015-10-15

Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

Imaging articular cartilage using second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Mansfield, Jessica C.; Winlove, C. Peter; Knapp, Karen; Matcher, Stephen J.

2006-02-01

Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.

Mutations in extracellular matrix genes NID1 and LAMC1 cause autosomal dominant Dandy-Walker malformation and occipital cephaloceles

PubMed Central

Darbro, Benjamin W.; Mahajan, Vinit B.; Gakhar, Lokesh; Skeie, Jessica M.; Campbell, Elizabeth; Wu, Shu; Bing, Xinyu; Millen, Kathleen J.; Dobyns, William B.; Kessler, John A.; Jalali, Ali; Cremer, James; Segre, Alberto; Manak, J. Robert; Aldinger, Kimerbly A.; Suzuki, Satoshi; Natsume, Nagato; Ono, Maya; Hai, Huynh Dai; Viet, Le Thi; Loddo, Sara; Valente, Enza M.; Bernardini, Laura; Ghonge, Nitin; Ferguson, Polly J.; Bassuk, Alexander G.

2013-01-01

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles (ADDWOC) and detected a mutation in the extracellular matrix protein encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1 binding partner. Structural modeling the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the extracellular matrix in the pathogenesis of Dandy-Walker spectrum disorders. PMID:23674478

Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research

PubMed Central

Akhmanova, Maria; Osidak, Egor; Domogatsky, Sergey; Rodin, Sergey; Domogatskaya, Anna

2015-01-01

Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems. PMID:26351461

Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach.

PubMed

Reichhardt, Courtney; Fong, Jiunn C N; Yildiz, Fitnat; Cegelski, Lynette

2015-01-01

Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using ¹³C CPMAS and ¹³C{(¹⁵N}, ¹⁵N{³¹P}, and ¹³C{³¹P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, ¹⁵N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems. Copyright © 2014. Published by Elsevier B.V.

A major protein component of the Bacillus subtilis biofilm matrix.

PubMed

Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

2006-02-01

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

PubMed

Escribano, J; Sánchez, M T; García-Aznar, J M

2015-11-07

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

NASA Astrophysics Data System (ADS)

Caroni, Pico; Carlson, Steven S.; Schweitzer, Erik; Kelly, Regis B.

1985-04-01

As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration1-5, considerable attention has been paid to unique molecules localized to this region6-11. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.

Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

PubMed

Poornejad, Nafiseh; Nielsen, Jeffery J; Morris, Ryan J; Gassman, Jason R; Reynolds, Paul R; Roeder, Beverly L; Cook, Alonzo D

2016-03-01

Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix. © The Author(s) 2015.

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.

Detection of extracellular matrix modification in cancer models with inverse spectroscopic optical coherence tomography

NASA Astrophysics Data System (ADS)

Spicer, Graham L. C.; Azarin, Samira M.; Yi, Ji; Young, Scott T.; Ellis, Ronald; Bauer, Greta M.; Shea, Lonnie D.; Backman, Vadim

2016-10-01

In cancer biology, there has been a recent effort to understand tumor formation in the context of the tissue microenvironment. In particular, recent progress has explored the mechanisms behind how changes in the cell-extracellular matrix ensemble influence progression of the disease. The extensive use of in vitro tissue culture models in simulant matrix has proven effective at studying such interactions, but modalities for non-invasively quantifying aspects of these systems are scant. We present the novel application of an imaging technique, Inverse Spectroscopic Optical Coherence Tomography, for the non-destructive measurement of in vitro biological samples during matrix remodeling. Our findings indicate that the nanoscale-sensitive mass density correlation shape factor D of cancer cells increases in response to a more crosslinked matrix. We present a facile technique for the non-invasive, quantitative study of the micro- and nano-scale structure of the extracellular matrix and its host cells.

Biomechanical cell regulatory networks as complex adaptive systems in relation to cancer.

PubMed

Feller, Liviu; Khammissa, Razia Abdool Gafaar; Lemmer, Johan

2017-01-01

Physiological structure and function of cells are maintained by ongoing complex dynamic adaptive processes in the intracellular molecular pathways controlling the overall profile of gene expression, and by genes in cellular gene regulatory circuits. Cytogenetic mutations and non-genetic factors such as chronic inflammation or repetitive trauma, intrinsic mechanical stresses within extracellular matrix may induce redirection of gene regulatory circuits with abnormal reactivation of embryonic developmental programmes which can now drive cell transformation and cancer initiation, and later cancer progression and metastasis. Some of the non-genetic factors that may also favour cancerization are dysregulation in epithelial-mesenchymal interactions, in cell-to-cell communication, in extracellular matrix turnover, in extracellular matrix-to-cell interactions and in mechanotransduction pathways. Persistent increase in extracellular matrix stiffness, for whatever reason, has been shown to play an important role in cell transformation, and later in cancer cell invasion. In this article we review certain cell regulatory networks driving carcinogenesis, focussing on the role of mechanical stresses modulating structure and function of cells and their extracellular matrices.

Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

PubMed

Kozel, Beth A; Ciliberto, Christopher H; Mecham, Robert P

2004-04-01

The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

NASA Astrophysics Data System (ADS)

Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-02-01

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix

PubMed Central

Ma, Luyan; Conover, Matthew; Lu, Haiping; Parsek, Matthew R.; Bayles, Kenneth; Wozniak, Daniel J.

2009-01-01

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell–cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications. PMID:19325879

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

PubMed

Ma, Luyan; Conover, Matthew; Lu, Haiping; Parsek, Matthew R; Bayles, Kenneth; Wozniak, Daniel J

2009-03-01

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

Cell-Extracellular Matrix Mechanobiology: Forceful Tools and Emerging Needs for Basic and Translational Research.

PubMed

Holle, Andrew W; Young, Jennifer L; Van Vliet, Krystyn J; Kamm, Roger D; Discher, Dennis; Janmey, Paul; Spatz, Joachim P; Saif, Taher

2018-01-10

Extracellular biophysical cues have a profound influence on a wide range of cell behaviors, including growth, motility, differentiation, apoptosis, gene expression, adhesion, and signal transduction. Cells not only respond to definitively mechanical cues from the extracellular matrix (ECM) but can also sometimes alter the mechanical properties of the matrix and hence influence subsequent matrix-based cues in both physiological and pathological processes. Interactions between cells and materials in vitro can modify cell phenotype and ECM structure, whether intentionally or inadvertently. Interactions between cell and matrix mechanics in vivo are of particular importance in a wide variety of disorders, including cancer, central nervous system injury, fibrotic diseases, and myocardial infarction. Both the in vitro and in vivo effects of this coupling between mechanics and biology hold important implications for clinical applications.

Cellulose biosynthesis by the beta-proteobacterium, Chromobacterium violaceum.

PubMed

Recouvreux, Derce O S; Carminatti, Claudimir A; Pitlovanciv, Ana K; Rambo, Carlos R; Porto, Luismar M; Antônio, Regina V

2008-11-01

The Chromobacterium violaceum ATCC 12472 genome was sequenced by The Brazilian National Genome Project Consortium. Previous annotation reported the presence of cellulose biosynthesis genes in that genome. Analysis of these genes showed that, as observed in other bacteria, they are organized in two operons. In the present work, experimental evidences of the presence of cellulose in the extracellular matrix of the biofilm produced by C. violaceum in static cultures are shown. Biofilm samples were enzymatically digested by cellulase, releasing glucose units, suggesting the presence of cellulose as an extracellular matrix component. Fluorescence microscopy observations showed that C. violaceum produces a cellulase-sensitive extracellular matrix composed of fibers able to bind calcofluor. C. violaceum grows on medium containing Congo red, forming brown-red colonies. Together, these results suggest that cellulase-susceptible matrix material is cellulose. Scanning electronic microscopy analysis showed that the extracellular matrix exhibited a network of microfibrils, typical of bacterial cellulose. Although cellulose production is widely distributed between several bacterial species, including at least the groups of Gram-negative proteobacteria alpha and gamma, we give for the first time experimental evidence for cellulose production in beta-proteobacteria.

Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury

NASA Technical Reports Server (NTRS)

Stauber, W. T.; Fritz, V. K.; Burkovskaia, T. E.; Il'ina-Kakueva, E. I.

1992-01-01

The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on Cosmos 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with nonenlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

PubMed

Somogyi, Eszter; Petersson, Ulrika; Hultenby, Kjell; Wendel, Mikael

2003-04-01

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.

Ultrasound Technologies for the Spatial Patterning of Cells and Extracellular Matrix Proteins and the Vascularization of Engineered Tissue

NASA Astrophysics Data System (ADS)

Garvin, Kelley A.

Technological advancements in the field of tissue engineering could save the lives of thousands of organ transplant patients who die each year while waiting for donor organs. Currently, two of the primary challenges preventing tissue engineers from developing functional replacement tissues and organs are the need to recreate complex cell and extracellular microenvironments and to vascularize the tissue to maintain cell viability and function. Ultrasound is a form of mechanical energy that can noninvasively and nondestructively interact with tissues at the cell and protein level. In this thesis, novel ultrasound-based technologies were developed for the spatial patterning of cells and extracellular matrix proteins and the vascularization of three-dimensional engineered tissue constructs. Acoustic radiation forces associated with ultrasound standing wave fields were utilized to noninvasively control the spatial organization of cells and cell-bound extracellular matrix proteins within collagen-based engineered tissue. Additionally, ultrasound induced thermal mechanisms were exploited to site-specifically pattern various extracellular matrix collagen microstructures within a single engineered tissue construct. Finally, ultrasound standing wave field technology was used to promote the rapid and extensive vascularization of three-dimensional tissue constructs. As such, the ultrasound technologies developed in these studies have the potential to provide the field of tissue engineering with novel strategies to spatially pattern cells and extracellular matrix components and to vascularize engineered tissue, and thus, could advance the fabrication of functional replacement tissues and organs in the field of tissue engineering.

Extracellular matrix scaffold as a tubular graft for ascending aorta aneurysm repair.

PubMed

Abu Saleh, Walid K; Al Jabbari, Odeaa; Grande-Allen, Jane; Ramchandani, Mahesh

2015-08-01

Although extracellular xenograft repair has produced encouraging results when applied to cardiac, valvular, and specific aortic defects, its employment as a tube graft to replace the ascending aorta has not been reported. We describe a patient who underwent resection and replacement of an infected ascending aortic graft with an extracellular matrix conduit. The patient did well, but 14 months later developed a pseudoaneurysm from the staple line used to construct the extracellular matrix conduit. The patient underwent a repeat sternotomy and removal of the graft. Because of the increased risk of graft failure, a homograft was felt to be more appropriate in this setting. Ultimately, we were unable to implant the homograft because it was too small for the aortic root; therefore we decided to construct a tubular graft from Cormatrix extracellular matrix (CorMatrix, Roswell, GA, USA). Fourteen months later, he presented with shortness of breath. Computed tomography scan revealed a 3.5 cm pseudoaneurysm of the ascending aorta. It appeared as if there was a disruption of the staple line in the extra cellular matrix graft. The plan was to replace it with a Dacron graft. The Cormatrix graft material was removed and sent for culture and histological analysis. A 28-mm Gel weave graft (Terumo Cardiovascular Systems, Ann Arbor, MI, USA) was implanted. The patient tolerated the procedure well with good hemodynamics. Our experience suggests that the superior strength, handling characteristics, and resistance to infection make extra cellular matrix scaffold a possible alternative conduit to cryopreserved homografts. Applicability as an aortic conduit merits further investigation to better understand behavior of extra cellular matrix in this situation. © 2015 Wiley Periodicals, Inc.

Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

PubMed Central

McCain, Megan L.; Yuan, Hongyan; Pasqualini, Francesco S.; Campbell, Patrick H.

2014-01-01

Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease. PMID:24682394

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

PubMed

Asmussen, Niels; Lin, Zhao; McClure, Michael J; Schwartz, Zvi; Boyan, Barbara D

2017-12-09

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs. Copyright © 2017. Published by Elsevier Inc.

Escherichia coli Biofilms Have an Organized and Complex Extracellular Matrix Structure

PubMed Central

Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S.; Dodson, Karen W.; Crowley, Jan R.; Heuser, John; Chapman, Matthew R.; Hadjifrangiskou, Maria; Henderson, Jeffrey P.; Hultgren, Scott J.

2013-01-01

ABSTRACT Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. PMID:24023384

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

PubMed Central

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-01-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method. PMID:24887553

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

NASA Astrophysics Data System (ADS)

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-06-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

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.

Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

PubMed

Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

1994-07-01

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

PubMed

Janson, David; Rietveld, Marion; Mahé, Christian; Saintigny, Gaëlle; El Ghalbzouri, Abdoelwaheb

2017-06-01

Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

PubMed Central

Glass, RH; Aggeler, J; Spindle, A; Pederson, RA; Werb, Z

1983-01-01

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0 PMID:6339525

Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion.

PubMed

Yan, Jing; Nadell, Carey D; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

2017-08-23

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species.Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

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.

Tendon Functional Extracellular Matrix

PubMed Central

Screen, H.R.C.; Birk, D.E.; Kadler, K.E.; Ramirez, F; Young, M.F.

2015-01-01

This article is one of a series, summarising views expressed at the Orthopaedic Research Society New Frontiers in Tendon Research Conference. This particular article reviews the three workshops held under the “Functional Extracellular Matrix” stream. The workshops focused on the roles of the tendon extracellular matrix, such as performing the mechanical functions of tendon, creating the local cell environment and providing cellular cues. Tendon is a complex network of matrix and cells, and its biological functions are influenced by widely-varying extrinsic and intrinsic factors such as age, nutrition, exercise levels and biomechanics. Consequently, tendon adapts dynamically during development, ageing and injury. The workshop discussions identified research directions associated with understanding cell-matrix interactions to be of prime importance for developing novel strategies to target tendon healing or repair. PMID:25640030

Mineralization of the Sea Urchin Skeleton

NASA Astrophysics Data System (ADS)

Wilt, F.

2001-12-01

The sea urchin possess a calcareous skeleton composed of over 99% magnesian calcite,an enveloping extracellular matrix, and an occluded protein matrix. The most intensively studied skeletal element is the spicule of the embryo. At the 32 cell stage of development a cohort of 4 cells becomes irrevocably dedicated to spicule formation. At the early gastrula stage the descendants of these founder cells form the primary mesenchyme (PMC). The PMCs fuse to form a multinucleated syncytium connected by cytoplasmic cables, and the calcitic skeleton is formed within these cables. Our primary concern is with the cellular and molecular mechanisms that support the formation of the mineralized spicules. The import of calcium into the PMCs results in appearance of intracellular vesicles containing precipitated calcium, which is neither very stable nor birefringent, and could be amorphous. The precipitated calcium is vectorially secreted into an extracellular space. This space is almost completely enclosed by cytoplasmic strands, and the mineral is encased in an extracellular matrix. Proteins destined for the extracellular matrix, and for inclusion in the spicule, are present in the Golgi membranes and in small intracellular vesicles. These vesicles apparently deliver the matrix proteins to the growing spicule. Our current view is that the matrix molecules are much more than a passive armature, but are actively involved in precipitation, secretion, and organization of the mineral phase.

The impact of quercetin on wound healing relates to changes in αV and β1 integrin expression.

PubMed

Doersch, Karen M; Newell-Rogers, M Karen

2017-08-01

Overly fibrotic wound healing can lead to excess scar formation, causing functional impairment and undesirable cosmetic results. However, there are few successful treatments available to prevent or remediate scars. This study sought to explore the molecular mechanisms by which quercetin, a naturally-occurring antifibrotic agent, diminishes scar formation. Using both mice and fibroblast cells, we examined quercetin's impact on fibrosis and the wound healing rate, and potential molecular mechanisms underlying the quercetin-mediated reduction of fibrosis. While cultured fibroblasts demonstrated normal growth in response to quercetin, quercetin increased surface αV integrin and decreased β1 integrin. These changes in surface integrin expression may impact factors that contribute to fibrosis including cell migration, proliferation, and extracellular matrix production. In both quercetin-treated and control mice, wounds healed in about 14 days. Masson's trichrome stain revealed diminished fibrosis at the wound site in quercetin-treated animals despite the normal healing rate, indicating the potential for better cosmetic results without delaying healing. An in vitro scratch wound model using cells plated on an artificial extracellular matrix demonstrated delayed closure following quercetin treatment. The extracellular matrix also ameliorated quercetin's effect on αV integrin. Thus, αV integrin recruitment in response to quercetin treatment may promote the quercetin-mediated decrease extracellular matrix because cells require less extracellular matrix to migrate into a wound. With added extracellular matrix, β1 integrin remained diminished in response to quercetin, indicating that quercetin's effect on β1 integrin expression is independent of extracellular matrix -mediated signaling and is likely driven by inhibition of the intracellular mechanisms driving β1 expression. These findings suggest that quercetin could alter the cells' interactions with the extracellular matrix through the regulation of integrin expression to promote a decrease in fibrosis. Furthermore, this work demonstrates that this naturally occurring and commercially available supplement could be used to improve wound healing by impacting integrin expression, leading to a lower extracellular matrix requirement to achieve healing. Impact statement Scar formation during wound healing can be problematic for patients but there are limited therapies available to treat or prevent excess fibrosis at wound sites. This work examines the impact of quercetin, a flavonoid that decreases fibrosis, on wound healing, and relates quercetin's effects to changes in integrin expression on the surface of fibroblast cells. To our knowledge, this is the first report that quercetin alters integrin expression or that this impact may be part of the mechanism by which quercetin prevents fibrosis. This work demonstrates that quercetin can be used to modulate integrin expression and that this effect may in turn reduce fibrosis during wound healing. Furthermore, this paper identifies the modulation of integrin expression as a possible therapeutic target in preventing scars. This information could be used to improve therapeutics to aid in the cosmetic and functional results following wound healing.

HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

PubMed

Lin, Michael K; Yang, Jin; Hsu, Chun Wei; Gore, Anuradha; Bassuk, Alexander G; Brown, Lewis M; Colligan, Ryan; Sengillo, Jesse D; Mahajan, Vinit B; Tsang, Stephen H

2018-05-05

High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture

PubMed Central

Valdetaro, Gisele P.; Aldrovani, Marcela; Padua, Ivan R. M.; Cristovam, Priscila C.; Gomes, José A. P.; Laus, José L.

2016-01-01

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure–induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties. PMID:28018719

Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

PubMed

Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

2016-01-21

Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prediction of Metastasis Using Second Harmonic Generation

DTIC Science & Technology

2016-07-01

extracellular matrix through which metastasizing cells must travel. We and others have demonstrated that tumor collagen structure, as measured with the...algorithm using separate training and validation sets, etc. Keywords: metastasis, overtreatment, extracellular matrix , collagen , second harmonic...optical process called second harmonic generation (SHG), influences tumor metastasis. This suggests that collagen structure may provide prognostic

Leg ulcer treatment outcomes with new ovine collagen extracellular matrix dressing: a retrospective case series.

PubMed

Bohn, Gregory A; Gass, Kimberly

2014-10-01

The purpose of this study was to describe the rate of closure observed in venous leg ulcers during treatment with ovine collagen extracellular matrix dressings and compression. Fourteen patients with 23 wounds were retrospectively evaluated with respect to healing rates, time to closure, and weekly facility charge fees.

Laser-assisted patch clamping: a methodology

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1997-01-01

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

PubMed

Liu, Chang; Tong, Huili; Li, Shufeng; Yan, Yunqin

2018-05-01

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity. © 2018 International Federation for Cell Biology.

Effects of boron derivatives on extracellular matrix formation.

PubMed

Benderdour, M; Van Bui, T; Hess, K; Dicko, A; Belleville, F; Dousset, B

2000-10-01

Boric acid solution (3%) dramatically improves wound healing through action on the extracellular matrix, a finding that has been obtained in vitro. Consequently, investigations are presently underway to produce boronated compounds having a therapeutical effectiveness similar to that of boric acid. On the basis of experimental results obtained with boric acid, we examined the effects of boron derivatives on extracellular matrix formation and degradation and analyzed their potential toxicity by using two biological models (chick embryo cartilage and human fibroblasts). The four boron derivatives tested in this study (triethanolamine borate; N-diethyl-phosphoramidate-propylboronique acid; 2,2 dimethylhexyl-1,3-propanediol-aminopropylboronate and 1,2 propanediol-aminopropylboronate) mimicked the effects of boric acid. They induced a decrease of intracellular concentrations in extracellular matrix macromolecules (proteoglycans, proteins)-associated with an increase of their release in culture medium and stimulated the activity of intra- and extracellular proteases. Similarly to boric acid, these actions occurred after exposure of the cells to concentrations of all boron derivatives without apparent toxic effects. The compounds were found to be more toxic than boric acid itself when concentrations were calculated according to their molecular weight. Nevertheless, these in vitro preliminary results demonstrate effects of boron derivatives that may be of therapeutic benefit in wound repair.

Bottom-Up and Top-Down Solid-State NMR Approaches for Bacterial Biofilm Matrix Composition

PubMed Central

Cegelski, Lynette

2015-01-01

The genomics and proteomics revolutions have been enormously successful in providing crucial “parts lists” for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this Perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The “sum-of-theparts” bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by E. coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in V. cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture. PMID:25797008

Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition.

PubMed

Cegelski, Lynette

2015-04-01

The genomics and proteomics revolutions have been enormously successful in providing crucial "parts lists" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The "sum-of-the-parts" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture. Copyright © 2015 Elsevier Inc. All rights reserved.

Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition

NASA Astrophysics Data System (ADS)

Cegelski, Lynette

2015-04-01

The genomics and proteomics revolutions have been enormously successful in providing crucial "parts lists" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The "sum-of-the-parts" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture.

Preliminary results of recurrent cubital tunnel syndrome treated with neurolysis and porcine extracellular matrix nerve wrap.

PubMed

Papatheodorou, Loukia K; Williams, Benjamin G; Sotereanos, Dean G

2015-05-01

To evaluate the clinical results of revision neurolysis and wrapping with porcine extracellular matrix (AxoGuard Nerve Protector, AxoGen Inc., Alachua, FL) for cubital tunnel syndrome after one previous surgical decompression. Twelve patients with recurrent cubital tunnel syndrome were treated with decompression, porcine extracellular matrix nerve wrap, and minimal medial epicondylectomy (if not previously performed). The average follow-up period was 41 months (range, 24-61 mo). All patients had recurrent symptoms after having previously undergone one surgical decompression. The mean patient age was 45 years (range, 30-58 y). All patients were evaluated subjectively and objectively (pain, satisfaction, static 2-point discrimination, grip strength, and pinch strength). A significant improvement was demonstrated in postoperative pain levels (from 8.5 to 1.7), grip strength (from 41% to 86% of the unaffected side), and pinch strength (from 64% to 83% of the unaffected side). Static 2-point discrimination improved from an average 10.4 mm preoperatively to 7.6 mm postoperatively. Eleven of 12 patients demonstrated 2 mm or more improvement in 2-point discrimination postoperatively. There were no complications related to the use of the porcine extracellular matrix for nerve wrapping. This study found that secondary decompression combined with porcine extracellular matrix nerve wrapping was an effective and safe treatment for patients with recurrent cubital tunnel syndrome. Therapeutic IV. Copyright © 2015 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

NASA Technical Reports Server (NTRS)

Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

2000-01-01

Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

PubMed

Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

2012-12-01

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

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

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissuesmore » in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.« less

Hypoxic Regulation of Functional Extracellular Matrix Elaboration by Nucleus Pulposus Cells in Long-Term Agarose Culture

PubMed Central

Gorth, Deborah J; Lothstein, Katherine E; Chiaro, Joseph A; Farrell, Megan J; Dodge, George R; Elliott, Dawn M; Malhotra, Neil R; Mauck, Robert L; Smith, Lachlan J

2015-01-01

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new, biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc. PMID:25640328

Large-scale production and isolation of Candida biofilm extracellular matrix.

PubMed

Zarnowski, Robert; Sanchez, Hiram; Andes, David R

2016-12-01

The extracellular matrix of biofilm is unique to the biofilm lifestyle, and it has key roles in community survival. A complete understanding of the biochemical nature of the matrix is integral to the understanding of the roles of matrix components. This knowledge is a first step toward the development of novel therapeutics and diagnostics to address persistent biofilm infections. Many of the assay methods needed for refined matrix composition analysis require milligram amounts of material that is separated from the cellular components of these complex communities. The protocol described here explains the large-scale production and isolation of the Candida biofilm extracellular matrix. To our knowledge, the proposed procedure is the only currently available approach in the field that yields milligram amounts of biofilm matrix. This procedure first requires biofilms to be seeded in large-surface-area roller bottles, followed by cell adhesion and biofilm maturation during continuous movement of the medium across the surface of the rotating bottle. The formed matrix is then separated from the entire biomass using sonication, which efficiently removes the matrix without perturbing the fungal cell wall. Subsequent filtration, dialysis and lyophilization steps result in a purified matrix product sufficient for biochemical, structural and functional assays. The overall protocol takes ∼11 d to complete. This protocol has been used for Candida species, but, using the troubleshooting guide provided, it could be adapted for other fungi or bacteria.

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

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

Hansen, R K; Bissell, M J

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellularmore » matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.« less

Material properties of biofilms – key methods for understanding permeability and mechanics

PubMed Central

Billings, Nicole; Birjiniuk, Alona; Samad, Tahoura S.; Doyle, Patrick S.; Ribbeck, Katharina

2015-01-01

Microorganisms can form biofilms, which are multicellular communities surrounded by a hydrated extracellular matrix of polymers. Central properties of the biofilm are governed by this extracellular matrix, which provides mechanical stability to the three-dimensional biofilm structure, regulates the ability of the biofilm to adhere to surfaces, and determines the ability of the biofilm to adsorb gasses, solutes, and foreign cells. Despite their critical relevance for understanding and eliminating of biofilms, the materials properties of the extracellular matrix are understudied. Here, we offer the reader a guide to current technologies that can be utilized to specifically assess the permeability and mechanical properties of the biofilm matrix and its interacting components. In particular, we highlight technological advances in instrumentation and interactions between multiple disciplines that have broadened the spectrum of methods available to conduct these studies. We review pioneering work that furthers our understanding of the material properties of biofilms. PMID:25719969

Engineering micropatterned surfaces to modulate the function of vascular stem cells.

PubMed

Li, Jennifer; Wu, Michelle; Chu, Julia; Sochol, Ryan; Patel, Shyam

2014-02-21

Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymer surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces. Copyright © 2014 Elsevier Inc. All rights reserved.

Material properties of biofilms—a review of methods for understanding permeability and mechanics

NASA Astrophysics Data System (ADS)

Billings, Nicole; Birjiniuk, Alona; Samad, Tahoura S.; Doyle, Patrick S.; Ribbeck, Katharina

2015-02-01

Microorganisms can form biofilms, which are multicellular communities surrounded by a hydrated extracellular matrix of polymers. Central properties of the biofilm are governed by this extracellular matrix, which provides mechanical stability to the 3D biofilm structure, regulates the ability of the biofilm to adhere to surfaces, and determines the ability of the biofilm to adsorb gases, solutes, and foreign cells. Despite their critical relevance for understanding and eliminating of biofilms, the materials properties of the extracellular matrix are understudied. Here, we offer the reader a guide to current technologies that can be utilized to specifically assess the permeability and mechanical properties of the biofilm matrix and its interacting components. In particular, we highlight technological advances in instrumentation and interactions between multiple disciplines that have broadened the spectrum of methods available to conduct these studies. We review pioneering work that furthers our understanding of the material properties of biofilms.

Specialisation of extracellular matrix for function in tendons and ligaments

PubMed Central

Birch, Helen L.; Thorpe, Chavaunne T.; Rumian, Adam P.

2013-01-01

Summary Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures. PMID:23885341

The Extracellular Protease Matrix Metalloproteinase-9 Is Activated by Inhibitory Avoidance Learning and Required for Long-Term Memory

ERIC Educational Resources Information Center

Nagy, Vanja; Bozdagi, Ozlem; Huntley, George W.

2007-01-01

Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the…

Altered Liver Proteoglycan/Glycosaminoglycan Structure as a Manifestation of Extracellular Matrix Remodeling upon BCG-induced Granulomatosis in Mice.

PubMed

Kim, L B; Shkurupy, V A; Putyatina, A N

2017-01-01

Experimental BCG-induced granulomatosis in mice was used to study changes in the dynamics of individual liver proteoglycan components reflecting phasic extracellular matrix remodeling, determined by the host-parasite interaction and associated with granuloma development. In the early BCG-granulomatosis period, the increase in individual proteoglycan components promotes granuloma formation, providing conditions for mycobacteria adhesion to host cells, migration of phagocytic cells from circulation, and cell-cell interaction leading to granuloma development and fibrosis. Later, reduced reserve capacity of the extracellular matrix, development of interstitial fibrosis and granuloma fibrosis can lead to trophic shortage for cells within the granulomas, migration of macrophages out of them, and development of spontaneous necrosis and apoptosis typical of tuberculosis.

Advanced techniques for in situ analysis of the biofilm matrix (structure, composition, dynamics) by means of laser scanning microscopy.

PubMed

Neu, Thomas R; Lawrence, John R

2014-01-01

The extracellular constituents in bioaggregates and biofilms can be imaged four dimensionally by using laser scanning microscopy. In this protocol we provide guidance on how to examine the various extracellular compartments in between microbial cells and communities associated with interfaces. The current options for fluorescence staining of matrix compounds and extracellular microhabitats are presented. Furthermore, practical aspects are discussed and useful notes are added. The chapter ends with a brief introduction to other approaches for EPS analysis and an outlook for future needs.

Effects of transforming growth factor-beta1 on cell motility, collagen gel contraction, myofibroblastic differentiation, and extracellular matrix expression of human adipose-derived stem cell.

PubMed

Kakudo, Natsuko; Kushida, Satoshi; Suzuki, Kenji; Ogura, Tsunetaka; Notodihardjo, Priscilla Valentin; Hara, Tomoya; Kusumoto, Kenji

2012-12-01

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, and their usefulness in plastic surgery has garnered attention in recent years. Although, there have been expectations that ASCs might function in wound repair and regeneration, no studies to date have examined the role of ASCs in the mechanism that promotes wound-healing. Transforming growth factor-beta1 (TGF-β1) is a strong candidate cytokine for the triggering of mesenchymal stem cell migration, construction of extracellular matrices, and differentiation of ASCs into myofibroblasts. Cell proliferation, motility, and differentiation, as well as extracellular matrix production, play an important role in wound-healing. We have evaluated the capacity of ASCs to proliferate and their potential to differentiate into phenotypic myofibroblasts, as well as their cell motility and collagen gel contraction ability, when cultured with TGF-β1. Cell motility was analyzed using a wound-healing assay. ASCs that differentiated into myofibroblasts expressed the gene for alpha-smooth muscle actin, and its protein expression was detected immunohistochemically. The extracellular matrix expression in ASCs was evaluated using real-time RT-PCR. Based on the results, we conclude that human ASCs have the potential for cell motility, extracellular matrix gene expression, gel contraction, and differentiation into myofibroblasts and, therefore, may play an important role in the wound-healing process.

Different properties of skin of different body sites: The root of keloid formation?

PubMed

Butzelaar, Liselotte; Niessen, Frank B; Talhout, Wendy; Schooneman, Dennis P M; Ulrich, Magda M; Beelen, Robert H J; Mink van der Molen, Aebele B

2017-09-01

The purpose of this study was to examine extracellular matrix composition, vascularization, and immune cell population of skin sites prone to keloid formation. Keloids remain a complex problem, posing esthetical as well as functional difficulties for those affected. These scars tend to develop at anatomic sites of preference. Mechanical properties of skin vary with anatomic location and depend largely on extracellular matrix composition. These differences in extracellular matrix composition, but also vascularization and resident immune cell populations might play a role in the mechanism of keloid formation. To examine this hypothesis, skin samples of several anatomic locations were taken from 24 human donors within zero to 36 hours after they had deceased. Collagen content and cross-links were determined through high-performance liquid chromatography. The expression of several genes, involved in extracellular matrix production and degradation, was measured by means of real-time PCR. (Immuno)histochemistry was performed to detect fibroblasts, collagen, elastin, blood vessels, Langerhans cells, and macrophages. Properties of skin of keloid predilections sites were compared to properties of skin from other locations (nonpredilection sites [NPS]). The results indicated that there are site specific variations in extracellular matrix properties (collagen and cross-links) as well as macrophage numbers. Moreover, predilection sites (PS) for keloid formation contain larger amounts of collagen compared to NPS, but decreased numbers of macrophages, in particular classically activated CD40 positive macrophages. In conclusion, the altered (histological, protein, and genetic) properties of skin of keloid PS may cause a predisposition for and contribute to keloid formation. © 2017 by the Wound Healing Society.

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

NASA Technical Reports Server (NTRS)

Ehrhart, E. J.; Gillette, E. L.; Barcellos-Hoff, M. H.; Chaterjee, A. (Principal Investigator)

1996-01-01

High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

PubMed

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

[Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation].

PubMed

Zheng, Zefeng; Shen, Weiliang; Le, Huihui; Dai, Xuesong; Ouyang, Hongwei; Chen, Weishan

2016-03-01

To investigate the effects of three-dimensional parallel collagen scaffold on the cell shape, arrangement and extracellular matrix formation of tendon stem cells. Parallel collagen scaffold was fabricated by unidirectional freezing technique, while random collagen scaffold was fabricated by freeze-drying technique. The effects of two scaffolds on cell shape and extracellular matrix formation were investigated in vitro by seeding tendon stem/progenitor cells and in vivo by ectopic implantation. Parallel and random collagen scaffolds were produced successfully. Parallel collagen scaffold was more akin to tendon than random collagen scaffold. Tendon stem/progenitor cells were spindle-shaped and unified orientated in parallel collagen scaffold, while cells on random collagen scaffold had disorder orientation. Two weeks after ectopic implantation, cells had nearly the same orientation with the collagen substance. In parallel collagen scaffold, cells had parallel arrangement, and more spindly cells were observed. By contrast, cells in random collagen scaffold were disorder. Parallel collagen scaffold can induce cells to be in spindly and parallel arrangement, and promote parallel extracellular matrix formation; while random collagen scaffold can induce cells in random arrangement. The results indicate that parallel collagen scaffold is an ideal structure to promote tendon repairing.

Interaction between the extracellular matrix and lymphatics - consequences for lymphangiogenesis and lymphatic function

PubMed Central

Wiig, Helge; Keskin, Doruk; Kalluri, Raghu

2014-01-01

The lymphatic system is important for body fluid balance as well as immunological surveillance. Due to the identification of new molecular markers during the last decade, there has been a recent dramatic increase in our knowledge on the molecular mechanisms involved in lymphatic vessel growth (lymphangiogenesis) and lymphatic function. Here we review data showing that although it is often overlooked, the extracellular matrix plays an important role in the generation of new lymphatic vessels as a response to physiological and pathological stimuli. Extracellular matrix-lymphatic interactions as well as biophysical characteristics of the stroma have consequences for tumor formation, growth and metastasis. During the recent years, anti-lymphangiogenesis has emerged as an additional therapeutic modality to the clinically applied anti-angiogenesis strategy. Oppositely, enhancement of lymphangiogenesis in situations of lymph accumulation is seen as a promising strategy to a set of conditions where few therapeutic avenues are available. Knowledge on the interaction between the extracellular matrix and the lymphatics may enhance our understanding of the underlying mechanisms and may ultimately lead to better therapies for conditions where reduced or increased lymphatic function is the therapeutic target PMID:20727409

Paracrine signaling in a bacterium.

PubMed

López, Daniel; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2009-07-15

Cellular differentiation is triggered by extracellular signals that cause target cells to adopt a particular fate. Differentiation in bacteria typically involves autocrine signaling in which all cells in the population produce and respond to the same signal. Here we present evidence for paracrine signaling in bacterial populations-some cells produce a signal to which only certain target cells respond. Biofilm formation in Bacillus involves two centrally important signaling molecules, ComX and surfactin. ComX triggers the production of surfactin. In turn, surfactin causes a subpopulation of cells to produce an extracellular matrix. Cells that produced surfactin were themselves unable to respond to it. Likewise, once surfactin-responsive cells commenced matrix production, they no longer responded to ComX and could not become surfactin producers. Insensitivity to ComX was the consequence of the extracellular matrix as mutant cells unable to make matrix responded to both ComX and surfactin. Our results demonstrate that extracellular signaling was unidirectional, with one subpopulation producing a signal and a different subpopulation responding to it. Paracrine signaling in a bacterial population ensures the maintenance, over generations, of particular cell types even in the presence of molecules that would otherwise cause those cells to differentiate into other cell types.

An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus.

PubMed

Dengler, Vanina; Foulston, Lucy; DeFrancesco, Alicia S; Losick, Richard

2015-12-01

Staphylococcus aureus is an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA, and polysaccharide. The exact compositions and roles of the different components are not fully understood. In this study, we investigated the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase and DNase treatments severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase-treated biofilm, clumping was partially restored, confirming the crucial role of eDNA in the interconnection of cells. On the basis of our results, we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH. Extracellular DNA (eDNA) is an important component of the biofilm matrix of diverse bacteria, but its role in biofilm formation is not well understood. Here we report that in Staphylococcus aureus, eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required to link cells together in the biofilm. These results confirm previous studies that showed that eDNA is an important component of the S. aureus biofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

PubMed Central

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J.; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I.

2015-01-01

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro. PMID:25736020

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

PubMed

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

2015-03-04

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

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.

Serum, liver, and lung levels of the major extracellular matrix components at the early stage of BCG-induced granulomatosis depending on the infection route.

PubMed

Kim, L B; Shkurupy, V A; Putyatina, A N

2015-01-01

Experiments on the model of mouse BCG-induced granulomatous showed that the content of glycosaminoglycans and proteoglycans in the extracellular matrix of the liver and lungs are changed at the early stages of inflammation (days 3 and 30 postinfection) before cell destruction in the organs begins. This is related to degradation of extracellular matrix structures. Their high content in the blood and interstitium probably contributes to the formation of granulomas, fibroblast proliferation and organ fibrosis. These processes depend on the infection route that determines different conditions for generalization of the inflammation process. Intravenous method of vaccine injection is preferable to use when designing the experiments simulating tuberculosis granulomatosis, especially for the analysis of its early stages.

Microfluidic vascularized bone tissue model with hydroxyapatite-incorporated extracellular matrix.

PubMed

Jusoh, Norhana; Oh, Soojung; Kim, Sudong; Kim, Jangho; Jeon, Noo Li

2015-10-21

Current in vitro systems mimicking bone tissues fail to fully integrate the three-dimensional (3D) microvasculature and bone tissue microenvironments, decreasing their similarity to in vivo conditions. Here, we propose 3D microvascular networks in a hydroxyapatite (HA)-incorporated extracellular matrix (ECM) for designing and manipulating a vascularized bone tissue model in a microfluidic device. Incorporation of HA of various concentrations resulted in ECM with varying mechanical properties. Sprouting angiogenesis was affected by mechanically modulated HA-extracellular matrix interactions, generating a model of vascularized bone microenvironment. Using this platform, we observed that hydroxyapatite enhanced angiogenic properties such as sprout length, sprouting speed, sprout number, and lumen diameter. This new platform integrates fibrin ECM with the synthetic bone mineral HA to provide in vivo-like microenvironments for bone vessel sprouting.

The Molecular Basis of Memory

PubMed Central

2012-01-01

We propose a tripartite biochemical mechanism for memory. Three physiologic components are involved, namely, the neuron (individual and circuit), the surrounding neural extracellular matrix, and the various trace metals distributed within the matrix. The binding of a metal cation affects a corresponding nanostructure (shrinking, twisting, expansion) and dielectric sensibility of the chelating node (address) within the matrix lattice, sensed by the neuron. The neural extracellular matrix serves as an electro-elastic lattice, wherein neurons manipulate multiple trace metals (n > 10) to encode, store, and decode coginive information. The proposed mechanism explains brains low energy requirements and high rates of storage capacity described in multiples of Avogadro number (NA = 6 × 1023). Supportive evidence correlates memory loss to trace metal toxicity or deficiency, or breakdown in the delivery/transport of metals to the matrix, or its degradation. Inherited diseases revolving around dysfunctional trace metal metabolism and memory dysfunction, include Alzheimer's disease (Al, Zn, Fe), Wilson’s disease (Cu), thalassemia (Fe), and autism (metallothionein). The tripartite mechanism points to the electro-elastic interactions of neurons with trace metals distributed within the neural extracellular matrix, as the molecular underpinning of “synaptic plasticity” affecting short-term memory, long-term memory, and forgetting. PMID:23050060

Engineering micropatterned surfaces to modulate the function of vascular stem cells

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

Li, Jennifer; Wu, Michelle; Chu, Julia

2014-02-21

Highlights: • We examine vascular stem cell function on microgrooved and micropost patterned polymer substrates. • 10 μm microgrooved surfaces significantly lower VSC proliferation but do not modulate calcified matrix deposition. • Micropost surfaces significantly lower VSC proliferation and decrease calcified matrix deposition. - Abstract: Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymermore » surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10 μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces.« less

New intracellular activities of matrix metalloproteinases shine in the moonlight.

PubMed

Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

2017-11-01

Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

PubMed

Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

2015-08-01

A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Aggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838).

PubMed

Morawski, M; Brückner, G; Jäger, C; Seeger, G; Künzle, H; Arendt, T

2010-02-03

The Madagascan tenrecs (Afrotheria), an ancient mammalian clade, are characterized by unique brain anatomy. Striking features are an expanded paleocortex but a small and poorly differentiated neocortex devoid of a distinct granular layer IV. To investigate the organization of cortical areas we analyzed extracellular matrix components in perineuronal nets (PNs) using antibodies to aggrecan, lectin staining and hyaluronan-binding protein. Selected subcortical regions were studied to correlate the cortical patterns with features in evolutionary conserved systems. In the neocortex, paleocortex and hippocampus PNs were associated with nonpyramidal neurons. Quantitative analysis in the cerebral cortex revealed area-specific proportions and laminar distribution patterns of neurons ensheathed by PNs. Cortical PNs showed divergent structural phenotypes. Diffuse PNs forming a cotton wool-like perisomatic rim were characteristic of the paleocortex. These PNs were associated with a dense pericellular plexus of calretinin-immunoreactive fibres. Clearly contoured PNs were devoid of a calretinin-positive plexus and predominated in the neocortex and hippocampus. The organization of the extracellular matrix in subcortical nuclei followed the widely distributed mammalian type. We conclude that molecular properties of the aggrecan-based extracellular matrix are conserved during evolution of mammals; however, the matrix scaffold is adapted to specific wiring patterns of cortical and subcortical neuronal networks. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

3D cancer cell migration in a confined matrix

NASA Astrophysics Data System (ADS)

Alobaidi, Amani; Sun, Bo

Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

Novel valve replacement with an extracellular matrix scaffold in an infant with single ventricle physiology.

PubMed

Guariento, Alvise; Burke, Redmond; Fedrigo, Marny; Angelini, Annalisa; Maschietto, Nicola; Vida, Vladimiro; Thiene, Gaetano; Stellin, Giovanni; Padalino, Massimo

2016-01-01

Valve replacement in children with functionally univentricular hearts remains challenging. The absence of small prostheses, the lack of growth, and the need for anticoagulation limit these procedures. We describe a 1-year follow-up of an extracellular matrix scaffold tube used as systemic atrio-ventricular valve in an infant. Copyright © 2015 Elsevier Inc. All rights reserved.

Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

PubMed Central

Seper, Andrea; Fengler, Vera H I; Roier, Sandro; Wolinski, Heimo; Kohlwein, Sepp D; Bishop, Anne L; Camilli, Andrew; Reidl, Joachim; Schild, Stefan

2011-01-01

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae. PMID:22032623

Kindler syndrome.

PubMed

Ashton, G H S

2004-03-01

Kindler syndrome is a rare, autosomal recessive skin fragility disorder characterized by blistering in infancy, followed by photosensitivity and progressive poikiloderma. Ultrastructural examination reveals marked basement membrane reduplication and variable levels of cleavage at the dermal-epidermal junction. The molecular pathology underlying Kindler syndrome has recently been shown to involve loss-of-function mutations in a novel gene, KIND1, encoding kindlin-1. Immunofluorescence, gene expression and cell biology studies have shown that kindlin-1 is expressed mainly in basal keratinocytes and plays a role in the attachment of the actin cytoskeleton via focal contacts to the extracellular matrix. Thus, Kindler syndrome is the first genodermatosis caused by a defect in actin-extracellular matrix linkage rather than the classic keratin-extracellular matrix linkage underlying the pathology of other inherited skin fragility disorders such as epidermolysis bullosa. This article reviews the clinical features as well as the molecular and cellular pathology of Kindler syndrome and highlights the importance of the new protein, kindlin-1, in cell-matrix adhesion and its intriguing link to photosensitivity.

Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

PubMed

Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

2015-10-01

Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

The Extracellular Matrix of Fungal Biofilms.

PubMed

Mitchell, Kaitlin F; Zarnowski, Robert; Andes, David R

A key feature of biofilms is their production of an extracellular matrix. This material covers the biofilm cells, providing a protective barrier to the surrounding environment. During an infection setting, this can include such offenses as host cells and products of the immune system as well as drugs used for treatment. Studies over the past two decades have revealed the matrix from different biofilm species to be as diverse as the microbes themselves. This chapter will review the composition and roles of matrix from fungal biofilms, with primary focus on Candida species, Saccharomyces cerevisiae, Aspergillus fumigatus, and Cryptococcus neoformans. Additional coverage will be provided on the antifungal resistance proffered by the Candida albicans matrix, which has been studied in the most depth. A brief section on the matrix produced by bacterial biofilms will be provided for comparison. Current tools for studying the matrix will also be discussed, as well as suggestions for areas of future study in this field.

Study of the relationship between mononuclear inflammatory infiltrate and Ki-67 and basement membrane and extracellular matrix protein expression in radicular cysts.

PubMed

Mourão, R V C; Júnior, E C Pinheiro; Barros Silva, P G; Turatti, E; Mota, M R L; Alves, A P N N

2016-05-01

To evaluate the relationship between mononuclear inflammatory infiltrate and the expression of a proliferative immunomarker (Ki-67) as well as to evaluate basement membrane and extracellular matrix proteins (laminin and collagen type IV) in radicular cysts and dentigerous cysts (DC). Immunohistochemical analyses were performed in heavily inflamed radicular cysts (HIRC), slightly inflamed radicular cysts (SIRC) and DC (n = 20) using Ki-67 (Dako(®) , 1 : 50), anticollagen type IV (DBS(®) , 1 : 40) and antilaminin (DBS(®) , 1 : 20). The data were analysed using anova/Tukey's test (Ki-67) and Kruskal-Wallis/Dunn's test (collagen type IV and laminin) (P < 0.05). The immunoexpression of Ki-67 was significantly greater in the SIRC group compared with the HIRC and DC (P = 0.0040). Likewise, the immunoexpression of collagen type IV in the basement membrane of the SIRC group was significantly more continuous (P = 0.0475) than in the HIRC group. DC had significantly less collagen type IV in extracellular matrix immunoexpression than HIRC and SIRC (P = 0.0246). Laminin was absent in the basement membrane in the SIRC and DC groups, and the extracellular matrix of the HIRC was weak and punctate. The presence of inflammatory factors in the radicular cyst wall modified the expression of proliferation factors in the epithelial lining and the expression of collagen type IV and laminin in the basement membrane, but did not modify extracellular matrix behaviour in radicular cysts. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

The extracellular matrix of rat pacinian corpuscles: an analysis of its fine structure.

PubMed

Dubový, P; Bednárová, J

1999-12-01

The Pacinian corpuscle consists of a sensory axon terminal that is enveloped by two different structures, the inner core and the capsule. Since proteoglycans are extremely water soluble and are extracted by conventional methods for electron microscopy, the current picture of the structural composition of the extracellular matrix in the inner core and the capsule of the Pacinian corpuscle is incomplete. To study the structural composition of the extracellular matrix of the Pacinian corpuscles, cationic dyes (ruthenium red, alcian blue, acridine orange) and tannic acid were applied simultaneously with the aldehyde fixation. The interosseal Pacinian corpuscles of the rat were fixed either in 2% formaldehyde and 1.5% glutaraldehyde, with the addition of one of these cationic dyes or, in Zamboni's fixative, with tannic acid added. The cationic dyes and tannic acid revealed a different structural pattern of proteoglycans in the extracellular matrix in the inner core and in the capsule of the rat Pacinian corpuscles. The inner core surrounding the sensory axon terminal is a compartment containing proteoglycans that were distributed not only in the extracellular matrix but also in the cytoplasm of the lamellae. In addition, this excitable domain was separated from the capsular fluid by a thick layer of proteoglycans on its surface. An enlarged interlamellar space of the capsule contained large amounts of proteoglycans that were removed by digestion with chondroitinase-ABC. Ruthenium red and alcian blue provided only electron dense granules, probably corresponding to collapsed monomeric proteoglycan molecules. Acridine orange and tannic acid preserved proteoglycans very well and made it possible to visualize them as "bottlebrush" structures in the electron microscope. These results show that the inner core and the capsule of rat Pacinian corpuscles have different structural patterns of proteoglycans, which are probably involved in different functions.

Inhibitory effect of OPC-15161, a component of fungus Thielavia minor, on proliferation and extracellular matrix production of rat cultured hepatic stellate cells.

PubMed

Sugawara, H; Ueno, T; Torimura, T; Inuzuka, S; Tanikawa, K

1998-03-01

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [3H]thymidine were investigated in the presence and absence of interleukin-1beta (IL-1beta). IL-1beta significantly increased the uptake of [3H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1beta, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [3H]-hydroxyproline in the presence and absence of transforming growth factor-beta1 (TGF-beta1). TGF-beta1 significantly increased the production of [3H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca2+ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP3) in the HSCs. IL-1beta induced the increase of intracellular Ca2+ and IP3 concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-1 5161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo.

Escherichia coli biofilms have an organized and complex extracellular matrix structure.

PubMed

Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S; Dodson, Karen W; Crowley, Jan R; Heuser, John; Chapman, Matthew R; Hadjifrangiskou, Maria; Henderson, Jeffrey P; Hultgren, Scott J

2013-09-10

Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy.

PubMed

Edwards, Amanda Nicole; Siuti, Piro; Bible, Amber N; Alexandre, Gladys; Retterer, Scott T; Doktycz, Mitchel J; Morrell-Falvey, Jennifer L

2011-01-01

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition. FEMS Microbiology Letters © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

Extracellular matrix components direct porcine muscle stem cell behavior

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

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

Regulation of Corneal Stroma Extracellular Matrix Assembly

PubMed Central

Chen, Shoujun; Mienaltowski, Michael J.; Birk, David E.

2014-01-01

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. PMID:25819456

Biomechanical regulation of cell orientation and fate

PubMed Central

Lopez, JI; Mouw, JK; Weaver, VM

2009-01-01

Biomechanical regulation of tumor phenotypes have been noted for several decades, yet the function of mechanics in the co-evolution of the tumor epithelium and altered cancer extracellular matrix has not been appreciated until fairly recently. In this review, we examine the dynamic interaction between the developing epithelia and the extracellular matrix, and discuss how similar interactions are exploited by the genetically modified epithelium during tumor progression. We emphasize the process of mechanoreciprocity, which is a phenomenon observed during epithelial transformation, in which tension generated within the extracellular microenvironment induce and cooperate with opposing reactive forces within transformed epithelium to drive tumor progression and metastasis. We highlight the importance of matrix remodeling, and present a new, emerging paradigm that underscores the importance of tissue morphology as a key regulator of epithelial cell invasion and metastasis. PMID:19029939

Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

PubMed

Sivakumar, Krishnakumar; Mukherjee, Manisha; Cheng, Hsin-I; Zhang, Yingdan; Ji, Lianghui; Cao, Bin

2015-03-01

Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations. © 2014 Wiley Periodicals, Inc.

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development

PubMed Central

Pandya, Mirali; Lin, Tiffani; Li, Leo; Allen, Michael J.; Jin, Tianquan; Luan, Xianghong; Diekwisch, Thomas G. H.

2017-01-01

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit. PMID:29089900

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

PubMed

Pandya, Mirali; Lin, Tiffani; Li, Leo; Allen, Michael J; Jin, Tianquan; Luan, Xianghong; Diekwisch, Thomas G H

2017-01-01

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8-16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

The Effects of Simulated Micro-gravity on Cultured Chicken Embryonic Chondrocytes

NASA Astrophysics Data System (ADS)

Li, X.; Zhang, X.; Yang, S.; Li, S.; Peidong, J.; Lin, Z.

T he effects of simulated microgravity on the microtubular system, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration, mitochondrial ATP synthase activity and oligomycin inhibition rate of cultured chicken embryonic chondrocytes were studied with a clinostat. The microtubular content decreased. The extracellualr matrix decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly. There was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. No significant changes happened in the mitochondrial ATP synthase activity and oligomycin inhibition rate. The possible mechanisms about them were discussed.

Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

PubMed

Halper, Jaroslava; Kjaer, Michael

2014-01-01

Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non-elastic extracellular matrixes, and interact closely with tropoelastin and integrins. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Fibulins are tightly connected with basement membranes, elastic fibers and other components of extracellular matrix and participate in formation of elastic fibers. Tenascins are ECM polymorphic glycoproteins found in many connective tissues in the body. Their expression is regulated by mechanical stress both during development and in adulthood. Tenascins mediate both inflammatory and fibrotic processes to enable effective tissue repair and play roles in pathogenesis of Ehlers-Danlos, heart disease, and regeneration and recovery of musculo-tendinous tissue. One of the roles of thrombospondin 1 is activation of TGFβ. Increased expression of thrombospondin and TGFβ activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially with physical activity, loading and post-injury. It plays a role in vascular wall remodeling and has been found in atherosclerotic plaques as well.

SXR, A Novel Target for Breast Cancer Therapeutics

DTIC Science & Technology

2007-04-01

similar, fat-soluble, 2-methyl-1,4-naphthoqui- nones, including phylloquinone (K1), menaquinones (K2), and menadi- one ( K3 ). Vitamin K1 and vitamin K2...xenobiotic receptor SXR mediates vitamin K2- activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells...2003. 89: p. 1-34. 10 Steroid and Xenobiotic Receptor SXR Mediates Vitamin K2- activated Transcription of Extracellular Matrix-related Genes and

Tenascin-C mimetic Peptide nanofibers direct stem cell differentiation to osteogenic lineage.

PubMed

Sever, Melike; Mammadov, Busra; Guler, Mustafa O; Tekinay, Ayse B

2014-12-08

Extracellular matrix contains various signals for cell surface receptors that regulate cell fate through modulation of cellular activities such as proliferation and differentiation. Cues from extracellular matrix components can be used for development of new materials to control the stem cell fate. In this study, we achieved control of stem cell fate toward osteogenic commitment by using a single extracellular matrix element despite the contradictory effect of mechanical stiffness. For this purpose, we mimicked bone extracellular matrix by incorporating functional sequence of fibronectin type III domain from native tenascin-C on self-assembled peptide nanofibers. When rat mesenchymal stem cells (rMSCs) were cultured on these peptide nanofibers, alkaline phosphatase (ALP) activity and alizarin red staining indicated osteogenic differentiation even in the absence of osteogenic supplements. Moreover, expression levels of osteogenic marker genes were significantly enhanced revealed by quantitative real-time polymerase chain reaction (qRT-PCR), which showed the remarkable bioactive role of this nanofiber system on osteogenic differentiation. Overall, these results showed that tenascin-C mimetic peptides significantly enhanced the attachment, proliferation, and osteogenic differentiation of rMSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation. Thus, these peptide nanofibers provide a promising new platform for bone regeneration.

Advances in our understanding of the Reinke space.

PubMed

Thibeault, Susan L

2005-06-01

Normal vocal fold vibration depends critically upon the composition of the Reinke space or the lamina propria extracellular matrix. Alterations in the normal composition of the extracellular matrix result in a loss of normal vibratory function. In this article, the present literature on the Reinke space in normal and disease states is reviewed including publications in the multidisciplinary fields of biomechanics, histology, molecular biology, and tissue engineering. With recent technology advances, the etiology for benign lesions has been investigated with computer models and bioreactors. Particular extracellular matrix constituents in various benign vocal fold lesions--fibronectin, fibromodulin and hyaluronan--appear to be involved in altering the viscoelastic properties of the Reinke space. Significant basic science approaches to the investigation of the characterization of the Reinke space in vocal fold scarring has produced several potential future treatment avenues. Tissue-engineering approaches for regeneration of the Reinke space are the most recent addition to the literature showing promising research directions. Voice disorders represent a significant clinical problem. Research attempting to discover the underlying molecular and genetic regulation and homeostasis of the extracellular matrix of the Reinke space are essential. Effective future clinical interventions must be based upon the knowledge of how genetic and biologic features are disturbed in vocal diseases and how they relate to vocal symptoms.

Hyaluronidase and Collagenase Increase the Transfection Efficiency of Gene Electrotransfer in Various Murine Tumors

PubMed Central

Golzio, Muriel; Sersa, Gregor; Escoffre, Jean-Michel; Coer, Andrej; Vidic, Suzana; Teissie, Justin

2012-01-01

Abstract One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy. PMID:21797718

Aberrant Epicardial Adipose Tissue Extracellular Matrix Remodeling in Patients with Severe Ischemic Cardiomyopathy: Insight from Comparative Quantitative Proteomics.

PubMed

Jiang, Ding-Sheng; Zeng, Hao-Long; Li, Rui; Huo, Bo; Su, Yun-Shu; Fang, Jing; Yang, Qing; Liu, Li-Gang; Hu, Min; Cheng, Cai; Zhu, Xue-Hai; Yi, Xin; Wei, Xiang

2017-03-03

There is ample evidence indicating that epicardial adipose tissue (EAT) volume and thickness is positively associated with coronary artery disease (CAD). However, the exact pathological changes in the human EAT after myocardial ischemia remains largely unclear. In the current study, we applied a comparative quantitative proteomics to elucidate the altered biological processes in the EAT of ischemic cardiomyopathy (ICM) patients. A total of 1649 proteins were successfully quantified in our study, among which 165 proteins were significantly changed (ratio <0.8 or >1.2 fold and p < 0.05 in both repetitions) in EAT of ICM individuals. Gene ontology (GO) enrichment analysis revealed that cardiac structure and cellular metabolism were over-represented among these regulated proteins. The hypertrophic cardiomyopathy, adrenergic signaling in cardiomyocytes, extracellular matrix (ECM)-receptor interaction, phagosome, Glycolysis/Gluconeogenesis, and PPAR signaling pathway were highlighted by the KEGG PATHWAY analysis. More importantly, we found that the proteins responsible for extracellular matrix organization were dramatically increased in EAT of ICM patients. In addition, the picrosirius red (PSR) staining results showed that the collagen fiber content was prominently increased, which indicated the EAT of ICM individuals underwent extracellular matrix remodeling and ERK1/2 activation maybe responsible for these pathological changes partially.

Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.

PubMed

Kyriakides, T R; Zhu, Y H; Yang, Z; Huynh, G; Bornstein, P

2001-10-01

The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-beta(1). The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivo evidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

Investigation of Aspergillus fumigatus biofilm formation by various “omics” approaches

PubMed Central

Muszkieta, Laetitia; Beauvais, Anne; Pähtz, Vera; Gibbons, John G.; Anton Leberre, Véronique; Beau, Rémi; Shibuya, Kazutoshi; Rokas, Antonis; Francois, Jean M.; Kniemeyer, Olaf; Brakhage, Axel A.; Latgé, Jean P.

2013-01-01

In the lung, Aspergillus fumigatus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix called biofilm (BF). This extracellular matrix embeds and glues hyphae together and protects the fungus from an outside hostile environment. This extracellular matrix is absent in fungal colonies grown under classical liquid shake conditions (PL), which were historically used to understand A. fumigatus pathobiology. Recent works have shown that the fungus in this aerial grown BF-like state exhibits reduced susceptibility to antifungal drugs and undergoes major metabolic changes that are thought to be associated to virulence. These differences in pathological and physiological characteristics between BF and liquid shake conditions suggest that the PL condition is a poor in vitro disease model. In the laboratory, A. fumigatus mycelium embedded by the extracellular matrix can be produced in vitro in aerial condition using an agar-based medium. To provide a global and accurate understanding of A. fumigatus in vitro BF growth, we utilized microarray, RNA-sequencing, and proteomic analysis to compare the global gene and protein expression profiles of A. fumigatus grown under BF and PL conditions. In this review, we will present the different signatures obtained with these three “omics” methods. We will discuss the advantages and limitations of each method and their complementarity. PMID:23407341

Targeting the extracellular matrix of ovarian cancer using functionalized, drug loaded lyophilisomes.

PubMed

van der Steen, Sophieke C H A; Raavé, René; Langerak, Sjoerd; van Houdt, Laurens; van Duijnhoven, Sander M J; van Lith, Sanne A M; Massuger, Leon F A G; Daamen, Willeke F; Leenders, William P; van Kuppevelt, Toin H

2017-04-01

Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy. Here, we report on the functionalization of drug-loaded lyophilisomes (albumin-based biocapsules) to specifically target the stroma of ovarian carcinomas with the potential to eliminate cancer cells. To achieve specific targeting, we conjugated single chain antibodies reactive with CS-E to lyophilisomes using a two-step approach comprising sortase-mediated ligation and bioorthogonal click chemistry. Antibody-functionalized lyophilisomes specifically targeted the ovarian cancer stroma through CS-E. In a CS-E rich micro-environment in vitro lyophilisomes induced cell death by extracellular release of doxorubicin which localized to the nucleus. Immunohistochemistry identified CS-E rich stroma in a variety of solid tumors other than ovarian cancer, including breast, lung and colon cancer indicating the potential versatility of matrix therapy and the use of highly sulfated chondroitin sulfates in cancer stroma as a micro-environmental hook for targeted drug delivery. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The extracellular matrix in myocardial injury, repair, and remodeling

PubMed Central

2017-01-01

The cardiac extracellular matrix (ECM) not only provides mechanical support, but also transduces essential molecular signals in health and disease. Following myocardial infarction, dynamic ECM changes drive inflammation and repair. Early generation of bioactive matrix fragments activates proinflammatory signaling. The formation of a highly plastic provisional matrix facilitates leukocyte infiltration and activates infarct myofibroblasts. Deposition of matricellular proteins modulates growth factor signaling and contributes to the spatial and temporal regulation of the reparative response. Mechanical stress due to pressure and volume overload and metabolic dysfunction also induce profound changes in ECM composition that contribute to the pathogenesis of heart failure. This manuscript reviews the role of the ECM in cardiac repair and remodeling and discusses matrix-based therapies that may attenuate remodeling while promoting repair and regeneration. PMID:28459429

Ultrastructure and biological function of matrix vesicles in bone mineralization.

PubMed

Hasegawa, Tomoka

2018-04-01

Bone mineralization is initiated by matrix vesicles, small extracellular vesicles secreted by osteoblasts, inducing the nucleation and subsequent growth of calcium phosphate crystals inside. Although calcium ions (Ca 2+ ) are abundant throughout the tissue fluid close to the matrix vesicles, the influx of phosphate ions (PO4 3- ) into matrix vesicles is a critical process mediated by several enzymes and transporters such as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis (ANK), and tissue nonspecific alkaline phosphatase (TNSALP). The catalytic activity of ENPP1 in osteoblasts generates inorganic pyrophosphate (PPi) intracellularly and extracellularly, and ANK may allow the intracellular PPi to pass through the plasma membrane to the outside of the osteoblasts. Although the extracellular PPi binds to growing hydroxyapatite crystals to prevent crystal overgrowth, TNSALP on the osteoblasts and matrix vesicles hydrolyzes PPi into PO4 3- monomers: the prevention of crystal growth is blocked, and PO4 3- monomers are supplied to matrix vesicles. In addition, PHOSPHO1 is thought to function inside matrix vesicles to catalyze phosphocoline, a constituent of the plasma membrane, consequently increasing PO4 3- in the vesicles. Accumulation of Ca 2+ and PO4 3- inside the matrix vesicles then initiates crystalline nucleation associated with the inner leaflet of the matrix vesicles. Calcium phosphate crystals elongate radially, penetrate the matrix vesicle's membrane, and finally grow out of the vesicles to form calcifying nodules, globular assemblies of needle-shaped mineral crystals retaining some of those transporters and enzymes. The subsequent growth of calcifying nodules appears to be regulated by surrounding organic compounds, finally leading to collagen mineralization.

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

PubMed

Fung, Ashley K; Paredes, J J; Andarawis-Puri, Nelly

2018-01-23

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing. Copyright © 2017. Published by Elsevier Ltd.

Ultrastructure of collagen fibers and distribution of extracellular matrix in the temporomandibular disk of the human fetus and adult.

PubMed

Takahashi, H; Sato, I

2001-12-01

We quantitatively examined the distribution of these differences in extracellular matrices (collagen types I, III, and fibronectin) and elastic fibers under confocal laser scanning microscopy and electron scanning microscopy in terms of their contribution to the mechanics of the TMJ during development and in adults. Elastic fibers were found in the anterior and posterior bands in adults aged 40 years, and a few elastic fibers in the anterior band of the disk in adults aged 80 to 90 years. The extracellular matrix contents of the TMJ disk are shown in various detected levels in the anterior, intermediate, posterior bands of TMJ disk. During development, collagen fibers are arranged in a complex fashion from 28 weeks' gestation. These ultrastructures of the embryonic TMJ are resembled to that of adults aged the 40s, however the difference in extracellular matrix distribution found in embryonic stages and adults. They might reflect the differences in function between mastication and sucking or the changes in shape and form as results of functional disorders of the TMJ.

Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo.

PubMed

Levay, Agata K; Peacock, Jacqueline D; Lu, Yinhui; Koch, Manuel; Hinton, Robert B; Kadler, Karl E; Lincoln, Joy

2008-10-24

Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx(-/-) mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx(-/-) mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

The effects of simulated microgravity on cultured chicken embryonic chondrocytes

NASA Astrophysics Data System (ADS)

Zhang, X.; Li, X. B.; Yang, S. Z.; Li, S. G.; Jiang, P. D.; Lin, Z. H.

2003-10-01

Using the cultured chicken embryonic chondrocytes as a model, the effects of simulated microgravity on the microtubular system of the cellular skeleton, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration and mitochondrial ATP synthase activity with its oligomycin inhibition rate were studied with a clinostat. The microtubular content was measured by a flow cytometer. The decrease of microtubular content showed the impairment of the cellular skeleton system. Observation on the extracellualr matrix by the scanning electron microscopy showed that it decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly than that of the control group. It can be concluded that the simulated microgravity can affect the secreting and assembly of the extracellular matrix. In contrast to the control, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular free calcium may be involved in the regulation of matrix calcification as the second signal transmitter. No significant changes happened in the mitochondrial ATP synthase activity and its oligomycin inhibition rate. Perhaps the energy metabolism wasn't affected by the simulated microgravity. The possible mechanisms about them were discussed.

Role of cells in freezing-induced cell-fluid-matrix interactions within engineered tissues.

PubMed

Seawright, Angela; Ozcelikkale, Altug; Dutton, Craig; Han, Bumsoo

2013-09-01

During cryopreservation, ice forms in the extracellular space resulting in freezing-induced deformation of the tissue, which can be detrimental to the extracellular matrix (ECM) microstructure. Meanwhile, cells dehydrate through an osmotically driven process as the intracellular water is transported to the extracellular space, increasing the volume of fluid for freezing. Therefore, this study examines the effects of cellular presence on tissue deformation and investigates the significance of intracellular water transport and cell-ECM interactions in freezing-induced cell-fluid-matrix interactions. Freezing-induced deformation characteristics were examined through cell image deformetry (CID) measurements of collagenous engineered tissues embedded with different concentrations of MCF7 breast cancer cells versus microspheres as their osmotically inactive counterparts. Additionally, the development of a biophysical model relates the freezing-induced expansion of the tissue due to the cellular water transport and the extracellular freezing thermodynamics for further verification. The magnitude of the freezing-induced dilatation was found to be not affected by the cellular water transport for the cell concentrations considered; however, the deformation patterns for different cell concentrations were different suggesting that cell-matrix interactions may have an effect. It was, therefore, determined that intracellular water transport during freezing was insignificant at the current experimental cell concentrations; however, it may be significant at concentrations similar to native tissue. Finally, the cell-matrix interactions provided mechanical support on the ECM to minimize the expansion regions in the tissues during freezing.

How bacteria hack the matrix and dodge the bullets of immunity.

PubMed

Paulsson, Magnus; Riesbeck, Kristian

2018-06-30

Haemophilus influenzae , Moraxella catarrhalis and Pseudomonas aeruginosa are common Gram-negative pathogens associated with an array of pulmonary diseases. All three species have multiple adhesins in their outer membrane, i.e. surface structures that confer the ability to bind to surrounding cells, proteins or tissues. This mini-review focuses on proteins with high affinity for the components of the extracellular matrix such as collagen, laminin, fibronectin and vitronectin. Adhesins are not structurally related and may be lipoproteins, transmembrane porins or large protruding trimeric auto-transporters. They enable bacteria to avoid being cleared together with mucus by attaching to patches of exposed extracellular matrix, or indirectly adhering to epithelial cells using matrix proteins as bridging molecules. As more adhesins are being unravelled, it is apparent that bacterial adhesion is a highly conserved mechanism, and that most adhesins target the same regions on the proteins of the extracellular matrix. The surface exposed adhesins are prime targets for new vaccines and the interactions between proteins are often possible to inhibit with interfering molecules, e.g heparin. In conclusion, this highly interesting research field of microbiology has unravelled host-pathogen interactions with high therapeutic potential. Copyright ©ERS 2018.

Brain Extracellular Space: The Final Frontier of Neuroscience.

PubMed

Nicholson, Charles; Hrabětová, Sabina

2017-11-21

Brain extracellular space is the narrow microenvironment that surrounds every cell of the central nervous system. It contains a solution that closely resembles cerebrospinal fluid with the addition of extracellular matrix molecules. The space provides a reservoir for ions essential to the electrical activity of neurons and forms an intercellular chemical communication channel. Attempts to reveal the size and structure of the extracellular space using electron microscopy have had limited success; however, a biophysical approach based on diffusion of selected probe molecules has proved useful. A point-source paradigm, realized in the real-time iontophoresis method using tetramethylammonium, as well as earlier radiotracer methods, have shown that the extracellular space occupies ∼20% of brain tissue and small molecules have an effective diffusion coefficient that is two-fifths that in a free solution. Monte Carlo modeling indicates that geometrical constraints, including dead-space microdomains, contribute to the hindrance to diffusion. Imaging the spread of macromolecules shows them increasingly hindered as a function of size and suggests that the gaps between cells are predominantly ∼40 nm with wider local expansions that may represent dead-spaces. Diffusion measurements also characterize interactions of ions and proteins with the chondroitin and heparan sulfate components of the extracellular matrix; however, the many roles of the matrix are only starting to become apparent. The existence and magnitude of bulk flow and the so-called glymphatic system are topics of current interest and controversy. The extracellular space is an exciting area for research that will be propelled by emerging technologies. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nanoscale characterization of effect of L-arginine on Streptococcus mutans biofilm adhesion by atomic force microscopy.

PubMed

Sharma, Shivani; Lavender, Stacey; Woo, JungReem; Guo, Lihong; Shi, Wenyuan; Kilpatrick-Liverman, LaTonya; Gimzewski, James K

2014-07-01

A major aetiological factor of dental caries is the pathology of the dental plaque biofilms. The amino acid L-arginine (Arg) is found naturally in saliva as a free molecule or as a part of salivary peptides and proteins. Plaque bacteria metabolize Arg to produce alkali and neutralize glycolytic acids, promoting a less cariogenous oral microbiome. Here, we explored an alternative and complementary mechanism of action of Arg using atomic force microscopy. The nanomechanical properties of Streptococcus mutans biofilm extracellular matrix were characterized under physiological buffer conditions. We report the effect of Arg on the adhesive behaviour and structural properties of extracellular polysaccharides in S. mutans biofilms. High-resolution imaging of biofilm surfaces can reveal additional structural information on bacterial cells embedded within the surrounding extracellular matrix. A dense extracellular matrix was observed in biofilms without Arg compared to those grown in the presence of Arg. S. mutans biofilms grown in the presence of Arg could influence the production and/or composition of extracellular membrane glucans and thereby affect their adhesion properties. Our results suggest that the presence of Arg in the oral cavity could influence the adhesion properties of S. mutans to the tooth surface. © 2014 The Authors.

Stereological analysis of the epiphyseal growth cartilage in the brachymorphic (bm/bm) mouse, with special reference to the distribution of matrix vesicles.

PubMed

Wikström, B; Hjerpe, A; Hultenby, K; Reinholt, F P; Engfeldt, B

1984-01-01

The brachymorphic (bm/bm) mutation in the mouse leads to disproportional dwarfism due to a disturbance of endochondral bone formation. The morphological characteristics of bm/bm epiphyseal growth cartilage are signs of cellular degeneration and disintegration and alteration of the composition of the extracellular matrix, with an abnormal mineralization pattern. The present stereological study of the bm/bm growth plate revealed a clearly altered distribution of matrix vesicles as compared with the controls. It was also demonstrated that the bm/bm matrix vesicles have an abnormal size distribution, with an increased mean caliper diameter. The biological significance of these findings is discussed in relation to the different hypotheses on the origin of matrix vesicles and their possible role in the mineralization process. The results support the opinion that extracellular matrix vesicles, at least partly, constitute cellular debris.

KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.

PubMed

Aguilar, Claudio; Vlamakis, Hera; Guzman, Alejandra; Losick, Richard; Kolter, Roberto

2010-05-18

Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

KinD Is a Checkpoint Protein Linking Spore Formation to Extracellular-Matrix Production in Bacillus subtilis Biofilms

PubMed Central

Aguilar, Claudio; Vlamakis, Hera; Guzman, Alejandra; Losick, Richard; Kolter, Roberto

2010-01-01

ABSTRACT Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed. PMID:20689749

Matrix Density-induced Mechanoregulation of Breast Cell Phenotype, Signaling, and Gene Expression through a FAK ERK Linkage

DTIC Science & Technology

2009-12-10

sites of integrin-clustering that link the actin cytoskeleton to the extracellular matrix (ECM; (Burridge et al., 1988)). The primary functions of...Hall, 1992). Furthermore, in fibroblasts, focal adhesion kinase (FAK), a key FA signaling molecule, is necessary for mechanosensing (Geiger et al...promotes FAK activation through phosphorylation on Y397 and Y925, followed by FAK- dependent extracellular signal-regulated kinase (ERK) phosphorylation

Collagens--structure, function, and biosynthesis.

PubMed

Gelse, K; Pöschl, E; Aigner, T

2003-11-28

The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramolecular processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutical use of members of this protein family discussed in other chapters of this issue.

Chondroitinase injection improves keloid pathology by reorganizing the extracellular matrix with regenerated elastic fibers.

PubMed

Ishiko, Toshihiro; Naitoh, Motoko; Kubota, Hiroshi; Yamawaki, Satoko; Ikeda, Mika; Yoshikawa, Katsuhiro; Fujita, Hiroshi; Yamaguchi, Hiroaki; Kurahashi, Yasuhiro; Suzuki, Shigehiko

2013-05-01

Keloids are a proliferative fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. Keloid lesions lack skin plasticity due to deficiencies in elastic fiber formation in the extracellular matrix. The loss of elastic fiber is caused by excessive accumulation of chondroitin sulfate (CS), a sulfated glycosaminoglycan. However, there is no radical cure for keloids. Using a model system, we show herein that treatment of keloid tissues with chondroitinase ABC, an enzyme that specifically digests CS, improves clinical features of keloids. Keloid tissues obtained from patients were grafted on nude mice, and chondroitinase ABC was injected into the grafted keloid tissues. Chondroitinase ABC treatment significantly reduced the volume of keloid implants concomitant with recovery of elastic fiber formation. These results suggest that chondroitinase ABC injection is an effective therapy for keloid. © 2013 Japanese Dermatological Association.

Dance band on the Titanic: biomechanical signaling in cardiac hypertrophy.

PubMed

Sussman, Mark A; McCulloch, Andrew; Borg, Thomas K

2002-11-15

Biomechanical signaling is a complex interaction of both intracellular and extracellular components. Both passive and active components are involved in the extracellular environment to signal through specific receptors to multiple signaling pathways. This review provides an overview of extracellular matrix, specific receptors, and signaling pathways for biomechanical stimulation in cardiac hypertrophy.

Regulation of corneal stroma extracellular matrix assembly.

PubMed

Chen, Shoujun; Mienaltowski, Michael J; Birk, David E

2015-04-01

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural alterations of the mucosa stroma in the Barrett's esophagus metaplasia-dysplasia-adenocarcinoma sequence.

PubMed

Bobryshev, Yuri V; Killingsworth, Murray C; Lord, Reginald V N

2012-09-01

Accumulating evidence suggests that the extracellular matrix play important roles in intercellular communications and contribute to the development of a number of diseases, including diseases of the gastrointestinal tract. The present study examined the structural characteristics and alterations of the extracellular matrix of the mucosa stroma in the Barrett's esophagus metaplasia-dysplasia-adenocarcinoma sequence. A total of 41 esophageal tissue specimens (15 esophageal adenocarcinoma, 10 Barrett's esophagus intestinal metaplasia, seven dysplasia and nine normal esophagus) were studied. The present study used transmission electron microscopy and computerized quantitative electron-microscopic analysis in order to investigate the characteristics of the extracellular matrix of the mucosa. The study revealed that marked structural alterations of the mucosa stroma, relating to changes in the distribution and appearance of collagen fibers as well as to changes in numbers of matrix microvesicles, occur in Barrett's esophagus and esophageal adenocarcinoma. It was found that there were 3.1 times more microvesicles in the stroma in Barrett's esophagus than in the stroma of the normal esophagus (P<0.0001) and that there were 5.8 times more microvesicles in esophageal adenocarcinoma than in the normal esophagus (P<0.0001). There were 1.9 times more microvesicles in esophageal adenocarcinoma than in Barrett's esophagus (P=0.0043). The study demonstrates distinctive alterations of the mucosa stroma extracellular matrix in the metaplasia-dysplasia-adenocarcinoma sequence. The findings suggest that the redistribution of collagen fibers and increases in numbers of matrix microvesicles may play roles in the formation of specialized intestinal metaplasia and the development of adenocarcinoma. © 2012 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

Community participation in biofilm matrix assembly and function.

PubMed

Mitchell, Kaitlin F; Zarnowski, Robert; Sanchez, Hiram; Edward, Jessica A; Reinicke, Emily L; Nett, Jeniel E; Mitchell, Aaron P; Andes, David R

2015-03-31

Biofilms of the fungus Candida albicans produce extracellular matrix that confers such properties as adherence and drug resistance. Our prior studies indicate that the matrix is complex, with major polysaccharide constituents being α-mannan, β-1,6 glucan, and β-1,3 glucan. Here we implement genetic, biochemical, and pharmacological approaches to unravel the contributions of these three constituents to matrix structure and function. Interference with synthesis or export of any one polysaccharide constituent altered matrix concentrations of each of the other polysaccharides. Each of these was also required for matrix function, as assessed by assays for sequestration of the antifungal drug fluconazole. These results indicate that matrix biogenesis entails coordinated delivery of the individual matrix polysaccharides. To understand whether coordination occurs at the cellular level or the community level, we asked whether matrix-defective mutant strains could be coaxed to produce functional matrix through biofilm coculture. We observed that mixed biofilms inoculated with mutants containing a disruption in each polysaccharide pathway had restored mature matrix structure, composition, and biofilm drug resistance. Our results argue that functional matrix biogenesis is coordinated extracellularly and thus reflects the cooperative actions of the biofilm community.

Community participation in biofilm matrix assembly and function

PubMed Central

Mitchell, Kaitlin F.; Zarnowski, Robert; Sanchez, Hiram; Edward, Jessica A.; Reinicke, Emily L.; Nett, Jeniel E.; Mitchell, Aaron P.; Andes, David R.

2015-01-01

Biofilms of the fungus Candida albicans produce extracellular matrix that confers such properties as adherence and drug resistance. Our prior studies indicate that the matrix is complex, with major polysaccharide constituents being α-mannan, β-1,6 glucan, and β-1,3 glucan. Here we implement genetic, biochemical, and pharmacological approaches to unravel the contributions of these three constituents to matrix structure and function. Interference with synthesis or export of any one polysaccharide constituent altered matrix concentrations of each of the other polysaccharides. Each of these was also required for matrix function, as assessed by assays for sequestration of the antifungal drug fluconazole. These results indicate that matrix biogenesis entails coordinated delivery of the individual matrix polysaccharides. To understand whether coordination occurs at the cellular level or the community level, we asked whether matrix-defective mutant strains could be coaxed to produce functional matrix through biofilm coculture. We observed that mixed biofilms inoculated with mutants containing a disruption in each polysaccharide pathway had restored mature matrix structure, composition, and biofilm drug resistance. Our results argue that functional matrix biogenesis is coordinated extracellularly and thus reflects the cooperative actions of the biofilm community. PMID:25770218

Human umbilical cord derived matrix: A scaffold suitable for tissue engineering application.

PubMed

Dan, Pan; Velot, Émilie; Mesure, Benjamin; Groshenry, Guillaume; Bacharouche, Jalal; Decot, Véronique; Menu, Patrick

2017-01-01

Human tissue derived natural extracellular matrix (ECM) has great potential in tissue engineering. We sought to isolate extracellular matrix derived from human umbilical cord and test its potential in tissue engineering. An enzymatic method was applied to isolate and solubilized complete human umbilical cord derived matrix (hUCM). The obtained solution was analyzed for growth factors, collagen and residual DNA contents, then used to coat 2D and 3D surfaces for cell culture application. The hUCM was successfully isolated with trypsin digestion to acquire a solution containing various growth factors and collagen but no residual DNA. This hUCM solution can form a coating on 2D and 3D substrates suitable cell culture. We developed a new matrix derived from human source that can be further used in tissue engineering.

In-depth proteomic analysis of shell matrix proteins of Pinctada fucata

PubMed Central

Liu, Chuang; Li, Shiguo; Kong, Jingjing; Liu, Yangjia; Wang, Tianpeng; Xie, Liping; Zhang, Rongqing

2015-01-01

The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment. PMID:26608573

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms.

PubMed

Castillo Pedraza, Midian C; Novais, Tatiana F; Faustoferri, Roberta C; Quivey, Robert G; Terekhov, Anton; Hamaker, Bruce R; Klein, Marlise I

2017-10-01

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA - ∆lytS and ∆lytT; LTA - ∆dltA and ∆dltD; and insoluble exopolysaccharide - ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

PubMed Central

Castillo Pedraza, Midian C.; Novais, Tatiana F.; Faustoferri, Roberta C.; Quivey, Robert G.; Terekhov, Anton; Hamaker, Bruce R.; Klein, Marlise I.

2018-01-01

Streptococcus mutans -derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ΔlytS and ΔlytT; LTA – ΔdltA and ΔdltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms. PMID:28946780

Ultrasonic and electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto a titanium plasma-spray surface.

PubMed

Fassina, Lorenzo; Saino, Enrica; Sbarra, Maria Sonia; Visai, Livia; Cusella De Angelis, Maria Gabriella; Mazzini, Giuliano; Benazzo, Francesco; Magenes, Giovanni

2009-06-01

Several studies suggest that the surface coating of titanium could play an important role in bone tissue engineering. In the present study, we have followed a particular biomimetic strategy where ultrasonically or electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix on a titanium plasma-spray surface. In comparison with control conditions, the ultrasonic stimulation (average power, 149 mW; frequency, 1.5 MHz) and the electromagnetic stimulation (magnetic field intensity, 2 mT; frequency, 75 Hz) caused higher cell proliferation, and increased surface coating with decorin, osteocalcin, osteopontin, and type I collagen together with higher incorporation of calcium and phosphorus inside the extracellular matrix. The immunofluorescence related to the preceding bone matrix proteins showed their colocalization in the cell-rich areas. The use of the two physical stimulations aimed at obtaining the coating of the rough titanium plasma-spray surface in terms of cell colonization and deposition of extracellular matrix. The superficially cultured biomaterial could be theoretically used, in clinical applications, as an implant for bone repair.

Extracellular Superoxide Dismutase Deficiency Exacerbates Pressure Overload–Induced Left Ventricular Hypertrophy and Dysfunction

PubMed Central

Lu, Zhongbing; Xu, Xin; Hu, Xinli; Zhu, Guangshuo; Zhang, Ping; van Deel, Elza D.; French, Joel P.; Fassett, John T.; Oury, Tim D.; Bache, Robert J.; Chen, Yingjie

2008-01-01

Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)–induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload. PMID:17998475

In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.

PubMed

Li, R; Rajan, R; Wong, W C V; Reid, D G; Duer, M J; Somovilla, V J; Martinez-Saez, N; Bernardes, G J L; Hayward, R; Shanahan, C M

2017-12-14

Non-enzymatic glycation of extracellular matrix with (U- 13 C 5 )-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH 3 -CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.

Collagen VI disorders: Insights on form and function in the extracellular matrix and beyond.

PubMed

Lamandé, Shireen R; Bateman, John F

2017-12-22

Mutations in the three canonical collagen VI genes, COL6A1, COL6A2 and COL6A3, cause a spectrum of muscle disease from Bethlem myopathy at the mild end to the severe Ullrich congenital muscular dystrophy. Mutations can be either dominant or recessive and the resulting clinical severity is influenced by the way mutations impact the complex collagen VI assembly process. Most mutations are found towards the N-terminus of the triple helical collagenous domain and compromise extracellular microfibril assembly. Outside the triple helix collagen VI is highly polymorphic and discriminating mutations from rare benign changes remains a major diagnostic challenge. Collagen VI deficiency alters extracellular matrix structure and biomechanical properties and leads to increased apoptosis and oxidative stress, decreased autophagy, and impaired muscle regeneration. Therapies that target these downstream consequences have been tested in a collagen VI null mouse and also in small human trials where they show modest clinical efficacy. An important role for collagen VI in obesity, cancer and diabetes is emerging. A major barrier to developing effective therapies is the paucity of information about how collagen VI deficiency in the extracellular matrix signals the final downstream consequences - the receptors involved and the intracellular messengers await further characterization. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Clinical Usage of an Extracellular, Collagen-rich Matrix: A Case Series.

PubMed

AbouIssa, Abdelfatah; Mari, Walid; Simman, Richard

2015-11-01

OASIS Ultra (Smith and Nephew, St. Petersburg, FL) is an extracellular, collagen-rich matrix derived from submucosa of porcine intestine. It is composed of collagen type I, glycosaminoglycan, and proteoglycans. This extracellular matrix (ECM) differs from the single layer in thickness and offers ease of handling and application. It also stimulates cell migration and structural support, provides moisture environment, decreases inflammation, and induces cell proliferation and cellular attachments. In this case series, the authors present their experience with this product in various clinical scenarios. The authors used the product in a variety of wounds with different etiologies to test the clinical outcome of the ECM. This was an observational case series with prospective review of 6 different patients with different types of wounds who received treatment with the ECM during their treatment. The product was applied on the following types of wounds: chronic venous ulcer, nonhealing Achilles tendon vasculitic wound, Marjolin's ulcer, posttraumatic wound, stage IV sacral-coccygeal pressure wound, and complicated transmetatarsal amputation of gangrenous left forefoot diabetic wound. All of these wounds healed within the expected time periods and without complications. In general, healing was achieved in 4-16 weeks using 1-12 applications of the ECM. Wounds with different etiologies were successfully treated with an extracellular, collagen-rich matrix. By replacing the lost ECM to guide cellular growth and migration, this product did ultimately hasten the healing process.

How Nucleus Mechanics and ECM Microstructure Influence the Invasion of Single Cells and Multicellular Aggregates.

PubMed

Giverso, Chiara; Arduino, Alessandro; Preziosi, Luigi

2018-05-01

In order to move in a three-dimensional extracellular matrix, the nucleus of a cell must squeeze through the narrow spacing among the fibers and, by adhering to them, the cell needs to exert sufficiently strong traction forces. If the nucleus is too stiff, the spacing too narrow, or traction forces too weak, the cell is not able to penetrate the network. In this article, we formulate a mathematical model based on an energetic approach, for cells entering cylindrical channels composed of extracellular matrix fibers. Treating the nucleus as an elastic body covered by an elastic membrane, the energetic balance leads to the definition of a necessary criterion for cells to pass through the regular network of fibers, depending on the traction forces exerted by the cells (or possibly passive stresses), the stretchability of the nuclear membrane, the stiffness of the nucleus, and the ratio of the pore size within the extracellular matrix with respect to the nucleus diameter. The results obtained highlight the importance of the interplay between mechanical properties of the cell and microscopic geometric characteristics of the extracellular matrix and give an estimate for a critical value of the pore size that represents the physical limit of migration and can be used in tumor growth models to predict their invasive potential in thick regions of ECM.

Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling.

PubMed

Li, Y Y; McTiernan, C F; Feldman, A M

2000-05-01

Myocardial fibrosis due to maladaptive extracellular matrix remodeling contributes to dysfunction of the failing heart. Further elucidation of the mechanism by which myocardial fibrosis and dilatation can be prevented or even reversed remains of great interest as a potential means to limit myocardial remodeling and dysfunction. Matrix metalloproteinases (MMPs) are the driving force behind extracellular matrix degradation during remodeling and are increased in the failing human heart. MMPs are regulated by a variety of growth factors, cytokines, and matrix fragments such as matrikines. In the present report, we discuss the regulation of MMPs, the role of MMPs in the development of cardiac fibrosis, and the modulation of MMP activity using gene transfer and knockout technologies. We also present recent findings from our laboratory on the regulation of the extracellular MMP inducer (EMMPRIN), MMPs, and transforming growth factor-beta(1) in the failing human heart before and after left ventricular assist device support, as well as the possibility of preventing ventricular fibrosis using different anti-MMP strategies. Several studies suggest that such modulation of MMP activity can alter ventricular remodeling, myocardial dysfunction, and the progression of heart failure. It is therefore suggested that the interplay of MMPs and their regulators is important in the development of the heart failure phenotype, and myocardial fibrosis in heart failure may be modified by modulating MMP activity.

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

PubMed Central

Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

2016-01-01

The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis. PMID:26811931

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

PubMed Central

Lambers, Christopher; Roth, Michael; Zhong, Jun; Campregher, Christoph; Binder, Petra; Burian, Bernhard; Petkov, Ventzislav; Block, Lutz-Henning

2013-01-01

Background Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor. Objective To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC. Methods PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580. Results ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1. Conclusion and Clinical Relevance ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension. PMID:24015303

[50 years of connective tissue research: from the French Connective Tissue Club to the French Society of Extracellular Matrix Biology].

PubMed

Maquart, François-Xavier; Borel, Jacques-Paul

2012-01-01

The history of connective tissue research began in the late 18th century. However, it is only 50 years later that the concept of connective tissue was shaped. It took another fifty years before biochemical knowledge of extracellular matrix macromolecules began to emerge in the first half of the 20th century. In 1962, thanks to Ladislas and Barbara Robert, back from the US, the first society called "French Connective Tissue Club" was created in Paris. The first board was constituted of Albert Delaunay, Suzanne Bazin and Ladislas Robert. Very quickly, under the influence of these pioneers, national and international meetings were organized and, in 1967, a "Federation of the European Connective Tissue Clubs" was created at the initiative of Ladislas Robert (Paris) and John Scott (Manchester). It spread rapidly to the major European nations. In 1982 the transformation of "Clubs" in "Societies" occurred, a name more in line with the requirements of the time. In 2008, the "French Connective Tissue Society" became the "French Society of Extracellular Matrix Biology" ("Société Française de Biologie de la Matrice Extracellulaire", SFBMEc), to better highlight the importance of the extracellular matrix in the biology of living organisms. The SFBMEc's mission today is to promote and develop scientific exchanges between academic, industrial, and hospital laboratories involved in research on the extracellular matrix. SFBMEc organizes or subsidizes scientific meetings and awards scholarships to Ph.D. students or post-docs to participate in international conferences. It includes 200 to 250 members from different disciplines, developing strong interactions between scientists, clinicians and pathologists. It is present all around the French territory in many research laboratories. During these last 50 years, the extraordinary advances made possible by the development of new investigation techniques, in particular molecular biology, cell and tissue imaging, molecular modeling, etc., have permitted a considerable increase of the knowledge in the field of connective tissue. © Société de Biologie, 2012.

Dynamic interactions between cells and their extracellular matrix mediate embryonic development.

PubMed

Goody, Michelle F; Henry, Clarissa A

2010-06-01

Cells and their surrounding extracellular matrix microenvironment interact throughout all stages of life. Understanding the continuously changing scope of cell-matrix interactions in vivo is crucial to garner insights into both congenital birth defects and disease progression. A current challenge in the field of developmental biology is to adapt in vitro tools and rapidly evolving imaging technology to study cell-matrix interactions in a complex 4-D environment. In this review, we highlight the dynamic modulation of cell-matrix interactions during development. We propose that individual cell-matrix adhesion proteins are best considered as complex proteins that can play multiple, often seemingly contradictory roles, depending upon the context of the microenvironment. In addition, cell-matrix proteins can also exert different short versus long term effects. It is thus important to consider cell behavior in light of the microenvironment because of the constant and dynamic reciprocal interactions occurring between them. Finally, we suggest that analysis of cell-matrix interactions at multiple levels (molecules, cells, tissues) in vivo is critical for an integrated understanding because different information can be acquired from all size scales. Copyright 2010 Wiley-Liss, Inc.

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.

Engineering a collagen matrix that replicates the biological properties of native extracellular matrix.

PubMed

Nam, Kwangwoo; Sakai, Yuuki; Funamoto, Seiichi; Kimura, Tsuyoshi; Kishida, Akio

2011-01-01

In this study, we aimed to replicate the function of native tissues that can be used in tissue engineering and regenerative medicine. The key to such replication is the preparation of an artificial collagen matrix that possesses a structure resembling that of the extracellular matrix. We, therefore, prepared a collagen matrix by fibrillogenesis in a NaCl/Na(2)HPO(4) aqueous solution using a dialysis cassette and investigated its biological behavior in vitro and in vivo. The in vitro cell adhesion and proliferation did not show any significant differences. The degradation rate in the living body could be controlled according to the preparation condition, where the collagen matrix with high water content (F-collagen matrix, >98%) showed fast degradation and collagen matrix with lower water content (T-collagen matrix, >80%) showed no degradation for 8 weeks. The degradation did not affect the inflammatory response at all and relatively faster wound healing response was observed. Comparing this result with that of collagen gel and decellularized cornea, it can be concluded that the structural factor is very important and no cell abnormal behavior would be observed for quaternary structured collagen matrix.

Solid-state NMR for bacterial biofilms

NASA Astrophysics Data System (ADS)

Reichhardt, Courtney; Cegelski, Lynette

2014-04-01

Bacteria associate with surfaces and one another by elaborating an extracellular matrix to encapsulate cells, creating communities termed biofilms. Biofilms are beneficial in some ecological niches, but also contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative measurements are needed to define the composition and architecture of bacterial biofilms to help drive the development of strategies to interfere with biofilm assembly. Solid-state nuclear magnetic resonance (NMR) is uniquely suited to the examination of insoluble and complex macromolecular and whole-cell systems. This article highlights three examples that implement solid-state NMR to deliver insights into bacterial biofilm composition and changes in cell-wall composition as cells transition to the biofilm lifestyle. Most recently, solid-state NMR measurements provided a total accounting of the protein and polysaccharide components in the extracellular matrix of an Escherichia coli biofilm and transformed our qualitative descriptions of matrix composition into chemical parameters that permit quantitative comparisons among samples. We present additional data for whole biofilm samples (cells plus the extracellular matrix) that complement matrix-only analyses. The study of bacterial biofilms by solid-state NMR is an exciting avenue ripe with many opportunities and we close the article by articulating some outstanding questions and future directions in this area.

The Lens Capsule

PubMed Central

Danysh, Brian P.; Duncan, Melinda K.

2009-01-01

The lens capsule is a modified basement membrane that completely surrounds the ocular lens. It is known that this extracellular matrix is important for both the structure and biomechanics of the lens in addition to providing informational cues to maintain lens cell phenotype. This review covers the development and structure of the lens capsule, lens diseases associated with mutations in extracellular matrix genes and the role of the capsule in lens function including those proposed for visual accommodation, selective permeability to infectious agents, and cell signaling. PMID:18773892

The role of the extracellular matrix in primary myelofibrosis

PubMed Central

Leiva, O; Ng, S K; Chitalia, S; Balduini, A; Matsuura, S; Ravid, K

2017-01-01

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm that arises from clonal proliferation of hematopoietic stem cells and leads to progressive bone marrow (BM) fibrosis. While cellular mutations involved in the development of PMF have been heavily investigated, noteworthy is the important role the extracellular matrix (ECM) plays in the progression of BM fibrosis. This review surveys ECM proteins contributors of PMF, and highlights how better understanding of the control of the ECM within the BM niche may lead to combined therapeutic options in PMF. PMID:28157219

Hyaluronic Acid is Overexpressed in Fibrotic Lung Tissue and Promotes Collagen Expression

DTIC Science & Technology

2008-04-01

cause of morbidity and mortality in scleroderma . The overexpression of collagen is accompanied by the overexpression of other extracellular matrix...7 Appendices…………………………………………………………………………… 7 3 INTRODUCTION Systemic scleroderma is a debilitating disease...excessive accumulation of extracellular matrix [ECM] proteins, particularly collagen I) is the major cause of morbidity and mortality in scleroderma . The

Towards evaluating post-irradiation tissue alterations

NASA Astrophysics Data System (ADS)

Daar, Eman; Bradley, D. A.; Alkhorayef, M.; Al-Mugren, K. S.; Abdallat, R. G.; Al-Dousari, H.

2017-08-01

There is apaucity of data concerning irradiation effects on the extracellular matrix and on organised tissues. Examples of such research are cited as are some of the limiting factors towards obtaining meaningful results. This would engender a range of research towards further improving the quality of life, most pointedly of those receiving radiotherapy. As cancer survivor rates increase, survivors are more likely to experience side effects of radiotherapy. This study examines the effects of radiotherapy doses on the extracellular matrix as hyaluronic acid (HA) and pericardium.

Isolation and characterization of chicken bile matrix metalloproteinase

USDA-ARS?s Scientific Manuscript database

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix (ECM) proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies but the significance of their expression in normal, he...

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix.

PubMed

Yu, Shan; Su, Tiantian; Wu, Huijun; Liu, Shiheng; Wang, Di; Zhao, Tianhu; Jin, Zengjun; Du, Wenbin; Zhu, Mei-Jun; Chua, Song Lin; Yang, Liang; Zhu, Deyu; Gu, Lichuan; Ma, Luyan Z

2015-12-01

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.

Use of Collagen Extracellular Matrix Dressing for the Treatment of a Recurrent Venous Ulcer in a 52-Year-Old Patient.

PubMed

González, Arturo

2016-01-01

This case study describes treatment for a 52-year-old man with a recurrent venous leg ulcer using a collagen dressing with extracellular matrix. The patient was admitted to the wound care service for a 3-week-old recurrent venous ulcer. Treatment included application of a collagen dressing with extracellular matrix twice weekly or as needed by the patient; application of a secondary dressing (4 × 4 gauze); and coverage with an expandable netting or gauze using a conforming stretch gauze bandage and latex-free dressing retention tape. The initial venous leg ulcer in this patient required 10 weeks to achieve closure. Ninety-eight percent resolution of the recurrent ulcer had occurred within 4 weeks of treatment, with complete closure at 7 weeks. The average healing time for recurrent venous ulcers is reported in the literature to be longer than initial venous ulcers. In the case provided, collagen ECM dressings promoted complete wound healing in 49 days.

A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

PubMed Central

Reticker-Flynn, Nathan E.; Braga Malta, David F.; Winslow, Monte M.; Lamar, John M.; Xu, Mary J.; Underhill, Gregory H.; Hynes, Richard O.; Jacks, Tyler E.; Bhatia, Sangeeta N.

2013-01-01

Extracellular matrix interactions play essential roles in normal physiology and many pathological processes. While the importance of ECM interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel screening platform capable of measuring phenotypic responses to combinations of ECM molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the ECM-dependent adhesion of tumor-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumor lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8, or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified ECM and integrin interactions that could serve as therapeutic targets. PMID:23047680

The interplay of extracellular matrix and microbiome in urothelial bladder cancer.

PubMed

Alfano, Massimo; Canducci, Filippo; Nebuloni, Manuela; Clementi, Massimo; Montorsi, Francesco; Salonia, Andrea

2016-02-01

Many pathological changes in solid tumours are caused by the accumulation of genetic mutations and epigenetic molecular alterations. In addition, tumour progression is profoundly influenced by the environment surrounding the transformed cells. The interplay between tumour cells and their microenvironment has been recognized as one of the key determinants of cancer development and is being extensively investigated. Data suggest that both the extracellular matrix and the microbiota represent microenvironments that contribute to the onset and progression of tumours. Through the introduction of omics technologies and pyrosequencing analyses, a detailed investigation of these two microenvironments is now possible. In urological research, assessment of their dysregulation has become increasingly important to provide diagnostic, prognostic and predictive biomarkers for urothelial bladder cancer. Understanding the roles of the extracellular matrix and microbiota, two key components of the urothelial mucosa, in the sequelae of pathogenic events that occur in the development and progression of urothelial carcinomas will be important to overcome the shortcomings in current bladder cancer treatment strategies.

Alginate Microcapsules Incorporating Hyaluronic Acid Recreate Closer in Vivo Environment for Mesenchymal Stem Cells.

PubMed

Cañibano-Hernández, Alberto; Saenz Del Burgo, Laura; Espona-Noguera, Albert; Orive, Gorka; Hernández, Rosa M; Ciriza, Jesús; Pedraz, Jose Luis

2017-07-03

The potential clinical application of alginate cell microencapsulation has advanced enormously during the past decade. However, the 3D environment created by alginate beads does not mimic the natural extracellular matrix surrounding cells in vivo, responsible of cell survival and functionality. As one of the most frequent macromolecules present in the extracellular matrix is hyaluronic acid, we have formed hybrid beads with alginate and hyaluronic acid recreating a closer in vivo cell environment. Our results show that 1% alginate-0.25% hyaluronic acid microcapsules retain 1.5% alginate physicochemical properties. Moreover, mesenchymal stem cells encapsulated in these hybrid beads show enhanced viability therapeutic protein release and mesenchymal stem cells' potential to differentiate into chondrogenic lineage. Although future studies with additional proteins need to be done in order to approach even more the extracellular matrix features, we have shown that hyaluronic acid protects alginate encapsulated mesenchymal stem cells by providing a niche-like environment and remaining them competent as a sustainable drug delivery system.

Contribution of α-smooth muscle actin and extracellular matrix to the in vitro reorganization of cardiomyocyte contractile system.

PubMed

Bildyug, Natalya; Bozhokina, Ekaterina; Khaitlina, Sofia

2016-04-01

Cardiomyocytes in culture undergo reversible rearrangement of their contractile apparatus with the conversion of typical myofibrils into the structures of non-muscle type and the loss of contractility. Along with these transformations, the cardiomyocytes gain the capacity to synthesize extracellular matrix. Here we show that during cultivation of rat neonatal cardiomyocytes, the inherent α-cardiac actin isoform is transiently replaced by α-smooth-muscle actin, whose expression is accompanied by transformation of myofibrils into stress-fiber-like structures. The following down-regulation of α-smooth muscle actin parallels restoration of myofibrillar system and correlates with the accumulation of extracellular collagen and laminin, initially missing from the cardiomyocytes culture. © 2016 International Federation for Cell Biology.

Matrix Remodeling in Pulmonary Fibrosis and Emphysema

PubMed Central

O’Reilly, Philip; Antony, Veena B.; Gaggar, Amit

2016-01-01

Pulmonary fibrosis and emphysema are chronic lung diseases characterized by a progressive decline in lung function, resulting in significant morbidity and mortality. A hallmark of these diseases is recurrent or persistent alveolar epithelial injury, typically caused by common environmental exposures such as cigarette smoke. We propose that critical determinants of the outcome of the injury-repair processes that result in fibrosis versus emphysema are mesenchymal cell fate and associated extracellular matrix dynamics. In this review, we explore the concept that regulation of mesenchymal cells under the influence of soluble factors, in particular transforming growth factor-β1, and the extracellular matrix determine the divergent tissue remodeling responses seen in pulmonary fibrosis and emphysema. PMID:26741177

Genetics of the extracellular matrix in aortic aneurysmal diseases.

PubMed

Lin, Chien-Jung; Lin, Chieh-Yu; Stitziel, Nathan O

2018-04-12

Aortic aneurysms are morbid conditions that can lead to rupture or dissection and are categorized as thoracic (TAA) or abdominal aortic aneurysms (AAA) depending on their location. While AAA shares overlapping risk factors with atherosclerotic cardiovascular disease, TAA exhibits strong heritability. Human genetic studies in the past two decades have successfully identified numerous genes involved in both familial and sporadic forms of aortic aneurysm. In this review we will discuss the genetic basis of aortic aneurysm, focusing on the extracellular matrix and how insights from these studies have informed our understanding of human biology and disease pathogenesis. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms

PubMed Central

Domenech, Mirian; Pedrero-Vega, Elena; Prieto, Alicia; García, Ernesto

2016-01-01

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that frequently colonizes the human nasopharynx; it is a common cause of chronic and recurrent otitis media in children and of exacerbations of chronic obstructive pulmonary disease. To date, no exopolysaccharide clearly contributing to NTHi biofilms has been identified. Consequently, there is some debate as to whether NTHi forms biofilms during colonization and infection. The present work shows that NTHi can form biofilms in vitro, producing an extracellular matrix composed of proteins, nucleic acids, and a β-glucan. Extracellular DNA, visualized by immunostaining and using fluorochromes, is an important component of this matrix and appears to be essential in biofilm maintenance. Extracellular RNA appears to be required only in the first steps of biofilm formation. Evidence of a matrix polysaccharide was obtained by staining with Calcofluor white M2R and by disaggregating biofilms with cellulase. Using strain 54997, residues of Glcp(1→4) in the NTHi biofilm were confirmed by gas-liquid chromatography-mass spectrometry. Evidence that N-acetyl-L-cysteine shows notable killing activity towards in vitro NTHi biofilm-forming bacteria is also provided. PMID:27805043

Bacterial Biofilms as Complex Communities

NASA Astrophysics Data System (ADS)

Vlamakis, Hera

2010-03-01

Many microbial populations form surface-associated multicellular communities known as biofilms. These multicellular communities are encased in a self-produced extracellular matrix composed of polysaccharides and proteins. Division of labor is a key feature of these communities and different cells serve distinct functions. We have found that in biofilms of the bacterium Bacillus subtilis, different cell types including matrix-producing and sporulating cells coexist and localize to distinct regions within the structured community. We were interested in understanding how these different cell types arise. Using fluorescence reporters under the control of promoters that are specific for distinct cell types we were able to follow the dynamics of differentiation throughout biofilm development. We found that a series of extracellular signals leads to differentiation of distinct cell types during biofilm formation. In addition, we found that extracellular matrix functions as a differentiation signal for timely sporulation within a biofilm and mutants unable to produce matrix were delayed in sporulation. Our results indicate that within a biofilm, cell-cell signaling is directional in that one cell type produces a signal that is sensed by another distinct cell type. Furthermore, once differentiated, cells become resistant to the action of other signaling molecules making it possible to maintain distinct cell populations over prolonged periods.

Semicarbazide-sensitive amine oxidase and extracellular matrix deposition by smooth-muscle cells

NASA Technical Reports Server (NTRS)

Langford, Shannon D.; Trent, Margaret B.; Boor, Paul J.

2002-01-01

We have recently reported in vivo disruption of collagen and elastin architecture within blood vessel walls resulting from the selective inhibition of the enzyme semicarbazide-sensitive amine oxidase (SSAO). This study further investigates the effects of SSAO inhibition on extracellular matrix deposition by smooth-muscle cells (SMCs) cultured from neonatal rat hearts. SMCs were characterized, SSAO activity was measured, and soluble and insoluble collagen and elastin in the extracellular matrix (ECM) were quantified. Cultured neonatal rat heart SMC exhibited a monotypic synthetic phenotype that likely represents a myofibroblast. Detectable levels of SSAO activity present throughout 30-d culture peaked at 7-14 d, coinciding with the production of ECM. The addition of enzyme inhibitors and alternate SSAO substrates (benzylamine) produced varied and, in some cases, marked changes in SSAO activity as well as in the composition of mature and soluble matrix components. Similar to our previous in vivo findings, in vitro SSAO inhibition produced aberrations in collagen and elastin deposition by heart SMC. Because changes in SSAO activity are associated with cardiovascular pathologic states, this enzyme may play a protective or modulating role by regulating ECM production during pathologic insult.

Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms.

PubMed

Domenech, Mirian; Pedrero-Vega, Elena; Prieto, Alicia; García, Ernesto

2016-11-02

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that frequently colonizes the human nasopharynx; it is a common cause of chronic and recurrent otitis media in children and of exacerbations of chronic obstructive pulmonary disease. To date, no exopolysaccharide clearly contributing to NTHi biofilms has been identified. Consequently, there is some debate as to whether NTHi forms biofilms during colonization and infection. The present work shows that NTHi can form biofilms in vitro, producing an extracellular matrix composed of proteins, nucleic acids, and a β-glucan. Extracellular DNA, visualized by immunostaining and using fluorochromes, is an important component of this matrix and appears to be essential in biofilm maintenance. Extracellular RNA appears to be required only in the first steps of biofilm formation. Evidence of a matrix polysaccharide was obtained by staining with Calcofluor white M2R and by disaggregating biofilms with cellulase. Using strain 54997, residues of Glcp(1→4) in the NTHi biofilm were confirmed by gas-liquid chromatography-mass spectrometry. Evidence that N-acetyl-L-cysteine shows notable killing activity towards in vitro NTHi biofilm-forming bacteria is also provided.

Titin-based stiffening of muscle fibers in Ehlers-Danlos Syndrome

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

Ottenheijm, Coen A.C.; Voermans, Nicol C.; Hudson, Bryan D.

Tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence leads to Ehlers-Danlos Syndrome (EDS). TNX-deficient EDS patients present with joint hypermobility and muscle weakness attributable to increased compliance of the extracellular matrix. We hypothesized that in response to the increased compliance of the extracellular matrix in TNX-deficient EDS patients, intracellular adaptations take place in the elastic properties of the giant muscle protein titin. We performed extensive single muscle fiber mechanical studies to determine active and passive properties in TNX-deficient EDS patients. Gel-electrophoresis, Western blotting, and microarray studies were used to evaluate titin expression and phosphorylation. X-ray diffraction was used tomore » measure myofilament lattice spacing. Passive tension of muscle fibers from TNX-deficient EDS patients was markedly increased. Myofilament extraction experiments indicated that the increased passive tension is attributable to changes in the properties of the sarcomeric protein titin. Transcript and protein data indicated no changes in titin isoform expression. Instead, differences in posttranslational modifications within titin's elastic region were found. In patients, active tension was not different at maximal activation level, but at submaximal activation level it was augmented attributable to increased calcium sensitivity. This increased calcium sensitivity might be attributable to stiffer titin molecules. In response to the increased compliance of the extracellular matrix in muscle of TNX-deficient EDS patients, a marked intracellular stiffening occurs of the giant protein titin. The stiffening of titin partly compensates for the muscle weakness in these patients by augmenting submaximal active tension generation.« less

Liarozole inhibits transforming growth factor-β3–mediated extracellular matrix formation in human three-dimensional leiomyoma cultures

PubMed Central

Levy, Gary; Malik, Minnie; Britten, Joy; Gilden, Melissa; Segars, James; Catherino, William H.

2014-01-01

Objective To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. Design Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. Setting Laboratory study. Patient(s) None. Intervention(s) Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. Main Outcome Measure(s) Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. Result(s) Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. Conclusion(s) Liarozole decreased TGF-β3 and TGF-β3–mediated extracellular matrix expression in a 3D uterine leiomyoma culture system. PMID:24825427

Nectin-like molecule 1 inhibits the migration and invasion of U251 glioma cells by regulating the expression of an extracellular matrix protein osteopontin.

PubMed

Yin, Bin; Li, Ke-han; An, Tai; Chen, Tao; Peng, Xiao-zhong

2010-06-01

To investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells. We infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. The restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade. A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

PubMed

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques.

PubMed Central

Woods, Alan A; Linton, Stuart M; Davies, Michael J

2003-01-01

Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix material obtained from advanced human atherosclerotic lesions are shown to contain elevated levels of oxidized amino acids [3,4-dihydroxyphenylalanine (DOPA), di-tyrosine, 2-hydroxyphenylalanine ( o-Tyr)] when compared with healthy (human and pig) arterial tissue. These matrix-derived materials account for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions. The detection of elevated levels of DOPA and o-Tyr, which have been previously attributed to the occurrence of oxygen-radical-mediated reactions, by HOCl treatment, suggests an alternative route to the formation of these materials in plaques. This is believed to involve the formation and subsequent decomposition of protein chloramines. PMID:12456264

Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

PubMed

Lyra-Leite, Davi M; Andres, Allen M; Petersen, Andrew P; Ariyasinghe, Nethika R; Cho, Nathan; Lee, Jezell A; Gottlieb, Roberta A; McCain, Megan L

2017-10-01

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. NEW & NOTEWORTHY A new methodology has been developed to measure O 2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses. Copyright © 2017 the American Physiological Society.

Effects of in vivo static compressive loading on aggrecan and type II and X collagens in the rat growth plate extracellular matrix.

PubMed

Cancel, Mathilde; Grimard, Guy; Thuillard-Crisinel, Delphine; Moldovan, Florina; Villemure, Isabelle

2009-02-01

Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In addition, growth plate extracellular matrix remodelling is essential to regulate the normal longitudinal bone growth process and to ensure physiological bone mineralization. In order to investigate the effects of static compression on growth plate extracellular matrix using an in vivo animal model, a loading device was used to precisely apply a compressive stress of 0.2 MPa for two weeks on the seventh caudal vertebra (Cd7) of rats during the pubertal growth spurt. Control, sham and loaded groups were studied. Growth modulation was quantified based on calcein labelling, and three matrix components (type II and X collagens, and aggrecan) were assessed using immunohistochemistry/safranin-O staining. As well, extracellular matrix components and enzymes (MMP-3 and -13, ADAMTS-4 and -5) were studied by qRT-PCR. Loading reduced Cd7 growth by 29% (p<0.05) and 15% (p=0.07) when compared to controls and shams respectively. No significant change could be observed in the mRNA expression of collagens and the proteolytic enzyme MMP-13. However, MMP-3 was significantly increased in the loaded group as compared to the control group (p<0.05). No change was observed in aggrecan and ADAMTS-4 and -5 expression. Low immunostaining for type II and X collagens was observed in 83% of the loaded rats as compared to the control rats. This in vivo study shows that, during pubertal growth spurt, two-week static compression reduced caudal vertebrae growth rates; this mechanical growth modulation occurred with decreased type II and X collagen proteins in the growth plate.

Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans

PubMed Central

Harrison, Paul F.; Lo, Tricia L.; Quenault, Tara; Dagley, Michael J.; Bellousoff, Matthew; Powell, David R.; Beilharz, Traude H.; Traven, Ana

2015-01-01

The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease. PMID:26474309

Thalidomide Accelerates the Degradation of Extracellular Matrix in Rat Hepatic Cirrhosis via Down-Regulation of Transforming Growth Factor-β1

PubMed Central

Meng, Qingshun; Liu, Jie; Wang, Chuanfang

2015-01-01

Purpose The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. Materials and Methods Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and α-smooth muscle actin (α-SMA) protein in the liver, transforming growth factor β1 (TGF-β1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-β1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. Results Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-β1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. Conclusion Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-β1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats. PMID:26446639

'Universal' microstructural patterns in cortical and trabecular, extracellular and extravascular bone materials: micromechanics-based prediction of anisotropic elasticity.

PubMed

Fritsch, Andreas; Hellmich, Christian

2007-02-21

Bone materials are characterized by an astonishing variability and diversity. Still, because of 'architectural constraints' due to once chosen material constituents and their physical interaction, the fundamental hierarchical organization or basic building plans of bone materials remain largely unchanged during biological evolution. Such universal patterns of microstructural organization govern the mechanical interaction of the elementary components of bone (hydroxyapatite, collagen, water; with directly measurable tissue-independent elastic properties), which are here quantified through a multiscale homogenization scheme delivering effective elastic properties of bone materials: at a scale of 10nm, long cylindrical collagen molecules, attached to each other at their ends by approximately 1.5nm long crosslinks and hosting intermolecular water inbetween, form a contiguous matrix called wet collagen. At a scale of several hundred nanometers, wet collagen and mineral crystal agglomerations interpenetrate each other, forming the mineralized fibril. At a scale of 5-10microm, the extracellular solid bone matrix is represented as collagen fibril inclusions embedded in a foam of largely disordered (extrafibrillar) mineral crystals. At a scale above the ultrastructure, where lacunae are embedded in extracellular bone matrix, the extravascular bone material is observed. Model estimates predicted from tissue-specific composition data gained from a multitude of chemical and physical tests agree remarkably well with corresponding acoustic stiffness experiments across a variety of cortical and trabecular, extracellular and extravascular materials. Besides from reconciling the well-documented, seemingly opposed concepts of 'mineral-reinforced collagen matrix' and 'collagen-reinforced mineral matrix' for bone ultrastructure, this approach opens new possibilities in the exploitation of computer tomographic data for nano-to-macro mechanics of bone organs.

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

PubMed Central

Tilghman, Robert W.; Cowan, Catharine R.; Mih, Justin D.; Koryakina, Yulia; Gioeli, Daniel; Slack-Davis, Jill K.; Blackman, Brett R.; Tschumperlin, Daniel J.; Parsons, J. Thomas

2010-01-01

Background The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: “rigidity dependent” (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models. PMID:20886123

Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of mineralized extracellular matrix.

PubMed

el-Ghannam, A; Ducheyne, P; Shapiro, I M

1997-02-01

The objective of the study was to examine the effect of alkali ion release, pH control and buffer capacity on the expression of the osteoblastic phenotype. In addition we determined the importance of modifications of the surface of porous bioactive glass (BG) on the activity of rat calvaria osteoblasts in vitro. We found that at a low tissue culture medium (TCM) volume to BG surface area (Vol/SA) ratio, the products of glass corrosion elevated the pH of the TCM to a value that adversely affected cellular activity; thus, the matrix synthesized by the cells was non-mineralized. On the other hand, when the Vol/SA was high and the buffer capacity of the medium was not exceeded, the cells generated a mineralized extracellular matrix. Addressing the second issue, we observed that modification of the composition of the BG surface markedly influenced osteoblast activity. BG that was coated with either a calcium phosphate-rich layer only or a serum protein layer changed the phenotypic characteristics of the osteoblasts. The presence of either of these surfaces lowered the alkaline phosphatase activity of the attached cells; this finding indicated that the osteoblast phenotype was not conserved. However, when the BG was coated with a bilayer of calcium phosphate and serum proteins, the alkaline phosphatase (AP) activity was elevated and the extracellular matrix contained characteristic bone markers. Our findings indicate that the calcium phosphate-rich layer promotes adsorption and concentration of proteins from the TCM, and it is utilized by the osteoblasts to form the mineralized extracellular matrix.

Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells

PubMed Central

Kenyon, Amy; Dhanani, Karim C. H.; Prinsloo, Earl; Edkins, Adrienne L.

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis. PMID:24466266

Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells.

PubMed

Hunter, Morgan C; O'Hagan, Kyle L; Kenyon, Amy; Dhanani, Karim C H; Prinsloo, Earl; Edkins, Adrienne L

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

Extracellular matrix structure governs invasion resistance in bacterial biofilms.

PubMed

Nadell, Carey D; Drescher, Knut; Wingreen, Ned S; Bassler, Bonnie L

2015-08-01

Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother-daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it.

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix.

PubMed

Valli, M; Barnes, A M; Gallanti, A; Cabral, W A; Viglio, S; Weis, M A; Makareeva, E; Eyre, D; Leikin, S; Antoniazzi, F; Marini, J C; Mottes, M

2012-11-01

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI. © 2011 John Wiley & Sons A/S.

In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

PubMed

Randrianjatovo-Gbalou, I; Rouquette, P; Lefebvre, D; Girbal-Neuhauser, E; Marcato-Romain, C-E

2017-05-01

This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control. © 2017 The Society for Applied Microbiology.

The Effects of Extracellular Matrix Proteins on Neutrophil-Endothelial Interaction ― A Roadway To Multiple Therapeutic Opportunities

PubMed Central

Padmanabhan, Jagannath; Gonzalez, Anjelica L.

2012-01-01

Polymorphoneuclear leukocytes or neutrophils, a major component of white blood cells, contribute to the innate immune response in humans. Upon sensing changes in the microenvironment, neutrophils adhere to the vascular wall, migrate through the endothelial cell (EC)-pericyte bilayer, and subsequently through the extracellular matrix to reach the site of inflammation. These cells are capable of destroying microbes, cell debris, and foreign proteins by oxidative and non-oxidative processes. While primarily mediators of tissue homeostasis, there are an increasing number of studies indicating that neutrophil recruitment and transmigration can also lead to host-tissue injury and subsequently inflammation-related diseases. Neutrophil-induced tissue injury is highly regulated by the microenvironment of the infiltrated tissue, which includes cytokines, chemokines, and the provisional extracellular matrix, remodeled through increased vascular permeability and other cellular infiltrates. Thus, investigation of the effects of matrix proteins on neutrophil-EC interaction and neutrophil transmigration may help identify the proteins that induce pro- or anti-inflammatory responses. This area of research presents an opportunity to identify therapeutic targets in inflammation-related diseases. This review will summarize recent literature on the role of neutrophils and the effects of matrix proteins on neutrophil-EC interactions, with focus on three different disease models: 1) atherosclerosis, 2) COPD, and 3) tumor growth and progression. For each disease model, inflammatory molecules released by neutrophils, important regulatory matrix proteins, current anti-inflammatory treatments, and the scope for further research will be summarized. PMID:22737047

A Rhizobium leguminosarum CHDL- (Cadherin-Like-) Lectin Participates in Assembly and Remodeling of the Biofilm Matrix

PubMed Central

Vozza, Nicolás F.; Abdian, Patricia L.; Russo, Daniela M.; Mongiardini, Elías J.; Lodeiro, Aníbal R.; Molin, Søren; Zorreguieta, Angeles

2016-01-01

In natural environments most bacteria live in multicellular structures called biofilms. These cell aggregates are enclosed in a self-produced polymeric extracellular matrix, which protects the cells, provides mechanical stability and mediates cellular cohesion and adhesion to surfaces. Although important advances were made in the identification of the genetic and extracellular factors required for biofilm formation, the mechanisms leading to biofilm matrix assembly, and the roles of extracellular proteins in these processes are still poorly understood. The symbiont Rhizobium leguminosarum requires the synthesis of the acidic exopolysaccharide and the PrsDE secretion system to develop a mature biofilm. PrsDE is responsible for the secretion of the Rap family of proteins that share one or two Ra/CHDL (cadherin-like-) domains. RapA2 is a calcium-dependent lectin with a cadherin-like β sheet structure that specifically recognizes the exopolysaccharide, either as a capsular polysaccharide (CPS) or in its released form [extracellular polysaccharide (EPS)]. In this study, using gain and loss of function approaches combined with phenotypic and microscopic studies we demonstrated that RapA lectins are involved in biofilm matrix development and cellular cohesion. While the absence of any RapA protein increased the compactness of bacterial aggregates, high levels of RapA1 expanded distances between cells and favored the production of a dense matrix network. Whereas endogenous RapA(s) are predominantly located at one bacterial pole, we found that under overproduction conditions, RapA1 surrounded the cell in a way that was reminiscent of the capsule. Accordingly, polysaccharide analyses showed that the RapA lectins promote CPS formation at the expense of lower EPS production. Besides, polysaccharide analysis suggests that RapA modulates the EPS size profile. Collectively, these results show that the interaction of RapA lectins with the polysaccharide is involved in rhizobial biofilm matrix assembly and remodeling. PMID:27790205

The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing.

PubMed

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla A; Diekwisch, Tom; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBN(Δ5-6) truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

PubMed Central

Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

2014-01-01

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins. PMID:24752318

MT1-MMP regulates the turnover and endocytosis of extracellular matrix fibronectin

PubMed Central

Shi, Feng; Sottile, Jane

2011-01-01

The extracellular matrix (ECM) is dynamically remodeled by cells during development, normal tissue homeostasis and in a variety of disease processes. We previously showed that fibronectin is an important regulator of ECM remodeling. The deposition and/or polymerization of fibronectin into the ECM controls the deposition and stability of other ECM molecules. In addition, agents that inhibit fibronectin polymerization promote the turnover of fibronectin fibrils and enhance ECM fibronectin endocytosis and intracellular degradation. Endocytosis of ECM fibronectin is regulated by β1 integrins, including α5β1 integrin. We have examined the role of extracellular proteases in regulating ECM fibronectin turnover. Our data show that membrane type matrix metalloproteinase 1 (MT1-MMP; also known as MMP14) is a crucial regulator of fibronectin turnover. Cells lacking MT1-MMP show reduced turnover and endocytosis of ECM fibronectin. MT1-MMP regulates ECM fibronectin remodeling by promoting extracellular cleavage of fibronectin and by regulating α5β1-integrin endocytosis. Our data also show that fibronectin polymerization stabilizes fibronectin fibrils and inhibits ECM fibronectin endocytosis by inhibiting α5β1-integrin endocytosis. These data are the first to show that an ECM protein and its modifying enzyme can regulate integrin endocytosis. These data also show that integrin trafficking plays a major role in modulating ECM fibronectin remodeling. The dual dependence of ECM fibronectin turnover on extracellular proteolysis and endocytosis highlights the complex regulatory mechanisms that control ECM remodeling to ensure maintenance of proper tissue function. PMID:22159414

Evidence for the in vivo polymerization of ependymin: a brain extracellular glycoprotein.

PubMed

Shashoua, V E; Hesse, G W; Milinazzo, B

1990-07-09

Ependymin, a glycoprotein of the brain extracellular fluid, has been implicated in synaptic changes associated with the consolidation process of long-term memory formation and the activity-dependent sharpening of connections of regenerating optic nerve. In vitro experiments have demonstrated that ependymin has the capacity to form fibrous insoluble polymers (FIP) when the solvent Ca2+ concentration is reduced by the addition of EGTA. Such products, once formed, do not dissolve in 2% sodium dodecyl sulfate (SDS) in 5 M urea. This property was used to develop a method for isolating brain FIP. A reproducible quantity of FIP was found in goldfish and mouse brain. This was highly concentrated in the synaptosomal fraction and had identical immunoreactivity properties to FIP obtained by the polymerization of pure ependymin in vitro as well as a cross-reactivity to other protein components of the extracellular matrix such as fibronectin and laminin. Labeling studies with [35S]methionine showed that labeled FIP aggregates are synthesized in vivo and become associated with the synaptosomal fraction. A comparison of the amino acid sequence of ependymin with those for proteins of the extracellular matrix indicated that common sequences 5-6 amino acids long exist in the molecules. These homologies may explain why antibodies to fibronectin, laminin and tubulin can recognize the FIP prepared from pure ependymin. These results suggest that ependymin can polymerize in vivo to form FIP aggregates which have similar immunoreactivity properties to major components of the brain extracellular matrix.

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus

PubMed Central

Vanderploeg, Eric J; Wilson, Christopher G; Imler, Stacy M; Ling, Carrie Hang-Yin; Levenston, Marc E

2012-01-01

A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue-level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue-level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra- and/or peri-cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies. PMID:22703476

Substrate-protecting antiproteolytic agents for the prevention of pathological degradation of connective tissues. A review.

PubMed

Robert, A-M

2012-02-01

Connective tissues play an important role in the physiological functions of the organism. The integrity of the macromolecular components of these tissues, also called extracellular matrix, is necessary for their functional efficiency. A number of proteinases present in the organism, and the activity of which increases with age and with several pathologies, specifically degrade the components of the extracellular matrix. For a long time, tentatives for the protection of the matrix-components against degradation were made with low molecular weight inhibitors, not very efficient in vivo and not devoid of inconveniencies. We initiated a different approach for the preservation of the macromolecules of the extracellular matrix against proteolytic degradation with substances which exert an intense antiproteolytic activity not only in vitro, but also in vivo. The particularity of these substances is the fact that they do not act on the enzymes, but combine with the macromolecules. This is the type of combination of substances with the macromolecules of the matrix that prevents their degradation by the proteinases. Because of this affinity of such antiproteolytic agents not for the enzymes but for the substrates, we called them "substrate protectors" (Robert et al., 1979). The aim of the present review is to summarise the essential of our experiments which led to the description of substrate protectors. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

PubMed

Xu, Q; Cao, X; Pan, J; Ye, Y; Xie, Y; Ohara, N; Ji, H

2015-01-01

PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

PubMed

Williams, Rachel C; Skelton, Andrew J; Todryk, Stephen M; Rowan, Andrew D; Preshaw, Philip M; Taylor, John J

2016-01-01

Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy).

Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

PubMed Central

Williams, Rachel C.; Skelton, Andrew J.; Todryk, Stephen M.; Rowan, Andrew D.; Preshaw, Philip M.; Taylor, John J.

2016-01-01

Introduction Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. Methods and Results We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. Conclusions We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy). PMID:26829555

Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides.

PubMed

Chew, Su Chuen; Kundukad, Binu; Seviour, Thomas; van der Maarel, Johan R C; Yang, Liang; Rice, Scott A; Doyle, Patrick; Kjelleberg, Staffan

2014-08-05

Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. Importance: Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical and industrial settings. One of the defining features of a biofilm is its extracellular matrix. The matrix has a heterogeneous structure and is formed from a secretion of various biopolymers, including proteins, extracellular DNA, and polysaccharides. It is generally known to interact with biofilm cells, thus affecting cell physiology and cell-cell communication. Despite the fact that the matrix may comprise up to 90% of the biofilm dry weight, how the matrix properties affect biofilm structure, maturation, and interspecies interactions remain largely unexplored. This study reveals that bacteria can use specific extracellular polymers to modulate the physical properties of their microenvironment. This in turn impacts biofilm structure, differentiation, and interspecies interactions. Copyright © 2014 Chew et al.

Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms.

PubMed

Domenech, Mirian; García, Ernesto; Prieto, Alicia; Moscoso, Miriam

2013-02-01

Biofilm matrices consist of a mixture of extracellular polymeric substances synthesized in large part by the biofilm-producing microorganisms themselves. These matrices are responsible for the cohesion and three-dimensional architecture of biofilms. The present study demonstrates the existence of a matrix composed of extracellular DNA, proteins and polysaccharides in the biofilm formed by the human pathogen Streptococcus pneumoniae. Extracellular DNA, visualized by fluorescent labelling, was an important component of this matrix. The existence of DNA-protein complexes associated with bacterial aggregates and other polymers was hypothesized based on the unexpected DNA binding activity of lysozyme LytC, a novel moonlighting protein. Actually, a 25-amino-acid-long peptide derived from LytC (positions 408 and 432 of the mature LytC) was also capable of efficiently binding to DNA. Moreover, the presence of intercellular DNA-LytC protein complexes in pneumococcal biofilms was demonstrated by confocal laser scanning microscopy. Evidence of extracellular polysaccharide different from the capsule was obtained by staining with Calcofluor dye and four types of lectin conjugated to Alexa fluorophores, and by incubation with glycoside hydrolases. The presence of residues of Glcp(1→4) and GlcNAc(1→4) (in its deacetylated form) in the pneumococcal biofilm was confirmed by GC-MS techniques. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Vesicoureteral reflux and the extracellular matrix connection

PubMed Central

Tokhmafshan, Fatima; Brophy, Patrick D.; Gbadegesin, Rasheed A.

2017-01-01

Primary vesicoureteral reflux (VUR) is a common pediatric condition due to a developmental defect in the ureterovesical junction. The prevalence of VUR among individuals with connective tissue disorders, as well as the importance of the ureter and bladder wall musculature for the anti-reflux mechanism, suggest that defects in the extracellular matrix (ECM) within the ureterovesical junction may result in VUR. This review will discuss the function of the smooth muscle and its supporting ECM microenvironment with respect to VUR, and explore the association of VUR with mutations in ECM-related genes. PMID:27139901

Role of Fatty Acid Binding Protein 5 (FABP5) in Breast Cancer Progression and Metastasis

DTIC Science & Technology

2013-04-01

invasiveness of S100A7 overexpressing ERa- and ERa? cells One of the hallmarks of tumor metastasis is its ability to degrade extracellular matrix to invade...increase in macrophages in doxycycline-inducedMMTV-mS100a7a15 compared with unin- duced mice (Fig. 2E). MMPs are known to degrade extracellular matrix (ECM...interplay with leptin and other adipocytokines. FEBS Lett 2009;583:259–65. 12. Ranger JJ, Levy DE, Shahalizadeh S, Hallett M, Muller WJ. Identifica- tion of

Stretchy Proteins on Stretchy Substrates: The Important Elements of Integrin-Mediated Rigidity Sensing

PubMed Central

Moore, Simon W.; Roca-Cusachs, Pere; Sheetz, Michael P.

2013-01-01

Matrix and tissue rigidity guides many cellular processes, including the differentiation of stem cells and the migration of cells in health and disease. Cells actively and transiently test rigidity using mechanisms limited by inherent physical parameters that include the strength of extracellular attachments, the pulling capacity on these attachments, and the sensitivity of the mechanotransduction system. Here we focus on rigidity sensing mediated through the integrin family of extracellular matrix receptors and linked proteins, and discuss the evidence supporting these proteins as mechanosensors. PMID:20708583

Extracellular redox state regulates features associated with prostate cancer cell invasion.

PubMed

Chaiswing, Luksana; Zhong, Weixiong; Cullen, Joseph J; Oberley, Larry W; Oberley, Terry D

2008-07-15

We have examined the possible role of extracellular reduction-oxidation (redox) state in regulation of biological/biochemical features associated with prostate cancer cell invasion. DU145, PC-3, and RWPE1-derived human prostate cancer (WPE1-NB26) cell lines were used for the present in vitro analysis. Increasing levels of nitric oxide using S-nitroso-N-acetylpenicillamine resulted in a decrease in cell invasion ability, whereas increasing levels of extracellular superoxide radical (O(2)(*-)) using xanthine/xanthine oxidase resulted in an increase in cell invasion ability in these three cell lines. WPE1-NB26 cells exhibited an increased glutathione/glutathione disulfide ratio in the medium in comparison with RWPE1 cells (immortalized but nonmalignant prostate epithelial cells), suggesting an alteration of extracellular redox state of WPE1-NB26 cells. We hypothesized that O(2)(*-) production at or near the plasma membrane or in the adjacent extracellular matrix at least partially regulated prostate cancer cell invasion. Using adenovirus-mediated extracellular superoxide dismutase (EC-SOD) gene transduction to enzymatically decrease O(2)(*-) levels, we showed that in the presence of heparin, adenovirus EC-SOD gene transduction resulted in an increase in the expression of EC-SOD outside the cells with resultant inhibition of cell invasion ability. This inhibition correlated with reduced metalloproteinase [matrix metalloproteinase (MMP) 2/membrane type 1-MMP] activities and increased levels of extracellular nitrite. Our results suggest a prominent role of extracellular redox status in regulation of cell invasion, which may provide opportunities for therapeutic interventions.

The Dynamic Sclera: Extracellular Matrix Remodeling in Normal Ocular Growth and Myopia Development

PubMed Central

Harper, Angelica R.; Summers, Jody A.

2014-01-01

Myopia is a common ocular condition, characterized by excessive elongation of the ocular globe. The prevalence of myopia continues to increase, particularly among highly educated groups, now exceeding 80% in some groups. In parallel with the increased prevalence of myopia, are increases in associated blinding ocular conditions including glaucoma, retinal detachment and macular degeneration, making myopia a significant global health concern. The elongation of the eye is closely related to the biomechanical properties of the sclera, which in turn are largely dependent on the composition of the scleral extracellular matrix. Therefore an understanding of the cellular and extracellular events involved in the regulation of scleral growth and remodeling during childhood and young adulthood will provide future avenues for the treatment of myopia and its associated ocular complications. PMID:25819458

Mimicking the extracellular matrix with functionalized, metal-assembled collagen peptide scaffolds.

PubMed

Hernandez-Gordillo, Victor; Chmielewski, Jean

2014-08-01

Natural and synthetic three-dimensional (3-D) scaffolds that mimic the microenvironment of the extracellular matrix (ECM), with growth factor storage/release and the display of cell adhesion signals, offer numerous advantages for regenerative medicine and in vitro morphogenesis and oncogenesis modeling. Here we report the design of collagen mimetic peptides (CMPs) that assemble into a highly crosslinked 3-D matrix in response to metal ion stimuli, that may be functionalized with His-tagged cargoes, such as green fluorescent protein (GFP-His8) and human epidermal growth factor (hEGF-His6). The bound hEGF-His6 was found to gradually release from the matrix in vitro and induce cell proliferation in the EGF-dependent cell line MCF10A. The additional incorporation of a cell adhesion sequence (RGDS) at the N-terminus of the CMP creates an environment that facilitated the organization of matrix-encapsulated MCF10A cells into spheroid structures, thus mimicking the ECM environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

The structure of cell-matrix adhesions: the new frontier.

PubMed

Hanein, Dorit; Horwitz, Alan Rick

2012-02-01

Adhesions between the cell and the extracellular matrix (ECM) are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These combined functions in force transduction, signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix. Despite advances in our understanding of the protein composition, interactions and regulation, our understanding of matrix adhesion structure and organization, how forces affect this organization, and how these changes dictate specific signaling events is limited. Insights across multiple structural levels are acutely needed to elucidate adhesion structure and ultimately the molecular basis of signaling and mechanotransduction. Here we describe the challenges and recent advances and prospects for unraveling the structure of cell-matrix adhesions and their response to force. Copyright © 2011 Elsevier Ltd. All rights reserved.

Omentin-1 prevents cartilage matrix destruction by regulating matrix metalloproteinases.

PubMed

Li, Zhigang; Liu, Baoyi; Zhao, Dewei; Wang, BenJie; Liu, Yupeng; Zhang, Yao; Li, Borui; Tian, Fengde

2017-08-01

Matrix metalloproteinases (MMPs) play a crucial role in the degradation of the extracellular matrix and pathological progression of osteoarthritis (OA). Omentin-1 is a newly identified anti-inflammatory adipokine. Little information regarding the protective effects of omentin-1 in OA has been reported before. In the current study, our results indicated that omentin-1 suppressed expression of MMP-1, MMP-3, and MMP-13 induced by the proinflammatory cytokine interleukin-1β (IL-1β) at both the mRNA and protein levels in human chondrocytes. Importantly, administration of omentin-1 abolished IL-1β-induced degradation of type II collagen (Col II) and aggrecan, the two major extracellular matrix components in articular cartilage, in a dose-dependent manner. Mechanistically, omentin-1 ameliorated the expression of interferon regulatory factor 1 (IRF-1) by blocking the JAK-2/STAT3 pathway. Our results indicate that omentin-1 may have a potential chondroprotective therapeutic capacity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Soil organic matter and the extracellular microbial matrix show contrasting responses to C and N availability

PubMed Central

Redmile-Gordon, M.A.; Evershed, R.P.; Hirsch, P.R.; White, R.P.; Goulding, K.W.T.

2015-01-01

An emerging paradigm in soil science suggests microbes can perform ‘N mining’ from recalcitrant soil organic matter (SOM) in conditions of low N availability. However, this requires the production of extracellular structures rich in N (including enzymes and structural components) and thus defies stoichiometric expectation. We set out to extract newly synthesised peptides from the extracellular matrix in soil and compare the amino acid (AA) profiles, N incorporation and AA dynamics in response to labile inputs of contrasting C/N ratio. Glycerol was added both with and without an inorganic source of N (10% 15N labelled NH4NO3) to a soil already containing a large pool of refractory SOM and incubated for 10 days. The resulting total soil peptide (TSP) and extracellular pools were compared using colorimetric methods, gas chromatography, and isotope ratio mass spectrometry. N isotope compositions showed that the extracellular polymeric substance (EPS) contained a greater proportion of products formed de novo than did TSP, with hydrophobic EPS-AAs (leucine, isoleucine, phenylalanine, hydroxyproline and tyrosine) deriving substantially more N from the inorganic source provided. Quantitative comparison between extracts showed that the EPS contained greater relative proportions of alanine, glycine, proline, phenylalanine and tyrosine. The greatest increases in EPS-peptide and EPS-polysaccharide concentrations occurred at the highest C/N ratios. All EPS-AAs responded similarly to treatment whereas the responses of TSP were more complex. The results suggest that extracellular investment of N (as EPS peptides) is a microbial survival mechanism in conditions of low N/high C which, from an evolutionary perspective, must ultimately lead to the tendency for increased N returns to the microbial biomass. A conceptual model is proposed that describes the dynamics of the extracellular matrix in response to the C/N ratio of labile inputs. PMID:26339106

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.

Extracellular matrix structure.

PubMed

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Biomineralization of the spicules of sea urchin embryos.

PubMed

Wilt, Fred H

2002-03-01

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.

Examination of the Abscission-Associated Transcriptomes for Soybean, Tomato, and Arabidopsis Highlights the Conserved Biosynthesis of an Extensible Extracellular Matrix and Boundary Layer.

PubMed

Kim, Joonyup; Sundaresan, Srivignesh; Philosoph-Hadas, Sonia; Yang, Ronghui; Meir, Shimon; Tucker, Mark L

2015-01-01

Abscission zone (AZ) development and the progression of abscission (detachment of plant organs) have been roughly separated into four stages: first, AZ differentiation; second, competence to respond to abscission signals; third, activation of abscission; and fourth, formation of a protective layer and post-abscission trans-differentiation. Stage three, activation of abscission, is when changes in the cell wall and extracellular matrix occur to support successful organ separation. Most abscission research has focused on gene expression for enzymes that disassemble the cell wall within the AZ and changes in phytohormones and other signaling events that regulate their expression. Here, transcriptome data for soybean, tomato and Arabidopsis were examined and compared with a focus not only on genes associated with disassembly of the cell wall but also on gene expression linked to the biosynthesis of a new extracellular matrix. AZ-specific up-regulation of genes associated with cell wall disassembly including cellulases (beta-1,4-endoglucanases, CELs), polygalacturonases (PGs), and expansins (EXPs) were much as expected; however, curiously, changes in expression of xyloglucan endotransglucosylase/hydrolases (XTHs) were not AZ-specific in soybean. Unexpectedly, we identified an early increase in the expression of genes underlying the synthesis of a waxy-like cuticle. Based on the expression data, we propose that the early up-regulation of an abundance of small pathogenesis-related (PR) genes is more closely linked to structural changes in the extracellular matrix of separating cells than an enzymatic role in pathogen resistance. Furthermore, these observations led us to propose that, in addition to cell wall loosening enzymes, abscission requires (or is enhanced by) biosynthesis and secretion of small proteins (15-25 kDa) and waxes that form an extensible extracellular matrix and boundary layer on the surface of separating cells. The synthesis of the boundary layer precedes what is typically associated with the post-abscission synthesis of a protective scar over the fracture plane. This modification in the abscission model is discussed in regard to how it influences our interpretation of the role of multiple abscission signals.

Bioengineering Human Myocardium on Native Extracellular Matrix

PubMed Central

Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

2015-01-01

Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable the bioengineering of functional human myocardial-like tissue of multiple complexities. PMID:26503464

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression.

PubMed

Christopher, R A; Judge, S R; Vincent, P A; Higgins, P J; McKeown-Longo, P J

1999-10-01

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

PubMed Central

Liskova, Jana; Babchenko, Oleg; Varga, Marian; Kromka, Alexander; Hadraba, Daniel; Svindrych, Zdenek; Burdikova, Zuzana; Bacakova, Lucie

2015-01-01

Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings. PMID:25670900

Platelets and Plasma Stimulate Sheep Rotator Cuff Tendon Tenocytes When Cultured in an Extracellular Matrix Scaffold

PubMed Central

Kelly, Brian A.; Proffen, Benedikt L.; Haslauer, Carla M.; Murray, Martha M.

2015-01-01

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: 1) Plasma (PPP), 2) Plasma and platelets (PAP), 3) Plasma and macrophages (PPPM), 4) Plasma, platelets and macrophages (PAPM), 5) Phosphate buffered saline (PBS), and 6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine if these changes in cellular function will translate into improved tendon healing. PMID:26419602

Usefulness and limitation of measurement methods for evaluation of tissue-engineered cartilage function and characterization using nanosecond pulsed laser

NASA Astrophysics Data System (ADS)

Ishihara, Miya; Sato, Masato; Kaneshiro, Nagatoshi; Mitani, Genya; Nagai, Toshihiro; Kutsuna, Toshiharu; Ishihara, Masayuki; Mochida, Joji; Kikuchi, Makoto

2007-02-01

There is a demand in the field of regenerative medicine for measurement technology that enables determination of functions and characterizations of engineered tissue. Regenerative medicine involving the articular cartilage in particular requires measurement of viscoelastic properties and characterization of the extracellular matrix, which plays a major role in articular cartilage. To meet this demand, we previously proposed a noninvasive method for determination of the viscoelasticity using laser-induced thermoelastic wave (1,2). We also proposed a method for characterization of the extracellular matrix using time-resolved autofluorescence spectroscopy, which could be performed simultaneously with laser-induced thermoelastic wave measurement(3). The purpose of this study was to verify the usefulness and limitation of these methods for evaluation of actual engineered cartilage. 3rd Q-SW Nd:YAG laser pulses, which are delivered through optical fiber, were used for the light source. Laser-induced thermoelastic waves were detected by a sensor consisting of a piezoelectric transducer, which was designed for use in arthroscopy(4). The time-resolved fluorescence spectroscopy was measured by a photonic multichannel analyzer with 4ch digital signal generator. Various tissue-engineered cartilages were developed as samples. Only a limited range of sample thickness could be measured, however, the measured viscoelastic parameters had a positive correlation with culture time, that is, the degree of formation of extracellular matrix(5,6). There were significant differences in the fluorescent parameters among the phenotypic expressions of cartilage because chondrocyte produces specific extracellular matrix as in collagen types depending on its phenotype.

Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

PubMed

Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

2016-04-01

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

PubMed

Herron, Todd J; Rocha, Andre Monteiro Da; Campbell, Katherine F; Ponce-Balbuena, Daniela; Willis, B Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

2016-04-01

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers. © 2016 American Heart Association, Inc.

Differential modulation of degradative and repair responses of interleukin-1-treated chondrocytes by platelet-derived growth factor.

PubMed Central

Harvey, A K; Stack, S T; Chandrasekhar, S

1993-01-01

Interleukin 1 (IL-1) plays a dual role in cartilage matrix degeneration by promoting extracellular proteinase action such as the matrix metalloproteinases (increased degradation) and by suppressing the synthesis of extracellular matrix molecules (inhibition of repair). Platelet-derived growth factor (PDGF) is a wound-healing hormone which is released along with IL-1 during the inflammatory response. Since previous studies have shown that PDGF enhances IL-1 alpha effects on metalloproteinase activity, in this report, we have examined whether PDGF modifies IL-1 beta effects on cartilage proteoglycan synthesis. Initially, we confirmed that rabbit articular chondrocytes treated with IL-1 beta + PDGF induced higher proteinase activity, in comparison with IL-1-treated cells. We further observed that the increased proteinase activity correlated with an increase in the synthesis of collagenase/stromelysin proteins and a corresponding increase in the steady-state mRNA levels for both the enzymes. Studies on IL-1 receptor expression suggested that PDGF caused an increase in IL-1 receptor expression which, by augmenting the IL-1 response, may have led to the increase in proteinase induction. Analysis of proteoglycan synthesis confirmed that IL-1 reduced the incorporation of sulphated proteoglycan, aggrecan, into the extracellular matrix of chondrocytes, whereas PDGF stimulated it. However, cells treated with IL-1 + PDGF synthesized normal levels of aggrecan. This is in contrast with cells treated with IL-1 + fibroblast growth factor, in which case only proteinase activity was potentiated. The results allow us to conclude that (a) the two effector functions that play a role in matrix remodelling, namely matrix lysis (proteinase induction) and matrix repair (proteoglycan synthesis), occur via distinct pathways and (b) PDGF may play a crucial role in cartilage repair by initially causing matrix degradation followed by promoting new matrix synthesis. Images Figure 1 Figure 2 Figure 5 Figure 6 PMID:8503839

Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

PubMed Central

Deora, Rajendar

2011-01-01

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the Gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs. PMID:21347299

Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito, Aedes aegypti.

PubMed

Kantor, A M; Dong, S; Held, N L; Ishimwe, E; Passarelli, A L; Clem, R J; Franz, A W E

2017-02-01

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut. © 2016 The Royal Entomological Society.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases.

PubMed

Rahbari, Nuh N; Kedrin, Dmitriy; Incio, Joao; Liu, Hao; Ho, William W; Nia, Hadi T; Edrich, Christina M; Jung, Keehoon; Daubriac, Julien; Chen, Ivy; Heishi, Takahiro; Martin, John D; Huang, Yuhui; Maimon, Nir; Reissfelder, Christoph; Weitz, Jurgen; Boucher, Yves; Clark, Jeffrey W; Grodzinsky, Alan J; Duda, Dan G; Jain, Rakesh K; Fukumura, Dai

2016-10-12

The survival benefit of anti-vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy. Copyright © 2016, American Association for the Advancement of Science.

Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation

PubMed Central

Styrczewska, Monika; Kostyn, Anna; Kulma, Anna; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Prescha, Anna; Czuj, Tadeusz; Szopa, Jan

2015-01-01

Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD), phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification. PMID:26347154

Optimization of Extracellular Matrix Synthesis and Accumulation by Human Articular Chondrocytes in 3-Dimensional Construct with Repetitive Hydrostatic Pressure.

PubMed

Ogura, Takahiro; Tsuchiya, Akihiro; Minas, Tom; Mizuno, Shuichi

2018-04-01

Objective The effects of hydrostatic pressure (HP) on the matrix synthesis by human articular chondrocytes have been reported elsewhere. In order to optimize the production of extracellular matrix, we aimed to clarify the effects of repetitive HP on metabolic function by human articular chondrocytes. Design The human articular chondrocytes were expanded and embedded within a collagen gel/sponge scaffold. We incubated these constructs with and without HP followed by atmospheric pressure (AP) and repeated the second HP followed by AP over 14 days. Genomic, biochemical, and histological evaluation were performed to compare the effects of each regimen on the constructs. Results The gene expressions of collagen type II and aggrecan core protein were significantly upregulated with repetitive HP regimens compared with a single HP or AP by 14 days ( P < 0.01 or 0.05). Matrix metalloptoteinase-13 (MMP-13) in AP was upregulated significantly compared to other HP regimens at day 14 ( P < 0.01). No significant difference was observed in tissue inhibitor of metalloproteinases-II. Immunohistology demonstrated that application of HP (both repetitive and single) promoted the accumulation of specific extracellular matrix and reduced a MMP-13. A single regimen of HP followed by AP significantly increased the amount of sulfated glycosaminoglycan than that of the AP, whereas repetitive HP remained similar level of that of the AP. Conclusions Repetitive HP had a greater effect on anabolic activity by chondrocytes than a single HP regimen, which will be advantageous for producing a matrix-rich cell construct.

Mifepristone inhibits extracellular matrix formation in uterine leiomyoma.

PubMed

Patel, Amrita; Malik, Minnie; Britten, Joy; Cox, Jeris; Catherino, William H

2016-04-01

To characterize the efficacy of mifepristone treatment on extracellular matrix (ECM) production in leiomyomas. Laboratory study. University research laboratory. None. Treatment of human immortalized two-dimensional (2D) and three-dimensional (3D) leiomyoma and myometrial cells with mifepristone and the progestin promegestone (R5020). Expression of COL1A1, fibronectin, versican variant V0, and dermatopontin in treated leiomyoma cells by Western blot analysis and confirmatory immunohistochemistry staining of treated 3D cultures. Treatment with progestin stimulated production of COL1A1, fibronectin, versican, and dermatopontin. Mifepristone treatment inhibited protein production of these genes, most notably with versican expression. Combination treatment with both the agonist and antagonist further inhibited protein expression of these genes. Immunohistochemistry performed on 3D cultures demonstrated generalized inhibition of ECM protein concentration. Our study demonstrated that the progesterone agonist R5020 directly stimulated extracellular matrix components COL1A1, fibronectin, versican, and dermatopontin production in human leiomyoma cells. Progesterone antagonist mifepristone decreased protein production of these genes to levels comparable with untreated leiomyoma cells. Published by Elsevier Inc.

The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

PubMed

Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

2011-10-01

Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Amyloid β-Protein as a Substrate Interacts with Extracellular Matrix to Promote Neurite Outgrowth

NASA Astrophysics Data System (ADS)

Koo, Edward H.; Park, Lisa; Selkoe, Dennis J.

1993-05-01

Progressive deposition of amyloid β-protein (Aβ) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic Aβ to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble Aβ may not accumulate in significant quantities in brain, we asked whether immobilized Aβ peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of β-amyloid. We detected no detrimental effects of Aβ substrate on neurite outgrowth. Rather, Aβ in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble Aβ in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of Aβ production by cultured neurons, our data suggest that Aβ plays a normal physiological role in brain by complexing with the extracellular matrix.

The Mechanobiology of Aging

PubMed Central

Walston, Jeremy; Wirtz, Denis

2016-01-01

Aging is a complex, multifaceted process that induces a myriad of physiological changes over an extended period of time. Aging is accompanied by major biochemical and biomechanical changes at macroscopic and microscopic length scales that affect not only tissues and organs but also cells and subcellular organelles. These changes include transcriptional and epigenetic modifications; changes in energy production within mitochondria; and alterations in the overall mechanics of cells, their nuclei, and their surrounding extracellular matrix. In addition, aging influences the ability of cells to sense changes in extracellular-matrix compliance (mechanosensation) and to transduce these changes into biochemical signals (mechanotransduction). Moreover, following a complex positive-feedback loop, aging is accompanied by changes in the composition and structure of the extracellular matrix, resulting in changes in the mechanics of connective tissues in older individuals. Consequently, these progressive dysfunctions facilitate many human pathologies and deficits that are associated with aging, including cardiovascular, musculoskeletal, and neurodegenerative disorders and diseases. Here, we critically review recent work highlighting some of the primary biophysical changes occurring in cells and tissues that accompany the aging process. PMID:26643020

Niche Extracellular Matrix Components and Their Influence on HSC.

PubMed

Domingues, Mélanie J; Cao, Huimin; Heazlewood, Shen Y; Cao, Benjamin; Nilsson, Susan K

2017-08-01

Maintenance of hematopoietic stem cells (HSC) takes place in a highly specialized microenvironment within the bone marrow. Technological improvements, especially in the field of in vivo imaging, have helped unravel the complexity of the niche microenvironment and have completely changed the classical concept from what was previously believed to be a static supportive platform, to a dynamic microenvironment tightly regulating HSC homeostasis through the complex interplay between diverse cell types, secreted factors, extracellular matrix molecules, and the expression of different transmembrane receptors. To add to the complexity, non-protein based metabolites have also been recognized as a component of the bone marrow niche. The objective of this review is to discuss the current understanding on how the different extracellular matrix components of the niche regulate HSC fate, both during embryonic development and in adulthood. Special attention will be provided to the description of non-protein metabolites, such as lipids and metal ions, which contribute to the regulation of HSC behavior. J. Cell. Biochem. 118: 1984-1993, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity.

PubMed

Ewald, Collin Y; Landis, Jess N; Porter Abate, Jess; Murphy, Coleen T; Blackwell, T Keith

2015-03-05

Interventions that delay ageing mobilize mechanisms that protect and repair cellular components, but it is unknown how these interventions might slow the functional decline of extracellular matrices, which are also damaged during ageing. Reduced insulin/IGF-1 signalling (rIIS) extends lifespan across the evolutionary spectrum, and in juvenile Caenorhabditis elegans also allows the transcription factor DAF-16/FOXO to induce development into dauer, a diapause that withstands harsh conditions. It has been suggested that rIIS delays C. elegans ageing through activation of dauer-related processes during adulthood, but some rIIS conditions confer robust lifespan extension unaccompanied by any dauer-like traits. Here we show that rIIS can promote C. elegans longevity through a program that is genetically distinct from the dauer pathway, and requires the Nrf (NF-E2-related factor) orthologue SKN-1 acting in parallel to DAF-16. SKN-1 is inhibited by IIS and has been broadly implicated in longevity, but is rendered dispensable for rIIS lifespan extension by even mild activity of dauer-related processes. When IIS is decreased under conditions that do not induce dauer traits, SKN-1 most prominently increases expression of collagens and other extracellular matrix genes. Diverse genetic, nutritional, and pharmacological pro-longevity interventions delay an age-related decline in collagen expression. These collagens mediate adulthood extracellular matrix remodelling, and are needed for ageing to be delayed by interventions that do not involve dauer traits. By genetically delineating a dauer-independent rIIS ageing pathway, our results show that IIS controls a broad set of protective mechanisms during C. elegans adulthood, and may facilitate elucidation of processes of general importance for longevity. The importance of collagen production in diverse anti-ageing interventions implies that extracellular matrix remodelling is a generally essential signature of longevity assurance, and that agents promoting extracellular matrix youthfulness may have systemic benefit.

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour.

PubMed

Grande, R; Di Giulio, M; Bessa, L J; Di Campli, E; Baffoni, M; Guarnieri, S; Cellini, L

2011-02-01

This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. Extracellular DNA was purified and characterized in a 2-day-old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2-day-old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I-treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro-organism defined as a 'quasi-species'. The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis.

PubMed

Shields, Robert C; Mokhtar, Norehan; Ford, Michael; Hall, Michael J; Burgess, J Grant; ElBadawey, Mohamed Reda; Jakubovics, Nicholas S

2013-01-01

The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.

Diffusion in Brain Extracellular Space

PubMed Central

Syková, Eva; Nicholson, Charles

2009-01-01

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is about 20% and the tortuosity about 1.6 (i.e. free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties are valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain. PMID:18923183

Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis

NASA Technical Reports Server (NTRS)

Ingber, D.

1991-01-01

Capillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future.

Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development.

PubMed

Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

2015-01-01

Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm.

Gene evolution and functions of extracellular matrix proteins in teeth

PubMed Central

Yoshizaki, Keigo; Yamada, Yoshihiko

2013-01-01

The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

Hacking the Matrix.

PubMed

Czerwinski, Michael; Spence, Jason R

2017-01-05

Recently in Nature, Gjorevski et al. (2016) describe a fully defined synthetic hydrogel that mimics the extracellular matrix to support in vitro growth of intestinal stem cells and organoids. The hydrogel allows exquisite control over the chemical and physical in vitro niche and enables identification of regulatory properties of the matrix. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutations in extracellular matrix genes NID1 and LAMC1 cause autosomal dominant Dandy-Walker malformation and occipital cephaloceles.

PubMed

Darbro, Benjamin W; Mahajan, Vinit B; Gakhar, Lokesh; Skeie, Jessica M; Campbell, Elizabeth; Wu, Shu; Bing, Xinyu; Millen, Kathleen J; Dobyns, William B; Kessler, John A; Jalali, Ali; Cremer, James; Segre, Alberto; Manak, J Robert; Aldinger, Kimerbly A; Suzuki, Satoshi; Natsume, Nagato; Ono, Maya; Hai, Huynh Dai; Viet, Le Thi; Loddo, Sara; Valente, Enza M; Bernardini, Laura; Ghonge, Nitin; Ferguson, Polly J; Bassuk, Alexander G

2013-08-01

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles and detected a mutation in the extracellular matrix (ECM) protein-encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1-binding partner. Structural modeling of the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the ECM in the pathogenesis of Dandy-Walker spectrum disorders. © 2013 WILEY PERIODICALS, INC.

[Conservative anal fistula treatment with collagenic plug and human fibrin sealant. Preliminary results].

PubMed

Gubitosi, A; Moccia, G; Malinconico, F A; Docimo, G; Ruggiero, R; Iside, G; Avenia, N; Docimo, L; Foroni, F; Gilio, F; Sparavigna, L; Agresti, M

2009-01-01

The authors, on the basis of a long clinical experience with human fibrin glue in general surgery, compared two different extracellular matrix (collagen), Surgisis and TissueDura, with human fibrin glue, applied during the operation, and sometimes in postoperative, to obtain the healing of perianal fistulas. The collagenic extracellular matrix provides, according to the rationale suggested, an optimal three-dimensional structure for the fibroblastic implant and neoangiogenesis, hence for the fistula "fibrotizzation" and closure. The encouraging results for transphincteric fistulas and a simple and easy technique push to researchers on samples statistically significant.

A fast and mild decellularization protocol for obtaining extracellular matrix.

PubMed

Mirzarafie, Ariana; Grainger, Rhian K; Thomas, Ben; Bains, William; Ustok, Fatma I; Lowe, Chris R

2014-04-01

Degradation of extracellular matrix (ECM) function with age is a major cause of loss of tissue function with age that we would wish to reverse. Tissue engineering to provide replacement tissue requires an ECM-mimicking scaffold for cell organization. The standard protocols for achieving this take 10 days and include steps that may change the protein structure of the ECM. Here we describe a much shorter protocol for decellularizing chicken muscle, skin, and tendon samples that achieves the same efficiency as the original protocol without protein cross-link interference. Our protocol can be completed in 72 hr.

Possible Involvement of Tight Junctions, Extracellular Matrix and Nuclear Receptors in Epithelial Differentiation

PubMed Central

Ichikawa-Tomikawa, Naoki; Sugimoto, Kotaro; Satohisa, Seiro; Nishiura, Keisuke; Chiba, Hideki

2011-01-01

Tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane. They consist of four kinds of transmembrane proteins (occludin, claudins, junctional adhesion molecules, and tricellulin) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function. The mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases. In this paper, we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions, extracellular matrix, and nuclear receptors in epithelial differentiation. PMID:22162632

Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

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

Nelson, Celeste M.; Bissell, Mina J.

2006-03-09

The microenvironment surrounding cells influences gene expression, such that a cell's behavior is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble cues released locally or by distant tissues. We describe the essential role of context and organ structure in directing mammary gland development and differentiated function, and in determining response to oncogenic insults including mutations. We expand on the concept of 'dynamic reciprocity' to present an integrated view of development, cancer, and aging, and posit that genes are like piano keys: while essential, it is the context that makes the music.

Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

PubMed Central

Brown, Helen L.; Hanman, Kate; Reuter, Mark; Betts, Roy P.; van Vliet, Arnoud H. M.

2015-01-01

Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments. PMID:26217328

Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment.

PubMed

Brown, Helen L; Hanman, Kate; Reuter, Mark; Betts, Roy P; van Vliet, Arnoud H M

2015-01-01

Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments.

ABCG1-mediated generation of extracellular cholesterol microdomains[S

PubMed Central

Freeman, Sebastian R.; Jin, Xueting; Anzinger, Joshua J.; Xu, Qing; Purushothaman, Sonya; Fessler, Michael B.; Addadi, Lia; Kruth, Howard S.

2014-01-01

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space. PMID:24212237

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy.

PubMed

Reichhardt, Courtney; Ferreira, Jose A G; Joubert, Lydia-Marie; Clemons, Karl V; Stevens, David A; Cegelski, Lynette

2015-11-01

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Small intestinal submucosa extracellular matrix (CorMatrix®) in cardiovascular surgery: a systematic review

PubMed Central

Mosala Nezhad, Zahra; Poncelet, Alain; de Kerchove, Laurent; Gianello, Pierre; Fervaille, Caroline; El Khoury, Gebrine

2016-01-01

Extracellular matrix (ECM) derived from small intestinal submucosa (SIS) is widely used in clinical applications as a scaffold for tissue repair. Recently, CorMatrix® porcine SIS-ECM (CorMatrix Cardiovascular, Inc., Roswell, GA, USA) has gained popularity for ‘next-generation’ cardiovascular tissue engineering due to its ease of use, remodelling properties, lack of immunogenicity, absorbability and potential to promote native tissue growth. Here, we provide an overview of the biology of porcine SIS-ECM and systematically review the preclinical and clinical literature on its use in cardiovascular surgery. CorMatrix® has been used in a variety of cardiovascular surgical applications, and since it is the most widely used SIS-ECM, this material is the focus of this review. Since CorMatrix® is a relatively new product for cardiovascular surgery, some clinical and preclinical studies published lack systematic reporting of functional and pathological findings in sufficient numbers of subjects. There are also emerging reports to suggest that, contrary to expectations, an undesirable inflammatory response may occur in CorMatrix® implants in humans and longer-term outcomes at particular sites, such as the heart valves, may be suboptimal. Large-scale clinical studies are needed driven by robust protocols that aim to quantify the pathological process of tissue repair. PMID:26912574

Updated biological roles for matrix metalloproteinases and new "intracellular" substrates revealed by degradomics.

PubMed

Butler, Georgina S; Overall, Christopher M

2009-11-24

Shotgun proteomics techniques are conceptually unbiased, but data interpretation and follow-up experiments are often constrained by dogma, established beliefs that are accepted without question, that can dilute the power of proteomics and hinder scientific progress. Proteomics and degradomics, the characterization of all proteases, inhibitors, and protease substrates by genomic and proteomic techniques, have exponentially expanded the known substrate repertoire of the matrix metalloproteinases (MMPs), even to include intracellular proteins with newly recognized extracellular functions. Thus, the dogma that MMPs are dowdy degraders of extracellular matrix has been resolutely overturned, and the metamorphosis of MMPs into modulators of multiple signaling pathways has been facilitated. Here we review progress made in the field of degradomics and present a current view of the MMP degradome.

CELLULAR CONTROL OF CONNECTIVE TISSUE MATRIX TENSION†

PubMed Central

Langevin, Helene M.; Nedergaard, Maiken; Howe, Alan

2013-01-01

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occurs during viscoelastic relaxation. We propose that this response of fibroblasts plays a role in regulating extracellular fluid flow into the tissue, and protects against swelling when the matrix is stretched. This article reviews the evidence supporting possible mechanisms underlying this response including autocrine purinergic signaling. We also discuss fibroblast regulation of connective tissue tension with respect to lymphatic flow, immune function and cancer. PMID:23444198

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

NASA Technical Reports Server (NTRS)

Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

1993-01-01

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

Relationship of biomarkers of extracellular matrix with myocardial function in Type 2 diabetes mellitus.

PubMed

Liu, Ju-Hua; Chen, Yan; Zhen, Zhe; Ho, Lai-Ming; Tsang, Anita; Yuen, Michele; Lam, Karen; Tse, Hung-Fat; Yiu, Kai-Hang

2017-07-01

The study evaluated the relationship of extracellular matrix and renin angiotensin system with myocardial dysfunction in Type 2 diabetes mellitus. All patients underwent resting and exercise echocardiography, including conventional parameters, E/E' ratio, global longitudinal strain and diastolic function reserve index. Plasma matrix metalloproteinase-1, TIMP-1, amino-terminal propeptide of type I and type III procollagen and renin angiotensin system activity were measured. As patients with diastolic dysfunction had a higher plasma level of TIMP-1 and propeptide of type III procollagen than those with no diastolic dysfunction. After multivariate adjustment, TIMP-1 associated with E/E' (both at rest and stress) and diastolic function reserve index. TIMP-1 is independently associated with myocardial diastolic dysfunction in patients with Type 2 diabetes mellitus.

Identification of an S100A8 Receptor Neuroplastin-β and its Heterodimer Formation with EMMPRIN.

PubMed

Sakaguchi, Masakiyo; Yamamoto, Mami; Miyai, Masashi; Maeda, Tatsuo; Hiruma, Junichiro; Murata, Hitoshi; Kinoshita, Rie; Winarsa Ruma, I Made; Putranto, Endy Widya; Inoue, Yusuke; Morizane, Shin; Huh, Nam-Ho; Tsuboi, Ryoji; Hibino, Toshihiko

2016-11-01

We previously reported a positive feedback loop between S100A8/A9 and proinflammatory cytokines mediated by extracellular matrix metalloproteinase inducer, an S100A9 receptor. Here, we identify neuroplastin-β as an unreported S100A8 receptor. Neuroplastin-β and extracellular matrix metalloproteinase inducer form homodimers and a heterodimer, and they are co-localized on the surface of cultured normal human keratinocytes. Knockdown of both receptors suppressed cell proliferation and proinflammatory cytokine induction. Upon stimulation with S100A8, neuroplastin-β recruited GRB2 and activated extracellular signal-regulated kinase, resulting in keratinocyte proliferation. Keratinocyte proliferation in response to inflammatory stimuli was accelerated in involucrin promoter-driven S100A8 transgenic mice. Further, S100A8 and S100A9 were strongly up-regulated and co-localized in lesional skin of atopic dermatitis patients. Our results indicate that neuroplastin-β and extracellular matrix metalloproteinase inducer form a functional heterodimeric receptor for S100A8/A9 heterodimer, followed by recruitment of specific adaptor molecules GRB2 and TRAF2, and this signaling pathway is involved in activation of both keratinocyte proliferation and skin inflammation in atopic skin. Suppression of this pathway might have potential for treatment of skin diseases associated with chronic inflammation such as atopic dermatitis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Characterization of Cell Surface and EPS Remodeling of Azospirillum brasilense Chemotaxis-like 1 Signal Transduction Pathway mutants by Atomic Force Microscopy

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

Billings, Amanda N; Siuti, Piro; Bible, Amber

2011-01-01

To compete in complex microbial communities, bacteria must quickly sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the modulation of multiple cellular responses, including motility, EPS production, and cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from Azospirillum brasilense was shown to modulate flocculation. In A. brasilense, cell surface properties, including EPS production, are thought to play a direct role in promoting flocculation. Using atomic force microscopy (AFM), we have detected distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains that are absent in the wild type strain.more » Whereas the wild type strain produces a smooth mucosal extracellular matrix, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition and lectin-binding assays suggest that the composition of EPS components in the extracellular matrix differs between the cheA1 and cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that mutations in the Che1 pathway that result in increased flocculation are correlated with distinctive changes in the extracellular matrix structure produced by the mutants, including likely changes in the EPS structure and/or composition.« less

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

PubMed

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

Mechanical phenotyping of cells and extracellular matrix as grade and stage markers of lung tumor tissues.

PubMed

Panzetta, Valeria; Musella, Ida; Rapa, Ida; Volante, Marco; Netti, Paolo A; Fusco, Sabato

2017-07-15

The mechanical cross-talk between cells and the extra-cellular matrix (ECM) regulates the properties, functions and healthiness of the tissues. When this is disturbed it changes the mechanical state of the tissue components, singularly or together, and cancer, along with other diseases, may start and progress. However, the bi-univocal mechanical interplay between cells and the ECM is still not properly understood. In this study we show how a microrheology technique gives us the opportunity to evaluate the mechanics of cells and the ECM at the same time. The mechanical phenotyping was performed on the surgically removed tissues of 10 patients affected by adenocarcinoma of the lung. A correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Our findings suggest a sort of asymmetric modification of the mechanical properties of the cells and the extra-cellular matrix in the tumor, being the more compliant cell even though it resides in a stiffer matrix. Overall, the simultaneous mechanical characterization of the tissues constituents (cells and ECM) provided new support for diagnosis and offered alternative points of analysis for cancer mechanobiology. When the integrity of the mechanical cross-talk between cells and the extra-cellular matrix is disturbed cancer, along with other diseases, may initiate and progress. Here, we show how a new technique gives the opportunity to evaluate the mechanics of cells and the ECM at the same time. It was applied on surgically removed tissues of 10 patients affected by adenocarcinoma of the lung and a correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The Extracellular Matrix of the Lateral Pharyngeal Wall in Obstructive Sleep Apnea

PubMed Central

Dantas, Danielle Andrade da Silva; Mauad, Thais; Silva, Luiz F. F.; Lorenzi-Filho, Geraldo; Formigoni, Gilberto G. S.; Cahali, Michel B.

2012-01-01

Study Objectives: To compare the components of the extracellular matrix in the lateral pharyngeal muscular wall in patients with and without obstructive sleep apnea (OSA). This may help to explain the origin of the increased collapsibility of the pharynx in patients with OSA. Design: Specimens from the superior pharyngeal constrictor muscle, obtained during pharyngeal surgeries, were evaluated using histochemical and immunohistochemical analyses to determine the fractional area of collagen types I and III, elastic fibers, versican, fibronectin, and matrix metalloproteinases 1 and 2 in the endomysium. Setting: Academic tertiary center. Patiens: A total of 51 nonobese adult patients, divided into 38 patients with OSA and 13 nonsnoring control subjects without OSA. Interventions: Postintervention study performed on tissues from patients after elective surgery. Measurements and Results: Pharyngeal muscles of patients with OSA had significantly more collagen type I than pharyngeal muscles in control subjects. Collagen type I was correlated positively and independently with age. The other tested components of the extracellular matrix did not differ significantly between groups. In a logistic regression, an additive effect of both the increase of collagen type I and the increase in age with the presence of OSA was observed (odds ratio (OR), 2.06; 95% confidence interval (CI), 1.17-3.63), when compared with the effect of increased age alone (OR, 1.11; 95% CI, 1.03-1.20). Conclusion: Collagen type I in the superior pharyngeal constrictor muscle was more prevalent in patients with OSA and also increased with age. It was hypothesized that this increase could delay contractile-relaxant responses in the superior pharyngeal constrictor muscle at the expiratory-inspiratory phase transition, thus increasing pharyngeal collapsibility. Citation: Dantas DAS; Mauad T; Silva LFF; Lorenzi-Filho G; Formigoni GGS; Cahali MB. The extracellular matrix of the lateral pharyngeal wall in obstructive sleep apnea. SLEEP 2012;35(4):483-490. PMID:22467986

The Ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing

PubMed Central

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla; Diekwisch, Thomas G.H.; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBNΔ5-6 truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. PMID:26899203

Extracellular matrix stiffness causes systematic variations in proliferation and chemosensitivity in myeloid leukemias.

PubMed

Shin, Jae-Won; Mooney, David J

2016-10-25

Extracellular matrix stiffness influences biological functions of some tumors. However, it remains unclear how cancer subtypes with different oncogenic mutations respond to matrix stiffness. In addition, the relevance of matrix stiffness to in vivo tumor growth kinetics and drug efficacy remains elusive. Here, we designed 3D hydrogels with physical parameters relevant to hematopoietic tissues and adapted them to a quantitative high-throughput screening format to facilitate mechanistic investigations into the role of matrix stiffness on myeloid leukemias. Matrix stiffness regulates proliferation of some acute myeloid leukemia types, including MLL-AF9 + MOLM-14 cells, in a biphasic manner by autocrine regulation, whereas it decreases that of chronic myeloid leukemia BCR-ABL + K-562 cells. Although Arg-Gly-Asp (RGD) integrin ligand and matrix softening confer resistance to a number of drugs, cells become sensitive to drugs against protein kinase B (PKB or AKT) and rapidly accelerated fibrosarcoma (RAF) proteins regardless of matrix stiffness when MLL-AF9 and BCR-ABL are overexpressed in K-562 and MOLM-14 cells, respectively. By adapting the same hydrogels to a xenograft model of extramedullary leukemias, we confirm the pathological relevance of matrix stiffness in growth kinetics and drug sensitivity against standard chemotherapy in vivo. The results thus demonstrate the importance of incorporating 3D mechanical cues into screening for anticancer drugs.

Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

PubMed

Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

2017-11-01

The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

PubMed

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-06-01

Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 μm; P =0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P =0.0015; interleukin-10: 7.6-fold, P =0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 μm; P =0.0306), increased collagen density (2.4-fold; P =0.0432), and increased number of M2 macrophages (2.1-fold; P =0.0335). CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. © 2017 American Heart Association, Inc.

Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells.

PubMed

Rodriguez, Elena M; Dunham, Elizabeth E; Martin, G Steven

2009-10-01

Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix. Copyright 2009 Wiley-Liss, Inc.

Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones.

PubMed

Malik, Minnie; Britten, Joy; Cox, Jeris; Patel, Amrita; Catherino, William H

2016-01-01

To determine the effect of GnRH analogues (GnRH-a) leuprolide acetate (LA) and cetrorelix acetate on gonadal hormone-regulated expression of extracellular matrix in uterine leiomyoma three-dimensional (3D) cultures. Laboratory study. University research laboratory. Women undergoing hysterectomy for symptomatic leiomyomas. The 3D cell cultures, protein analysis, Western blot, immunohistochemistry. Expression of extracellular matrix proteins, collagen 1, fibronectin, and versican in leiomyoma cells 3D cultures exposed to E2, P, LA, cetrorelix acetate, and combinations for 24- and 72-hour time points. The 3D leiomyoma cultures exposed to E2 for 24 hours demonstrated an increased expression of collagen-1 and fibronectin, which was maintained for up to 72 hours, a time point at which versican was up-regulated significantly. Although P up-regulated collagen-1 protein (1.29 ± 0.04) within 24 hours of exposure, significant increase in all extracellular matrix (ECM) proteins was observed when the gonadal hormones were used concomitantly. Significant decrease in the amount of ECM proteins was observed on use of GnRH-a, LA and cetrorelix, with 24-hour exposure. Both the compounds also significantly decreased ECM protein concentration despite the presence of E2 or both gonadal hormones. This study demonstrates that GnRH-a directly affect the gonadal hormone-regulated collagen-1, fibronectin, and versican production in their presence. These findings suggest that localized therapy with GnRH-a may inhibit leiomyoma growth even in the presence of endogenous gonadal hormone exposure, thereby providing a mechanism to eliminate the hypoestrogenic side effects associated with GnRH-a therapy. Published by Elsevier Inc.

Assessment of Myocardial Remodeling Using an Elastin/Tropoelastin Specific Agent with High Field Magnetic Resonance Imaging (MRI).

PubMed

Protti, Andrea; Lavin, Begoña; Dong, Xuebin; Lorrio, Silvia; Robinson, Simon; Onthank, David; Shah, Ajay M; Botnar, Rene M

2015-08-13

Well-defined inflammation, proliferation, and maturation phases orchestrate the remodeling of the injured myocardium after myocardial infarction (MI) by controlling the formation of new extracellular matrix. The extracellular matrix consists mainly of collagen but also fractions of elastin. It is thought that elastin is responsible for maintaining elastic properties of the myocardium, thus reducing the risk of premature rupture. An elastin/tropoelastin-specific contrast agent (Gd-ESMA) was used to image tropoelastin and mature elastin fibers for in vivo assessment of extracellular matrix remodeling post-MI. Gd-ESMA enhancement was studied in a mouse model of myocardial infarction using a 7 T MRI scanner and results were compared to those achieved after injection of a nonspecific control contrast agent, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). In the infarcted tissue, Gd-ESMA uptake (measured as R1 relaxation rate) steadily increased from day 3 to day 21 as a result of the synthesis of elastin/tropoelastin. R1 values were in good agreement with histological findings. A similar R1 behavior was observed in the remote myocardium. No mature cross-linked elastin was found at any time point. In contrast, Gd-DTPA uptake was only observed in the infarct with no changes in R1 values between 3 and 21 days post-MI. We demonstrate the feasibility of in vivo imaging of extracellular matrix remodeling post-MI using a tropoelastin/elastin binding MR contrast agent, Gd-ESMA. We found that tropoelastin is the main contributor to the increased MRI signal at late stages of MI where its augmentation in areas of infarction was in good agreement with the R1 increase. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Pirfenidone inhibits transforming growth factor β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Park, Il-Ho; Lee, Heung-Man

2015-01-01

Pirfenidone has been shown to have antifibrotic and anti-inflammatory effects in the lungs. The purpose of this study was to evaluate the inhibitory effects of pirfenidone on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix accumulation. We also determined the molecular mechanisms of pirfenidone in nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from nasal polyps from eight patients who had chronic rhinosinusitis with nasal polyp. Pirfenidone was used to treat TGF-β1-induced NPDFs. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Fibroblast migration was evaluated with scratch assays. Expression levels of α-smooth muscle actin (SMA), fibronectin, and phosphorylated Smad2/3 were determined by Western blot and/or reverse transcription-polymerase chain reaction and immunofluorescent staining. Total collagen production was analyzed with the Sircol collagen assay and contractile activity was measured by a collagen gel contraction assay. Pirfenidone (0-2 mg/mL) has no significant cytotoxic effects in TGF-β1-induced NPDFs. Migration of NPDFs was significantly inhibited by pirfenidone treatment. The expression levels of α-SMA and fibronectin were significantly reduced in pirfenidone-treated NPDFs. Collagen contraction and production were also significantly decreased by pirfenidone treatment. Finally, pirfenidone significantly inhibited phosphorylation of the Smad2/3 pathway in TGF-β1-induced NPDFs. Pirfenidone has an inhibitory effect on TGF-β1-induced migration, myofibroblast differentiation (α-SMA), extracellular matrix accumulation, and collagen contraction by blocking the phosphorylation of Smad2/3 pathways in NPDFs. Thus, pirfenidone may inhibit TGF-β1-induced extracellular matrix by regulating Smad2/3.

Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in Geobacter sulfurreducens▿ †

PubMed Central

Rollefson, Janet B.; Stephen, Camille S.; Tien, Ming; Bond, Daniel R.

2011-01-01

Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-d-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane. PMID:21169487

TM4SF5 promotes metastatic behavior of cells in 3D extracellular matrix gels by reducing dependency on environmental cues

PubMed Central

Nam, Seo Hee; Cheong, Jin-Gyu; Jeong, Doyoung; Lee, Seo-Jin; Pan, Cheol-Ho; Jung, Jae Woo; Kim, Hye-Jin; Ryu, Jihye; Kim, Ji Eon; Kim, Somi; Cho, Chang Yun; Kang, Min-Kyung; Lee, Kyung-Min; Lee, Jung Weon

2017-01-01

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells. PMID:29137358

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

PubMed Central

1995-01-01

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation. PMID:7593177

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.

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture*

PubMed Central

Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

2010-01-01

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment. PMID:19887451

Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture.

PubMed

Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-Ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

2010-01-15

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

Fibroblasts and the extracellular matrix in right ventricular disease.

PubMed

Frangogiannis, Nikolaos G

2017-10-01

Right ventricular failure predicts adverse outcome in patients with pulmonary hypertension (PH), and in subjects with left ventricular heart failure and is associated with interstitial fibrosis. This review manuscript discusses the cellular effectors and molecular mechanisms implicated in right ventricular fibrosis. The right ventricular interstitium contains vascular cells, fibroblasts, and immune cells, enmeshed in a collagen-based matrix. Right ventricular pressure overload in PH is associated with the expansion of the fibroblast population, myofibroblast activation, and secretion of extracellular matrix proteins. Mechanosensitive transduction of adrenergic signalling and stimulation of the renin-angiotensin-aldosterone cascade trigger the activation of right ventricular fibroblasts. Inflammatory cytokines and chemokines may contribute to expansion and activation of macrophages that may serve as a source of fibrogenic growth factors, such as transforming growth factor (TGF)-β. Endothelin-1, TGF-βs, and matricellular proteins co-operate to activate cardiac myofibroblasts, and promote synthesis of matrix proteins. In comparison with the left ventricle, the RV tolerates well volume overload and ischemia; whether the right ventricular interstitial cells and matrix are implicated in these favourable responses remains unknown. Expansion of fibroblasts and extracellular matrix protein deposition are prominent features of arrhythmogenic right ventricular cardiomyopathies and may be implicated in the pathogenesis of arrhythmic events. Prevailing conceptual paradigms on right ventricular remodelling are based on extrapolation of findings in models of left ventricular injury. Considering the unique embryologic, morphological, and physiologic properties of the RV and the clinical significance of right ventricular failure, there is a need further to dissect RV-specific mechanisms of fibrosis and interstitial remodelling. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

Experimental Detection and Visualization of the Extracellular Matrix in Macrocolony Biofilms.

PubMed

Serra, Diego O; Hengge, Regine

2017-01-01

By adopting elaborate three-dimensional morphologies that vary according to their extracellular matrix composition, macrocolony biofilms offer a unique opportunity to interrogate about the roles of specific matrix components in shaping biofilm architecture. Here, we describe two methods optimized for Escherichia coli that profit from morphology and the high level of structural organization of macrocolonies to gain insight into the production and assembly of amyloid curli and cellulose-the two major biofilm matrix elements of E. coli-in biofilms. The first method, the macrocolony morphology assay, is based on the ability of curli and cellulose-either alone or in combination-to generate specific morphological and Congo Red-staining patterns in E. coli macrocolonies, which can then be used as a direct visual readout for the production of these matrix components. The second method involves thin sectioning of macrocolonies, which along with in situ staining of amyloid curli and cellulose and microscopic imaging allows gaining fine details of the spatial arrangement of both matrix elements inside macrocolonies. Beyond their current use with E. coli and related curli and cellulose-producing Enterobacteriaceae, both the methods offer the potential to be adapted to other bacterial species.

Fibrinogen inhibits fibroblast-mediated contraction of collagen

PubMed Central

Nien, Yih-Dar; Han, Yuan-Ping; Tawil, Bill; Chan, Linda S.; Tuan, Tai-Lan; Garner, Warren L.

2008-01-01

Extracellular matrix changes in composition and organization as it transitions from the provisional matrix of the fibrin/platelet plug to collagen scar in healed wounds. The manner in which individual matrix proteins affect these activities is not well established. In this article we describe the interactions of two important extracellular matrix components, fibrin and collagen, using an in vitro model of wound contraction, the fibroblast-populated collagen lattice. We utilized different fibrinogen sources and measured tissue reorganization in floating and tensioned collagen lattices. Our results showed that both fibrin and fibrinogen decreased the contraction of fibroblast populated collagen lattices in a dose-dependent manner. Polymerization of fibrinogen to fibrin using thrombin had no effect on this inhibition. Further, there was no effect due to changes in protein concentration, alternate components of the fibrin sealant, or the enzymatic action of thrombin. These results suggest that the initial stability of the fibrin provisional matrix is due to the fibrin, because this protein appears to inhibit contraction of the matrix. This may be important in the early phases of wound healing when clot stability is vital for hemostasis. Later, as fibrin is replaced by collagen, wound contraction can occur. PMID:12950643

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

PubMed

Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

2016-05-04

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications

PubMed Central

Fortunato, Tiago M.; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A.; Pula, Giordano

2016-01-01

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

A Novel Human Adipocyte-derived Basement Membrane for Tissue Engineering Applications

NASA Astrophysics Data System (ADS)

Damm, Aaron

Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

Differentiating Pseudomonas sp. strain ADP cells in suspensions and biofilms using Raman spectroscopy and scanning electron microscopy.

PubMed

Henry, Victoria A; Jessop, Julie L P; Peeples, Tonya L

2017-02-01

High quality spectra of Pseudomonas sp. strain ADP in the planktonic and biofilm state were obtained using Raman microspectroscopy. These spectra enabled the identification of key differences between free and biofilm cells in the fingerprint region of Raman spectra in the nucleic acid, carbohydrate, and protein regions. Scanning electron microscopy (SEM) enabled detailed visualization of ADP biofilm with confirmation of associated extracellular matrix structure. Following extraction and Raman analysis of extracellular polymeric substances, Raman spectral differences between free and biofilm cells were largely attributed to the contribution of extracellular matrix components produced in mature biofilms. Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species. Graphical Abstract Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species.

Neural ECM proteases in learning and synaptic plasticity.

PubMed

Tsilibary, Effie; Tzinia, Athina; Radenovic, Lidija; Stamenkovic, Vera; Lebitko, Tomasz; Mucha, Mariusz; Pawlak, Robert; Frischknecht, Renato; Kaczmarek, Leszek

2014-01-01

Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink.

PubMed

Pati, Falguni; Cho, Dong-Woo

2017-01-01

Bioprinting provides an exciting opportunity to print and pattern all the components that make up a tissue-cells and extracellular matrix (ECM) material-in three dimensions (3D) to generate tissue analogues. A large number of materials have been used for making bioinks; however, majority of them cannot represent the complexity of natural ECM and thus are unable to reconstitute the intrinsic cellular morphologies and functions. We present here a method for making of bioink from decellularized extracellular matrices (dECMs) and a protocol for bioprinting of cell-laden constructs with this novel bioink. The dECM bioink is capable of providing an optimized microenvironment that is conducive to the growth of 3D structured tissue. We have prepared bioinks from different tissues, including adipose, cartilage and heart tissues and achieved high cell viability and functionality of the bioprinted tissue structures using our novel bioink.

Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

PubMed

Kong, Li; Zhao, Yun-Peng; Tian, Qing-Yun; Feng, Jian-Quan; Kobayashi, Tatsuya; Merregaert, Joseph; Liu, Chuan-Ju

2016-08-01

Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone-related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.-Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor. © FASEB.

Activation of Parathyroid Hormone 2 Receptor Induces Decorin Expression and Promotes Wound Repair

PubMed Central

Sato, Emi; Zhang, Ling-juan; Dorschner, Robert A.; Adase, Christopher A.; Choudhury, Biswa P.; Gallo, Richard L.

2018-01-01

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39−/− mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R−/− mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair. PMID:28454729

Upregulation of EMMPRIN (OX47) in Rat Dorsal Root Ganglion Contributes to the Development of Mechanical Allodynia after Nerve Injury.

PubMed

Wang, Qun; Sun, Yanyuan; Ren, Yingna; Gao, Yandong; Tian, Li; Liu, Yang; Pu, Yanan; Gou, Xingchun; Chen, Yanke; Lu, Yan

2015-01-01

Matrix metalloproteinases (MMPs) are widely implicated in inflammation and tissue remodeling associated with various neurodegenerative diseases and play an important role in nociception and allodynia. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) plays a key regulatory role for MMP activities. However, the role of EMMPRIN in the development of neuropathic pain is not clear. Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunofluorescence were performed to determine the changes of messenger RNA and protein of EMMPRIN/OX47 and their cellular localization in the rat dorsal root ganglion (DRG) after nerve injury. Paw withdrawal threshold test was examined to evaluate the pain behavior in spinal nerve ligation (SNL) model. The lentivirus containing OX47 shRNA was injected into the DRG one day before SNL. The expression level of both mRNA and protein of OX47 was markedly upregulated in ipsilateral DRG after SNL. OX47 was mainly expressed in the extracellular matrix of DRG. Administration of shRNA targeted against OX47 in vivo remarkably attenuated mechanical allodynia induced by SNL. In conclusion, peripheral nerve injury induced upregulation of OX47 in the extracellular matrix of DRG. RNA interference against OX47 significantly suppressed the expression of OX47 mRNA and the development of mechanical allodynia. The altered expression of OX47 may contribute to the development of neuropathic pain after nerve injury.

Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone–related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor

PubMed Central

Kong, Li; Zhao, Yun-Peng; Tian, Qing-Yun; Feng, Jian-Quan; Kobayashi, Tatsuya; Merregaert, Joseph; Liu, Chuan-Ju

2016-01-01

Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone–related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo. In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.—Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone–related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor. PMID:27075243

High attenuation areas on chest computed tomography in community-dwelling adults: the MESA study.

PubMed

Podolanczuk, Anna J; Oelsner, Elizabeth C; Barr, R Graham; Hoffman, Eric A; Armstrong, Hilary F; Austin, John H M; Basner, Robert C; Bartels, Matthew N; Christie, Jason D; Enright, Paul L; Gochuico, Bernadette R; Hinckley Stukovsky, Karen; Kaufman, Joel D; Hrudaya Nath, P; Newell, John D; Palmer, Scott M; Rabinowitz, Dan; Raghu, Ganesh; Sell, Jessica L; Sieren, Jered; Sonavane, Sushil K; Tracy, Russell P; Watts, Jubal R; Williams, Kayleen; Kawut, Steven M; Lederer, David J

2016-11-01

Evidence suggests that lung injury, inflammation and extracellular matrix remodelling precede lung fibrosis in interstitial lung disease (ILD). We examined whether a quantitative measure of increased lung attenuation on computed tomography (CT) detects lung injury, inflammation and extracellular matrix remodelling in community-dwelling adults sampled without regard to respiratory symptoms or smoking.We measured high attenuation areas (HAA; percentage of lung voxels between -600 and -250 Hounsfield Units) on cardiac CT scans of adults enrolled in the Multi-Ethnic Study of Atherosclerosis.HAA was associated with higher serum matrix metalloproteinase-7 (mean adjusted difference 6.3% per HAA doubling, 95% CI 1.3-11.5), higher interleukin-6 (mean adjusted difference 8.8%, 95% CI 4.8-13.0), lower forced vital capacity (FVC) (mean adjusted difference -82 mL, 95% CI -119--44), lower 6-min walk distance (mean adjusted difference -40 m, 95% CI -1--80), higher odds of interstitial lung abnormalities at 9.5 years (adjusted OR 1.95, 95% CI 1.43-2.65), and higher all cause-mortality rate over 12.2 years (HR 1.58, 95% CI 1.39-1.79).High attenuation areas are associated with biomarkers of inflammation and extracellular matrix remodelling, reduced lung function, interstitial lung abnormalities, and a higher risk of death among community-dwelling adults. Copyright ©ERS 2016.

Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

PubMed

Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

2017-01-01

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

PubMed

Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

2014-01-01

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the -112/-61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications.

Chicken bile Matrix metalloproteinase; its characterization and significance

USDA-ARS?s Scientific Manuscript database

Previous studies from our lab had shown that the avian bile was rich in matrix metalloproteinase (MMP), enzymes implicated in the degradation of extracellular matrices (ECM) such as collagens and proteoglycans. We hypothesized that bile MMP may be evolutionarily associated with the digestion of ECM ...

Novel entries in a fungal biofilm matrix encyclopedia

USDA-ARS?s Scientific Manuscript database

Virulence of Candida albicans is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we conduc...

Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

PubMed

Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

2016-01-01

The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

PubMed

Kubow, Kristopher E; Vukmirovic, Radmila; Zhe, Lin; Klotzsch, Enrico; Smith, Michael L; Gourdon, Delphine; Luna, Sheila; Vogel, Viola

2015-08-14

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

Interaction of electromagnetic fields with chondrocytes in gel culture

NASA Astrophysics Data System (ADS)

Grodzinsky, Alan J.; Buschmann, Michael D.; Gluzband, Yehezkiel A.

1992-01-01

The specific objectives of this research period were: (1) to quantify the effect of applied electric fields on chondrocyte metabolism, using a range of stimulation frequencies and amplitudes; (2) to compare the chondrocyte biosynthetic response to applied fields at early times in agarose gel culture before an extracellular matrix has accumulated and at later times after significant deposition of matrix around and between the cells; and (3) to begin to interpret the biosynthetic response to applied fields in terms of models of physical mechanisms. The results of these studies suggest that electric fields applied to chondrocytes in agarose can modulate the synthesis of proteoglycans and protein constituents. Biosynthesis may be inhibited or stimulated depending on the amplitude of the applied current density. In addition, the presence of extracellular matrix may enhance the ability of normal chondrocytes and cells in intact cartilage to respond to electric fields, although the presence of matrix was not required for the stimulatory response to be observed with Swarm rat chondrosarcoma cells.

Evaluation of matrix metalloproteinase-9 expressions in nasopharyngeal carcinoma patients

NASA Astrophysics Data System (ADS)

Farhat; Asnir, R. A.; Yudhistira, A.; Daulay, E. R.; Puspitasari, D.; Yulius, S.

2018-03-01

Nasopharyngeal carcinoma (NPC) is one of head and neck cancer with a poor prognosis because of the position of the tumor adjacent to the skull base and vital structures. Degradation of extracellular matrix that will cause tumor cells to invade surrounding tissues, vascular or lymphatic vessels. One that plays a role in the extracellular matrix degradation process is matrix metalloproteinase-9 (MMP-9). MMP-9 plays a role in tumor invasion process, metastasis and induction of tumor tissue vascularization. To determine the expression of MMP-9 in patients with nasopharyngeal carcinoma, a descriptive study was conducted by examining immunohistochemistry MMP-9 in 30 NPC tissues that had never received radiotherapy, chemotherapy or combination. Frequency distribution of NPC patient mostly in the age group 41-50 years old and 51-60 years were nine people (30.0%); men (73.3%) and non-keratinizing squamous cell carcinoma (53.3%) histopathology type. The overexpression of MMP-9 in patients with nasopharyngeal carcinoma were mostly found in advance stage.

Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

PubMed

Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

2016-01-01

Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Effects of Light Stress on Extracellular Cycling in a Cyanobacterial Biofilm Community

NASA Astrophysics Data System (ADS)

Stuart, R.; Mayali, X.; Pett-Ridge, J.; Weber, P. K.; Thelen, M.; Bebout, B.; Lipton, M. S.

2015-12-01

Cyanobacterial carbon excretion is crucial to carbon cycling in many microbial communities, but the nature and bioavailability of the carbon excreted is dependent on physiological function, which is often unknown. Cyanobacteria are the dominant primary producers in hypersaline mats and there is large reservoir of carbon in the extracellular matrix, but the nature and flux is understudied. In a previous study, we examined the macromolecular composition of the matrix of microbial mats from Elkhorn Slough in Monterey Bay, California and a unicyanobacterial culture, ESFC-1, isolated from the those mats, and found evidence for cyanobacterial degradation and re-uptake of extracellular organic matter. In this work, we further explore mechanisms of this degradation and re-uptake by examining effects of light using a combination of high-resolution imaging mass spectrometry (NanoSIMS) and metaproteomics of extracellular proteins. Based on these findings, we propose that mat Cyanobacteria store and recycle organic material from the mat extracellular matrix. Cyanobacteria can account for 70-90% of the biomass in the upper phototrophic layer of the mats, so their re-uptake of organic carbon and nitrogen has the potential to re-define organic matter availability in these systems. This work has implications for cyanobacterial adaptation to dynamic environments like microbial mats, where uptake of carbon and nitrogen in variable forms may be necessary to persist. This research was supported by the U.S. Department of Energy Office of Science, Office of Biological and Environmental Research Genomic Science program under FWP SCW1039. Work at LLNL was performed under the auspices of the U.S. Department of Energy under Contract DE-AC52-07NA27344.

Inhibition of MMP-13 with modified polymer particles

NASA Astrophysics Data System (ADS)

Tran, Hai; Bratlie, Kaitlin M.

2016-06-01

Matrix metalloproteinases (MMPs) are proteases that destroy the extracellular matrix and have important roles in the foreign body response, wound healing, and disease. Of particular importance is the chronic wound environment in which MMP activity is increased, resulting in destruction of the de novo extracellular matrix. One potential treatment of these wounds would be to use dressings that are capable of inhibiting MMP activity. In this study, we examined the effect of seven polymer modifiers (2-amino-3-guanidinopropionic acid, arginine, carnitine, citrulline, creatine, 3-guanidino propionic acid, and Nw-nitro-L-arginine) on MMP-13 activity. MMP-13 is a collagenase that is present in chronic wounds and is zinc dependent. Our results showed that these polymer modifiers were able to inhibit MMP-13 activity to varying degrees. The mechanism of inhibition appears to be binding zinc to the modifiers.

Cellular control of connective tissue matrix tension.

PubMed

Langevin, Helene M; Nedergaard, Maiken; Howe, Alan K

2013-08-01

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occurs during viscoelastic relaxation. We propose that this response of fibroblasts plays a role in regulating extracellular fluid flow into the tissue, and protects against swelling when the matrix is stretched. This article reviews the evidence supporting possible mechanisms underlying this response including autocrine purinergic signaling. We also discuss fibroblast regulation of connective tissue tension with respect to lymphatic flow, immune function, and cancer. Copyright © 2013 Wiley Periodicals, Inc.

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

PubMed

Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-07-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

Does the Loss of Stromal Caveolin-1 Remodel the Tumor Microenvironment by Activating Src-Mediated PEAK1 and PI3K Pathways

DTIC Science & Technology

2016-09-01

Inhibition of MAP kinase pathway prevents plasma protrusions Next we used a selective inhibitor of MAP kinases , PD98059, to address whether we can...from human patients harbor AKT1 and that AKT1 kinase activity is sustained in these particles, nominating them as active signaling platforms...with the extracellular matrix (ECM) and extracellular molecules (2). Though many classic extracellular signaling molecules (e.g., hormones, peptide

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

PubMed Central

2012-01-01

Background Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure. Methods We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells. Results Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested. Conclusions We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific. PMID:22631225

Astrocytes and extracellular matrix in extrasynaptic volume transmission.

PubMed

Vargová, Lýdia; Syková, Eva

2014-10-19

Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer's disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron-glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Lessons on the pathogenesis of aneurysm from heritable conditions

PubMed Central

Lindsay, Mark E.; Dietz, Harry C.

2013-01-01

Aortic aneurysm is common, accounting for 1–2% of all deaths in industrialized countries. Early theories of the causes of human aneurysm mostly focused on inherited or acquired defects in components of the extracellular matrix in the aorta. Although several mutations in the genes encoding extracellular matrix proteins have been recognized, more recent discoveries have shown important perturbations in cytokine signalling cascades and intracellular components of the smooth muscle contractile apparatus. The modelling of single-gene heritable aneurysm disorders in mice has shown unexpected involvement of the transforming growth factor-β cytokine pathway in aortic aneurysm, highlighting the potential for new therapeutic strategies. PMID:21593863

Back to basics--how the evolution of the extracellular matrix underpinned vertebrate evolution.

PubMed

Huxley-Jones, Julie; Pinney, John W; Archer, John; Robertson, David L; Boot-Handford, Raymond P

2009-04-01

The extracellular matrix (ECM) is a complex substrate that is involved in and influences a spectrum of behaviours such as growth and differentiation and is the basis for the structure of tissues. Although a characteristic of all metazoans, the ECM has elaborated into a variety of tissues unique to vertebrates, such as bone, tendon and cartilage. Here we review recent advances in our understanding of the molecular evolution of the ECM. Furthermore, we demonstrate that ECM genes represent a pivotal family of proteins the evolution of which appears to have played an important role in the evolution of vertebrates.

Stretching the boundaries of extracellular matrix research.

PubMed

Hynes, Richard O

2014-12-01

Extracellular matrix (ECM) proteins constitute >1% of the proteome and interact with many modifiers and growth factors to affect most aspects of cellular behaviour during development and normal physiology, as well as in diseases such as fibroses, cancer and many genetic disorders. In addition to biochemical signals provided to cells by ECM proteins, important cell–ECM interactions involve bidirectional mechanotransduction influences, which are dependent on the physical structure and organization of the ECM. These are beginning to be understood using twenty-first-century approaches, including biophysics, nanotechnology, biological engineering and modern microscopy. Articles in this issue of Nature Reviews Molecular Cell Biology review progress in our understanding of the ECM.

Extracellular matrix functionalized microcavities to control hematopoietic stem and progenitor cell fate.

PubMed

Kurth, Ina; Franke, Katja; Pompe, Tilo; Bornhäuser, Martin; Werner, Carsten

2011-06-14

Polymeric microcavities functionalized with extracellular matrix components were used as an experimental in vitro model to investigate principles of hematopoietic stem and progenitor cell (HSPC) fate control. Using human CD133+ HSPC we could demonstrate distinct differences in HSPC cycling and differentiation dependence on the adhesion ligand specificity (i.e., heparin, collagen I) and cytokine levels. The presented microcavity platform provides a powerful in vitro approach to explore the role of exogenous cues in HSPC fate decisions and can therefore be instrumental to progress in stem cell biology and translational research toward new therapies. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering a Biocompatible Scaffold with Either Micrometre or Nanometre Scale Surface Topography for Promoting Protein Adsorption and Cellular Response

PubMed Central

Le, Xuan; Poinern, Gérrard Eddy Jai; Ali, Nurshahidah; Berry, Cassandra M.; Fawcett, Derek

2013-01-01

Surface topographical features on biomaterials, both at the submicrometre and nanometre scales, are known to influence the physicochemical interactions between biological processes involving proteins and cells. The nanometre-structured surface features tend to resemble the extracellular matrix, the natural environment in which cells live, communicate, and work together. It is believed that by engineering a well-defined nanometre scale surface topography, it should be possible to induce appropriate surface signals that can be used to manipulate cell function in a similar manner to the extracellular matrix. Therefore, there is a need to investigate, understand, and ultimately have the ability to produce tailor-made nanometre scale surface topographies with suitable surface chemistry to promote favourable biological interactions similar to those of the extracellular matrix. Recent advances in nanoscience and nanotechnology have produced many new nanomaterials and numerous manufacturing techniques that have the potential to significantly improve several fields such as biological sensing, cell culture technology, surgical implants, and medical devices. For these fields to progress, there is a definite need to develop a detailed understanding of the interaction between biological systems and fabricated surface structures at both the micrometre and nanometre scales. PMID:23533416

Ultrastructure of the extracellular matrix of bovine dura mater, optic nerve sheath and sclera.

PubMed

Raspanti, M; Marchini, M; Della Pasqua, V; Strocchi, R; Ruggeri, A

1992-10-01

The sclera, the outermost sheath of the optic nerve and the dura mater have been investigated histologically and ultrastructurally. Although these tissues appear very similar under the light microscope, being dense connective tissues mainly composed of collagen bundles and a limited amount of cells and elastic fibres, they exhibit subtle differences on electron microscopy. In the dura and sclera collagen appears in the form of large, nonuniform fibrils, similar to those commonly found in tendons, while in the optic nerve sheath the fibrils appear smaller and uniform, similar to those commonly observed in reticular tissues, vessel walls and skin. Freeze-fracture also reveals these fibrils to have different subfibrillar architectures, straight or helical, which correspond to 2 distinct forms of collagen fibril previously described (Raspanti et al. 1989). The other extracellular matrix components also vary with the particular collagen fibril structure. Despite their common embryological derivation, the dura mater, optic nerve sheath and sclera exhibit diversification of their extracellular matrix consistent with the mechanical loads to which these tissues are subjected. Our observations indicate that the outermost sheath of the optic nerve resembles the epineurium of peripheral nerves rather than the dura to which it is commonly likened.

Human dermal and gingival fibroblasts in a three-dimensional culture: a comparative study on matrix remodeling.

PubMed

Chaussain Miller, C; Septier, D; Bonnefoix, M; Lecolle, S; Lebreton-Decoster, C; Coulomb, B; Pellat, B; Godeau, G

2002-03-01

Free-floating collagen lattice is considered a useful tool for assessing wound healing in vitro. This work compared extracellular matrix remodeling in collagen lattices populated by gingival or dermal fibroblasts. For 21 days we followed gel contraction and changes in cell number of collagen lattices seeded with l.5 x 10(5) fibroblasts of each tissue. We also used indirect immunodetection to study extracellular matrix components, metalloproteinases (MMPs), and their tissues inhibitors (TIMPs). In addition, the presence of MMPs and TIMPs in the culture media was analyzed by zymography and western blotting. No significant difference was found concerning gel contraction and changes in cell number. We observed the early expression of fibrillin I and collagen type III, apparently codistributed and at the end of the gel contraction their disappearance. Concomitantly we demonstrated the expression of MMPs and TIMPs, initially localized in cellular cytoplasm, then spreading in the extracellular compartment, and even found in the culture medium. This remodeling was more rapid and intense with gingival fibroblasts than dermal fibroblasts. In conclusion, gingival fibroblasts seem more efficient at remodeling the connective tissue than dermal fibroblasts and could lead to the better wound healing observed in vivo.

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation.

PubMed

Yu, L; Grist, S M; Nasseri, S S; Cheng, E; Hwang, Y-C E; Ni, C; Cheung, K C

2015-03-01

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.

Lamins at the crossroads of mechanosignaling

PubMed Central

Osmanagic-Myers, Selma; Dechat, Thomas

2015-01-01

The intermediate filament proteins, A- and B-type lamins, form the nuclear lamina scaffold adjacent to the inner nuclear membrane. B-type lamins confer elasticity, while A-type lamins lend viscosity and stiffness to nuclei. Lamins also contribute to chromatin regulation and various signaling pathways affecting gene expression. The mechanical roles of lamins and their functions in gene regulation are often viewed as independent activities, but recent findings suggest a highly cross-linked and interdependent regulation of these different functions, particularly in mechanosignaling. In this newly emerging concept, lamins act as a “mechanostat” that senses forces from outside and responds to tension by reinforcing the cytoskeleton and the extracellular matrix. A-type lamins, emerin, and the linker of the nucleoskeleton and cytoskeleton (LINC) complex directly transmit forces from the extracellular matrix into the nucleus. These mechanical forces lead to changes in the molecular structure, modification, and assembly state of A-type lamins. This in turn activates a tension-induced “inside-out signaling” through which the nucleus feeds back to the cytoskeleton and the extracellular matrix to balance outside and inside forces. These functions regulate differentiation and may be impaired in lamin-linked diseases, leading to cellular phenotypes, particularly in mechanical load-bearing tissues. PMID:25644599

Mechanoregulatory tumor-stroma crosstalk in pancreatic cancer: Measurements of the effects of extracellular matrix mechanics on tumor growth behavior, and vice-versa, to inform therapeutics

NASA Astrophysics Data System (ADS)

Celli, Jonathan; Jones, Dustin; El-Hamidi, Hamid; Cramer, Gwendolyn; Hanna, William; Caide, Andrew; Jafari, Seyedehrojin

The rheological properties of the extracellular matrix (ECM) have been shown to play key roles in regulating tumor growth behavior through mechanotranduction pathways. The role of the mechanical microenvironment may be particularly important tumors of the pancreas, noted for an abundance of rigid fibrotic stroma, implicated in therapeutic resistance. At the same time, cancer cells and their stromal partners (e.g. tumor associated fibroblasts) continually alter the mechanical microenvironment in response to extracellular physical and biochemical cues as part of a two-way mechanoregulatory dialog. Here, we describe experimental studies using 3D pancreatic cell cultures with customized mechanical properties, combined with optical microrheology to provide insight into tumor-driven matrix remodeling. Quantitative microscopy provides measurements of phenotypic changes accompanying systematic variation of ECM composition in collagen and laminin-rich basement membrane admixtures, while analysis of the trajectories of passive tracer particles embedded in ECM report dynamic changes in heterogeneity, microstructure and local shear modulus accompanying both ECM stiffening (fibrosis) processes, and ECM degradation near invading cells. We gratefully acknowledge funding from the National Cancer Institute, R00CA155045 (PI: Celli).

Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

PubMed

Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2004-10-01

Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline.

PubMed Central

Miyata, S; Monnier, V

1992-01-01

Pyrraline is one of the major Maillard compounds resulting from the reaction of glucose with amino compounds at slightly acidic pH. For in vivo studies, monoclonal pyrraline antibodies were raised after immunization of Balb/c mice with keyhole limpet hemocyamin-caproyl pyrraline conjugate. Of 660 hybridoma clones from one donor, 260 produced an antibody to the free hapten, two of which named Pyr-A and Pyr-B also cross-reacted with L-lysyl pyrraline. Using Pyr-B antibody and an ELISA, a gradual increase in pyrraline immunoreactivity was observed in serum albumin incubated with glucose or 3-deoxyglucosone. Plasma pyrraline levels increased fourfold (P less than 0.001) in Sprague-Dawley rats upon induction of diabetes with streptozotocin and were twofold increased in randomly selected plasmas from diabetic humans. Highly specific pyrraline immunoreactivity was detected in sclerosed glomeruli from diabetic and old normal kidneys as well as in renal arteries with arteriolosclerosis and in perivascular and peritubular sclerosed extracellular matrix and basement membranes. The preferential localization of pyrraline immunoreactivity in the extracellular matrix strengthens the notion that the advanced glycosylation reaction may contribute to decreased turnover and thickening of the extracellular matrix in diabetes and aging. Images PMID:1556177

Assembly and remodeling of the fibrillar fibronectin extracellular matrix during gastrulation and neurulation in Xenopus laevis.

PubMed

Davidson, Lance A; Keller, Raymond; DeSimone, Douglas W

2004-12-01

Fibronectin, a major component of the extracellular matrix is critical for processes of cell traction and cell motility. Whole-mount confocal imaging of the three-dimensional architecture of the extracellular matrix is used to describe dynamic assembly and remodeling of fibronectin fibrils during gastrulation and neurulation in the early frog embryo. As previously reported, fibrils first appear under the prospective ectoderm. We describe here the first evidence for regulated assembly of fibrils along the somitic mesoderm/endoderm boundary as well as at the notochord/somitic mesoderm boundary and clearing of fibrils from the dorsal and ventral surfaces of the notochord that occurs over the course of a few hours. As gastrulation proceeds, fibrils are restored to the dorsal surface of the notochord, where the notochord contacts the prospective floor plate. As the neural folds form, fibrils are again remodeled as deep neural plate cells move medially. The process of neural tube closure leaves a region of the ectoderm overlying the neural crest transiently bare of fibrils. Fibrils are assembled surrounding the dorsal surface of the neural tube as the neural tube lumen is restored. Copyright (c) 2004 Wiley-Liss, Inc.

Role of tumor–host interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors

PubMed Central

Pluen, Alain; Boucher, Yves; Ramanujan, Saroja; McKee, Trevor D.; Gohongi, Takeshi; di Tomaso, Emmanuelle; Brown, Edward B.; Izumi, Yotaro; Campbell, Robert B.; Berk, David A.; Jain, Rakesh K.

2001-01-01

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor–host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors. PMID:11274375

Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes

PubMed Central

Cuadrado, Irene; Castejon, Borja; Martin, Ana M.; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis

2016-01-01

Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR. PMID:27649573

Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes.

PubMed

Cuadrado, Irene; Castejon, Borja; Martin, Ana M; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis; Zaragoza, Carlos

2016-01-01

Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

PubMed Central

Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng

2014-01-01

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl−/−; or Yes, Src, and Fyn knockout mice (YSF−/−)] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl−/− MEF showed impaired matrix endocytosis, YSF−/− MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9. PMID:25080486

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics.

PubMed

Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng; Shah, Vijay H

2014-10-01

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl(-/-); or Yes, Src, and Fyn knockout mice (YSF(-/-))] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl(-/-) MEF showed impaired matrix endocytosis, YSF(-/-) MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9. Copyright © 2014 the American Physiological Society.

Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

NASA Technical Reports Server (NTRS)

Mooney, D. J.; Langer, R.; Ingber, D. E.

1995-01-01

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS).

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

PubMed

Yang, Yuanheng; Lin, Hang; Shen, He; Wang, Bing; Lei, Guanghua; Tuan, Rocky S

2018-03-15

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

PubMed Central

Boncompagni, Simona; Arthurton, Lewis; Akujuru, Eugene; Pearson, Timothy; Steverding, Dietmar; Protasi, Feliciano; Mutungi, Gabriel

2015-01-01

A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway. PMID:25846902

A Comparative Study of Collagen Matrix Density Effect on Endothelial Sprout Formation Using Experimental and Computational Approaches.

PubMed

Shamloo, Amir; Mohammadaliha, Negar; Heilshorn, Sarah C; Bauer, Amy L

2016-04-01

A thorough understanding of determining factors in angiogenesis is a necessary step to control the development of new blood vessels. Extracellular matrix density is known to have a significant influence on cellular behaviors and consequently can regulate vessel formation. The utilization of experimental platforms in combination with numerical models can be a powerful method to explore the mechanisms of new capillary sprout formation. In this study, using an integrative method, the interplay between the matrix density and angiogenesis was investigated. Owing the fact that the extracellular matrix density is a global parameter that can affect other parameters such as pore size, stiffness, cell-matrix adhesion and cross-linking, deeper understanding of the most important biomechanical or biochemical properties of the ECM causing changes in sprout morphogenesis is crucial. Here, we implemented both computational and experimental methods to analyze the mechanisms responsible for the influence of ECM density on the sprout formation that is difficult to be investigated comprehensively using each of these single methods. For this purpose, we first utilized an innovative approach to quantify the correspondence of the simulated collagen fibril density to the collagen density in the experimental part. Comparing the results of the experimental study and computational model led to some considerable achievements. First, we verified the results of the computational model using the experimental results. Then, we reported parameters such as the ratio of proliferating cells to migrating cells that was difficult to obtain from experimental study. Finally, this integrative system led to gain an understanding of the possible mechanisms responsible for the effect of ECM density on angiogenesis. The results showed that stable and long sprouts were observed at an intermediate collagen matrix density of 1.2 and 1.9 mg/ml due to a balance between the number of migrating and proliferating cells. As a result of weaker connections between the cells and matrix, a lower collagen matrix density (0.7 mg/ml) led to unstable and broken sprouts. However, higher matrix density (2.7 mg/ml) suppressed sprout formation due to the high level of matrix entanglement, which inhibited cell migration. This study also showed that extracellular matrix density can influence sprout branching. Our experimental results support this finding.

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

PubMed

deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

2003-02-01

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function. Copyright 2003 Elsevier Science (USA)

A Candida Biofilm-Induced Pathway for Matrix Glucan Delivery: Implications for Drug Resistance

PubMed Central

Taff, Heather T.; Nett, Jeniel E.; Zarnowski, Robert; Ross, Kelly M.; Sanchez, Hiram; Cain, Mike T.; Hamaker, Jessica; Mitchell, Aaron P.; Andes, David R.

2012-01-01

Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics. PMID:22876186

Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

PubMed Central

Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

2010-01-01

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a β-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix. PMID:20088866

Vitamins E and C - effects on matrix components in the vascular system

USDA-ARS?s Scientific Manuscript database

The connective tissue in the vascular system, consisting mainly of vascular smooth muscle cells (VSMC) and the interstitial extracellular matrix (ECM), plays important roles in the maintenance of an intact vascular wall as well as in the repair of atherosclerotic lesions during disease development. ...

The influence of extracelluar matrix on intramuscular and extramuscular adipogenesis

USDA-ARS?s Scientific Manuscript database

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed. Engelbreth-Holm-Swarm (EHS) substratum and laminin per se markedly incre...

Biofilm Matrix Proteins.

PubMed

Fong, Jiunn N C; Yildiz, Fitnat H

2015-04-01

Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins, and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this article, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation.

Nonlinear mechanical response of the extracellular matrix: learning from articular cartilage

NASA Astrophysics Data System (ADS)

Kearns, Sarah; Das, Moumita

2015-03-01

We study the mechanical structure-function relations in the extracellular matrix (ECM) with focus on nonlinear shear and compression response. As a model system, our study focuses on the ECM in articular cartilage tissue which has two major mechanobiological components: a network of the biopolymer collagen that acts as a stiff, reinforcing matrix, and a flexible aggrecan network that facilitates deformability. We model this system as a double network hydrogel made of interpenetrating networks of stiff and flexible biopolymers respectively. We study the linear and nonlinear mechanical response of the model ECM to shear and compression forces using a combination of rigidity percolation theory and energy minimization approaches. Our results may provide useful insights into the design principles of the ECM as well as biomimetic hydrogels that are mechanically robust and can, at the same time, easily adapt to cues in their surroundings.

Microscopic and histochemical manifestations of hyaline cartilage dynamics.

PubMed

Malinin, G I; Malinin, T I

1999-01-01

Structure and function of hyaline cartilages has been the focus of many correlative studies for over a hundred years. Much of what is known regarding dynamics and function of cartilage constituents has been derived or inferred from biochemical and electron microscopic investigations. Here we show that in conjunction with ultrastructural, and high-magnification transmission light and polarization microscopy, the well-developed histochemical methods are indispensable for the analysis of cartilage dynamics. Microscopically demonstrable aspects of cartilage dynamics include, but are not limited to, formation of the intracellular liquid crystals, phase transitions of the extracellular matrix and tubular connections between chondrocytes. The role of the interchondrocytic liquid crystals is considered in terms of the tensegrity hypothesis and non-apoptotic cell death. Phase transitions of the extracellular matrix are discussed in terms of self-alignment of chondrons, matrix guidance pathways and cartilage growth in the absence of mitosis. The possible role of nonenzymatic glycation reactions in cartilage dynamics is also reviewed.

Capturing extracellular matrix properties in vitro: Microengineering materials to decipher cell and tissue level processes.

PubMed

Abdeen, Amr A; Lee, Junmin; Kilian, Kristopher A

2016-05-01

Rapid advances in biology have led to the establishment of new fields with tremendous translational potential including regenerative medicine and immunoengineering. One commonality to these fields is the need to extract cells for manipulation in vitro; however, results obtained in laboratory cell culture will often differ widely from observations made in vivo. To more closely emulate native cell biology in the laboratory, designer engineered environments have proved a successful methodology to decipher the properties of the extracellular matrix that govern cellular decision making. Here, we present an overview of matrix properties that affect cell behavior, strategies for recapitulating important parameters in vitro, and examples of how these properties can affect cell and tissue level processes, with emphasis on leveraging these tools for immunoengineering. © 2016 by the Society for Experimental Biology and Medicine.

Extracellular matrix and its receptors in Drosophila neural development

PubMed Central

Broadie, Kendal; Baumgartner, Stefan; Prokop, Andreas

2011-01-01

Extracellular matrix (ECM) and matrix receptors are intimately involved in most biological processes. The ECM plays fundamental developmental and physiological roles in health and disease, including processes underlying the development, maintenance and regeneration of the nervous system. To understand the principles of ECM-mediated functions in the nervous system, genetic model organisms like Drosophila provide simple, malleable and powerful experimental platforms. This article provides an overview of ECM proteins and receptors in Drosophila. It then focuses on their roles during three progressive phases of neural development: 1) neural progenitor proliferation, 2) axonal growth and pathfinding and 3) synapse formation and function. Each section highlights known ECM and ECM-receptor components and recent studies done in mutant conditions to reveal their in vivo functions, all illustrating the enormous opportunities provided when merging work on the nervous system with systematic research into ECM-related gene functions. PMID:21688401

Regulation of the basement membrane by epithelia generated forces

NASA Astrophysics Data System (ADS)

Tanner, Kandice

2012-12-01

Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre-invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor.

Fibromodulin deficiency reduces collagen structural network but not glycosaminoglycan content in a syngeneic model of colon carcinoma.

PubMed

Olsson, P Olof; Kalamajski, Sebastian; Maccarana, Marco; Oldberg, Åke; Rubin, Kristofer

2017-01-01

Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.

Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

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

Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito

2008-08-29

Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assaymore » revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.« less

Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation

PubMed Central

Mazza, Giuseppe; Rombouts, Krista; Rennie Hall, Andrew; Urbani, Luca; Vinh Luong, Tu; Al-Akkad, Walid; Longato, Lisa; Brown, David; Maghsoudlou, Panagiotis; Dhillon, Amar P.; Fuller, Barry; Davidson, Brian; Moore, Kevin; Dhar, Dipok; De Coppi, Paolo; Malago, Massimo; Pinzani, Massimo

2015-01-01

Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development. PMID:26248878

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix

PubMed Central

Williams, B. Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-01-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

Control of cell fate by the formation of an architecturally complex bacterial community.

PubMed

Vlamakis, Hera; Aguilar, Claudio; Losick, Richard; Kolter, Roberto

2008-04-01

Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells.

Control of cell fate by the formation of an architecturally complex bacterial community

PubMed Central

Vlamakis, Hera; Aguilar, Claudio; Losick, Richard; Kolter, Roberto

2008-01-01

Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells. PMID:18381896

Mechanistic understanding of nanoparticles' interactions with extracellular matrix: the cell and immune system.

PubMed

Engin, Ayse Basak; Nikitovic, Dragana; Neagu, Monica; Henrich-Noack, Petra; Docea, Anca Oana; Shtilman, Mikhail I; Golokhvast, Kirill; Tsatsakis, Aristidis M

2017-06-24

Extracellular matrix (ECM) is an extraordinarily complex and unique meshwork composed of structural proteins and glycosaminoglycans. The ECM provides essential physical scaffolding for the cellular constituents, as well as contributes to crucial biochemical signaling. Importantly, ECM is an indispensable part of all biological barriers and substantially modulates the interchange of the nanotechnology products through these barriers. The interactions of the ECM with nanoparticles (NPs) depend on the morphological characteristics of intercellular matrix and on the physical characteristics of the NPs and may be either deleterious or beneficial. Importantly, an altered expression of ECM molecules ultimately affects all biological processes including inflammation. This review critically discusses the specific behavior of NPs that are within the ECM domain, and passing through the biological barriers. Furthermore, regenerative and toxicological aspects of nanomaterials are debated in terms of the immune cells-NPs interactions.

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

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

Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observedmore » increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.« less

The extracellular matrix Component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms.

PubMed

Billings, Nicole; Millan, MariaRamirez; Caldara, Marina; Rusconi, Roberto; Tarasova, Yekaterina; Stocker, Roman; Ribbeck, Katharina

2013-01-01

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive "non-producing" cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.

Visualization of extracellular matrix components within sectioned Salmonella biofilms on the surface of human gallstones.

PubMed

Marshall, Joanna M; Flechtner, Alan D; La Perle, Krista M; Gunn, John S

2014-01-01

Chronic carriage of Salmonella Typhi is mediated primarily through the formation of bacterial biofilms on the surface of cholesterol gallstones. Biofilms, by definition, involve the formation of a bacterial community encased within a protective macromolecular matrix. Previous work has demonstrated the composition of the biofilm matrix to be complex and highly variable in response to altered environmental conditions. Although known to play an important role in bacterial persistence in a variety of contexts, the Salmonella biofilm matrix remains largely uncharacterized under physiological conditions. Initial attempts to study matrix components and architecture of the biofilm matrix on gallstone surfaces were hindered by the auto-fluorescence of cholesterol. In this work we describe a method for sectioning and direct visualization of extracellular matrix components of the Salmonella biofilm on the surface of human cholesterol gallstones and provide a description of the major matrix components observed therein. Confocal micrographs revealed robust biofilm formation, characterized by abundant but highly heterogeneous expression of polysaccharides such as LPS, Vi and O-antigen capsule. CsgA was not observed in the biofilm matrix and flagellar expression was tightly restricted to the biofilm-cholesterol interface. Images also revealed the presence of preexisting Enterobacteriaceae encased within the structure of the gallstone. These results demonstrate the use and feasibility of this method while highlighting the importance of studying the native architecture of the gallstone biofilm. A better understanding of the contribution of individual matrix components to the overall biofilm structure will facilitate the development of more effective and specific methods to disrupt these bacterial communities.

Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

PubMed

Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

2015-01-01

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

Quiescence and activation of stem and precursor cell populations in the subependymal zone of the mammalian brain are associated with distinct cellular and extracellular matrix signals

USDA-ARS?s Scientific Manuscript database

The subependymal zone (SEZ) of the lateral ventricles is one of the areas of the adult brain where new neurons are continuously generated from neural stem cells (NSCs), via rapidly dividing precursors. This neurogenic niche is a complex cellular and extracellular microenvironment, highly vascularize...

The Cleavage Effect of Mesenchymal Stem Cell and Its Derived Matrix Metalloproteinase‐2 on Extracellular α‐Synuclein Aggregates in Parkinsonian Models

PubMed Central

Oh, Se Hee; Kim, Ha Na; Park, Hyun Jung; Shin, Jin Young; Kim, Dong Yeol

2016-01-01

Abstract Ample evidence has suggested that extracellular α‐synuclein aggregates would play key roles in the pathogenesis and progression of Parkinsonian disorders (PDs). In the present study, we investigated whether mesenchymal stem cells (MSCs) and their derived soluble factors could exert neuroprotective effects via proteolysis of extracellular α‐synuclein. When preformed α‐synuclein aggregates were incubated with MSC‐conditioned medium, α‐synuclein aggregates were disassembled, and insoluble and oligomeric forms of α‐synuclein were markedly decreased, thus leading to a significant increase in neuronal viability. In an animal study, MSC or MSC‐conditioned medium treatment decreased the expression of α‐synuclein oligomers and the induction of pathogenic α‐synuclein with an attenuation of apoptotic cell death signaling. Furthermore, we identified that matrix metalloproteinase‐2 (MMP‐2), a soluble factor derived from MSCs, played an important role in the degradation of extracellular α‐synuclein. Our data demonstrated that MSCs and their derived MMP‐2 exert neuroprotective properties through proteolysis of aggregated α‐synuclein in PD‐related microenvironments. Stem Cells Translational Medicine 2017;6:949–961 PMID:28297586

Cellular senescence of human mammary epithelial cells (HMEC) is associated with an altered MMP-7/HB-EGF signaling and increased formation of elastin-like structures.

PubMed

Bertram, Catharina; Hass, Ralf

2009-10-01

The extracellular matrix (ECM) and a complex interplay of cell-to-cell and cell-to-matrix (ECM) interactions provide important platforms to determine cellular senescence and a potentially tumorigenic transformation of normal human mammary epithelial cells (HMEC). An enhanced formation of extracellular filaments, consisting of elastin-like structures, in senescent post-selection HMEC populations was paralleled by a significantly increased expression of its precursor protein tropoelastin and matched with a markedly elevated activity of the cross-linking enzyme family of lysyl oxidases (LOX). RNAi experiments revealed both the ECM metalloproteinase MMP-7 and the growth factor HB-EGF as potential effectors of an increased tropoelastin expression. Moreover, co-localization of MMP-7 and HB-EGF as well as a concomittant downstream signaling via Fra-1 indicated a possible association between the reduced MMP-7 enzyme activity and an impaired HB-EGF processing, resulting in an enhanced tropoelastin synthesis during senescence of HMEC. In agreement with previous work, these findings suggested an important influence of the extracellular proteinase MMP-7 on the aging process of HMEC, affecting both extracellular remodeling as well as intracellular signaling pathways.

Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

PubMed Central

Ali, Saniya; Saik, Jennifer E.; Gould, Dan J.; Dickinson, Mary E.

2013-01-01

Abstract Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis. PMID:23914330

Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

PubMed Central

Smith, Michael L; Gourdon, Delphine; Little, William C; Kubow, Kristopher E; Eguiluz, R. Andresen; Luna-Morris, Sheila; Vogel, Viola

2007-01-01

Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes. PMID:17914904

Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K.

PubMed

Ali Mohammed, Marwan Mansoor; Nerland, Audun H; Al-Haroni, Mohammed; Bakken, Vidar

2013-01-01

Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.

Stepwise Evolution of Coral Biomineralization Revealed with Genome-Wide Proteomics and Transcriptomics

PubMed Central

Sawada, Hitoshi; Satoh, Noriyuki

2016-01-01

Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs) in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1) formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2) additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3) calcification controlled by coral-specific SOMPs. PMID:27253604

Mapping the Dynamics of Shear Stress—Induced Structural Changes in Endothelial Cells

PubMed Central

Mott, Rosalind E.; Helmke, Brian P.

2009-01-01

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and extracellular matrix on a time scale of minutes. Using multi-wavelength 4-D fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and of GFP-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in both subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly towards the downstream direction within one minute after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and extracellular matrix are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction. PMID:17855768

Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

PubMed

Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

2018-08-01

Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

Shell Extracts from the Marine Bivalve Pecten maximus Regulate the Synthesis of Extracellular Matrix in Primary Cultured Human Skin Fibroblasts

PubMed Central

Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

2014-01-01

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the −112/−61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications. PMID:24949635

In vitro enhancement of extracellular matrix formation as natural bioscaffold for stem cell culture

NASA Astrophysics Data System (ADS)

Naroeni, Aroem; Shalihah, Qonitha; Meilany, Sofy

2017-02-01

Growing cells in plastic with liquid media for in vitro study is very common but far from physiological. The use of scaffold materials is more biocompatible. Extracellular matrix provides tissue integrity which acts as a native scaffold for cell attachment and interaction, as well as it serves as a reservoir for growth factors. For this reason, we have developed natural scaffold from mice fibroblast to form a natural scaffold for stem cell culture. Fibroblasts were cultured under crowded condition and lysed to form natural scaffold. The natural scaffold formation was observed using immunofluorescence which then will be used and tested for stem cell propagation and differentiation.

DNA-templated assembly of viral protein hydrogel

NASA Astrophysics Data System (ADS)

Xu, Xin; Tao, Ailin; Xu, Yun

2014-11-01

Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs.Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02414a

Hypoxia and the extracellular matrix: drivers of tumour metastasis

PubMed Central

Gilkes, Daniele M.; Semenza, Gregg L.; Wirtz, Denis

2014-01-01

Of the deaths attributed to cancer, 90% are due to metastasis, and treatments that prevent or cure metastasis remain elusive. Emerging data indicate that hypoxia and the extracellular matrix (ECM) might have crucial roles in metastasis. During tumour evolution, changes in the composition and the overall content of the ECM reflect both its biophysical and biological properties and these strongly influence tumour and stromal cell properties, such as proliferation and motility. Originally thought of as independent contributors to metastatic spread, recent studies have established a direct link between hypoxia and the composition and the organization of the ECM, which suggests a new model in which multiple microenvironmental signals might converge to synergistically influence metastatic outcome. PMID:24827502

The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.

PubMed

Ahmed, Ahmed Ashour; Mills, Anthony D; Ibrahim, Ashraf E K; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E; Iyer, N Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D; Earl, Helena M; Laskey, Ronald A; Caldas, Carlos; Brenton, James D

2007-12-01

The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Extracellular matrix motion and early morphogenesis

PubMed Central

Loganathan, Rajprasad; Rongish, Brenda J.; Smith, Christopher M.; Filla, Michael B.; Czirok, Andras; Bénazéraf, Bertrand

2016-01-01

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale ‘total’ cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. PMID:27302396

Heterogeneous Pathology of Melasma and Its Clinical Implications.

PubMed

Kwon, Soon-Hyo; Hwang, Young-Ji; Lee, Soo-Keun; Park, Kyoung-Chan

2016-05-26

Melasma is a commonly acquired hypermelanosis that affects sun-exposed areas of the skin, with frequent facial involvement. Its histologic manifestations are evident in the epidermis, extracellular matrix, and dermis. In addition to epidermal pigmentation, pathologic findings of melasma include extracellular matrix abnormality, especially solar elastosis. The disrupted basement membrane has been described in melasma with variable incidences. In the dermis, an increase in vascularity and an increase in the number of mast cells were observed, indicating that dermal factors have critical roles in the pathogenesis of melasma, despite the fact that melasma is characterized by epidermal hyperpigmentation. This review discusses such histologic characteristics of melasma, with consideration to their implications for melasma treatment.

Heterogeneous Pathology of Melasma and Its Clinical Implications

PubMed Central

Kwon, Soon-Hyo; Hwang, Young-Ji; Lee, Soo-Keun; Park, Kyoung-Chan

2016-01-01

Melasma is a commonly acquired hypermelanosis that affects sun-exposed areas of the skin, with frequent facial involvement. Its histologic manifestations are evident in the epidermis, extracellular matrix, and dermis. In addition to epidermal pigmentation, pathologic findings of melasma include extracellular matrix abnormality, especially solar elastosis. The disrupted basement membrane has been described in melasma with variable incidences. In the dermis, an increase in vascularity and an increase in the number of mast cells were observed, indicating that dermal factors have critical roles in the pathogenesis of melasma, despite the fact that melasma is characterized by epidermal hyperpigmentation. This review discusses such histologic characteristics of melasma, with consideration to their implications for melasma treatment. PMID:27240341

Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome*

PubMed Central

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L.; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J.; Bateman, John F.; Wilson, Richard

2013-01-01

The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations. PMID:23530037

Matrix Metalloproteinase-1 Activation Contributes to Airway Smooth Muscle Growth and Asthma Severity

PubMed Central

Naveed, Shams-un-nisa; Clements, Debbie; Jackson, David J.; Philp, Christopher; Billington, Charlotte K.; Soomro, Irshad; Reynolds, Catherine; Harrison, Timothy W.; Johnston, Sebastian L.; Shaw, Dominick E.

2017-01-01

Rationale: Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of people with asthma. Objectives: To investigate whether MMP-1 could be activated by mast cells and increase asthma severity. Methods: Patients with stable asthma and healthy control subjects underwent spirometry, methacholine challenge, and bronchoscopy, and their airway smooth muscle cells were grown in culture. A second asthma group and control subjects had symptom scores, spirometry, and bronchoalveolar lavage before and after rhinovirus-induced asthma exacerbations. Extracellular matrix was prepared from decellularized airway smooth muscle cultures. MMP-1 protein and activity were assessed. Measurements and Main Results: Airway smooth muscle cells generated pro–MMP-1, which was proteolytically activated by mast cell tryptase. Airway smooth muscle treated with activated mast cell supernatants produced extracellular matrix, which enhanced subsequent airway smooth muscle growth by 1.5-fold (P < 0.05), which was dependent on MMP-1 activation. In asthma, airway pro–MMP-1 was 5.4-fold higher than control subjects (P = 0.002). Mast cell numbers were associated with airway smooth muscle proliferation and MMP-1 protein associated with bronchial hyperresponsiveness. During exacerbations, MMP-1 activity increased and was associated with fall in FEV1 and worsening asthma symptoms. Conclusions: MMP-1 is activated by mast cell tryptase resulting in a proproliferative extracellular matrix. In asthma, mast cells are associated with airway smooth muscle growth, MMP-1 levels are associated with bronchial hyperresponsiveness, and MMP-1 activation are associated with exacerbation severity. Our findings suggest that airway smooth muscle/mast cell interactions contribute to asthma severity by transiently increasing MMP activation, airway smooth muscle growth, and airway responsiveness. PMID:27967204

Stimulation of matrix metalloproteinases by black-pigmented Bacteroides in human pulp and periodontal ligament cell cultures.

PubMed

Chang, Yu-Chao; Lai, Chung-Chih; Yang, Shun-Fa; Chan, You; Hsieh, Yih-Shou

2002-02-01

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes capable of degrading most components of the extracellular matrix. Recently, evidence has shown that MMPs may play a role in tissue degradation in inflamed dental pulp. To date very little is known regarding the mechanism of extracellular matrix destruction at the site of bacterial infection. The purpose of this study was to determine the effects of the supernatants from Porphyromonas endodontalis and Porphyromonas gingivalis on the production and secretion of MMPs by primary human pulp and periodontal ligament (PDL) cell cultures in vitro. The results were evaluated by substrate gel zymography from long-term cultures. The main gelatinase secreted by human pulp and PDL cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period, P. endodontalis and P. gingivalis were found to elevate MMP-2 production both in human pulp and PDL cell cultures. In addition, the stimulation was in a dose- and time-dependent manner. Both human pulp and PDL cells, however, treated with either P. endodontalis or P. gingivalis had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These results indicate that black-pigmented Bacteroides species play an important role in tissue destruction and disintegration of extracellular matrix in pulpal and periapical diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of microbial-induced pulpal and periapical lesion. An understanding of the actions of these black-pigmented Bacteroides species on pulp and PDL cells may result in new therapies to augment current treatment of pulpal and periapical lesions.

The phenotype of cancer cell invasion controlled by fibril diameter and pore size of 3D collagen networks.

PubMed

Sapudom, Jiranuwat; Rubner, Stefan; Martin, Steve; Kurth, Tony; Riedel, Stefanie; Mierke, Claudia T; Pompe, Tilo

2015-06-01

The behavior of cancer cells is strongly influenced by the properties of extracellular microenvironments, including topology, mechanics and composition. As topological and mechanical properties of the extracellular matrix are hard to access and control for in-depth studies of underlying mechanisms in vivo, defined biomimetic in vitro models are needed. Herein we show, how pore size and fibril diameter of collagen I networks distinctively regulate cancer cell morphology and invasion. Three-dimensional collagen I matrices with a tight control of pore size, fibril diameter and stiffness were reconstituted by adjustment of concentration and pH value during matrix reconstitution. At first, a detailed analysis of topology and mechanics of matrices using confocal laser scanning microscopy, image analysis tools and force spectroscopy indicate pore size and not fibril diameter as the major determinant of matrix elasticity. Secondly, by using two different breast cancer cell lines (MDA-MB-231 and MCF-7), we demonstrate collagen fibril diameter--and not pore size--to primarily regulate cell morphology, cluster formation and invasion. Invasiveness increased and clustering decreased with increasing fibril diameter for both, the highly invasive MDA-MB-231 cells with mesenchymal migratory phenotype and the MCF-7 cells with amoeboid migratory phenotype. As this behavior was independent of overall pore size, matrix elasticity is shown to be not the major determinant of the cell characteristics. Our work emphasizes the complex relationship between structural-mechanical properties of the extracellular matrix and invasive behavior of cancer cells. It suggests a correlation of migratory and invasive phenotype of cancer cells in dependence on topological and mechanical features of the length scale of single fibrils and not on coarse-grained network properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteomic Discovery and Development of a Multiplexed Targeted MRM-LC-MS/MS Assay for Urine Biomarkers of Extracellular Matrix Disruption in Mucopolysaccharidoses I, II, and VI.

PubMed

Heywood, Wendy E; Camuzeaux, Stephane; Doykov, Ivan; Patel, Nina; Preece, Rhian-Lauren; Footitt, Emma; Cleary, Maureen; Clayton, Peter; Grunewald, Stephanie; Abulhoul, Lara; Chakrapani, Anupam; Sebire, Neil J; Hindmarsh, Peter; de Koning, Tom J; Heales, Simon; Burke, Derek; Gissen, Paul; Mills, Kevin

2015-12-15

The mucopolysaccharidoses (MPS) are lysosomal storage disorders that result from defects in the catabolism of glycosaminoglycans. Impaired muscle, bone, and connective tissue are typical clinical features of MPS due to disruption of the extracellular matrix. Markers of MPS disease pathology are needed to determine disease severity and monitor effects of existing and emerging new treatments on disease mechanisms. Urine samples from a small cohort of MPS-I, -II, and -VI patients (n = 12) were analyzed using label-free quantative proteomics. Fifty-three proteins including many associated with extracellular matrix organization were differently expressed. A targeted multiplexed peptide MRM LC-MS/MS assay was used on a larger validation cohort of patient samples (MPS-I n = 18, MPS-II n = 12, MPS-VI n = 6, control n = 20). MPS-I and -II groups were further subdivided according to disease severity. None of the markers assessed were altered significantly in the mild disease groups compared to controls. β-galactosidase, a lysosomal protein, was elevated 3.6-5.7-fold significantly (p < 0.05) in all disease groups apart from mild MPS-I and -II. Collagen type Iα, fatty-acid-binding-protein 5, nidogen-1, cartilage oligomeric matrix protein, and insulin-like growth factor binding protein 7 concentrations were elevated in severe MPS I and II groups. Cartilage oligomeric matrix protein, insulin-like growth factor binding protein 7, and β-galactosidase were able to distinguish the severe neurological form of MPS-II from the milder non-neurological form. Protein Heg1 was significantly raised only in MPS-VI. This work describes the discovery of new biomarkers of MPS that represent disease pathology and allows the stratification of MPS-II patients according to disease severity.

Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia

PubMed Central

McKee, Marc D.; Hoac, Betty; Addison, William N.; Barros, Nilana M.T.; Millán, José Luis; Chaussain, Catherine

2013-01-01

As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential to dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition such that teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibres (Sharpey's fibres) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here with clinical examples given, namely tissue-nonspecific alkaline phosphatase (TNAP) and phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH), respectively, where levels of local and systemic circulating mineralization determinants are perturbed. In XLH, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating SIBLING proteins such as matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), and the phosphorylated peptides proteolytically released from them such as the acidic serine- and aspartate-rich motif (ASARM) peptide, may accumulate locally to impair mineralization in this disease. PMID:23931057

Microfabrication of extracellular matrix structures using multipohoton-excited photochemistry: Application to modeling ovarian tissue in vitro

NASA Astrophysics Data System (ADS)

Ajeti, Visar

The extracellular matrix plays a crucial role in tissue development, differentiation and homeostasis by providing the necessary biophysical and biochemical cues for the cells. In tumors, the composition and the structure of the microenvironment is thought to be manipulated by the cancers cells to support proliferative growth and enhanced migration as means of facilitated metastasis. Current in vitro tools to address these mechanistic events in tumor progression are lacking in part due to the difficulty in recapitulating the complexity of the composition and nanoarchitecture of the tumor microenvironment. In this thesis, we explore the feasibility of multiphoton-excited photochemistry as a fabrication tool for generating in vitro scaffolds that are highly repeatable, biologically relevant and relatively affordable in a research setting. The power of this technique lays in the capabilities of crosslinking whole extracellular matrix proteins in three dimensions (3D) to recreate key topographical features of the tissue with sub-micron resolution and high fidelity. The technological developments we present here enable direct translation of matrix topographies by using the high resolution image data of the tissue samples as a fabrication template. To this effect, we have applied the fabrication technique to generate gradients of crosslinked proteins as means of studying the role of haptotaxis in ovarian and breast cancers. Our findings show that cancer cells modulate their migration velocity and persistence in response to the changes in the composition of the extracellular matrix. In addition, we have examined structural features of the stroma in relation to cancer migration dynamics. We find that by recreating highly aligned nanoarchitectural features prevalent in cancer stroma, we see permissive and enhanced cell migration with cell morphologies similar to in vivo. We believe multiphoton fabrication to be an enabling tool in the next generation of tissue scaffolding. Having the means to carefully recreate complex topographies can ultimately enhance our knowledge of cancer migration in 3D environments as well as provide valued insights in developing novel therapies aim at treating this disease.

Skeletal Muscle Regeneration in a Rat (Rattus norvegicus) Model with CorMatrix and Adipose Derived Stem Cells

DTIC Science & Technology

2015-07-16

outcome or training benefit the DoD/USAF? Yes. This study provided evidence that extracellular matrix made from swine small intestinal submucosa does...Isometric functional testing was implemented prior to euthanasia 2 FDGXXX at 10 months to further evaluate healing at later time points

Imaging of matrix-disorder in normal and pathological human dermis using nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Zhuo, Shuangmu; Chen, Jianxin; Xie, Shusen; Zheng, Liqin; Jiang, Xingshan

2009-11-01

In dermis, collagen and elastin are important structural proteins of extracellular maxtrix. The matrix-disorder is associated with various physiologic processes, such as localized scleroderma, anetoderma, photoaging. In this work, we demonstrate the capability of nonlinear optical microscopy in imaging structural proteins in normal and pathological human dermis.

Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

PubMed

Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

2017-01-05

Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative analysis of collagens and fibronectin expression in human right ventricular hypertrophy.

PubMed

Peters, T H; Sharma, H S; Yilmaz, E; Bogers, A J

1999-06-30

One of the main features in human tetralogy of Fallot (TF) is right ventricular hypertrophy (RVH) due to pressure (sub-pulmonary stenosis) and volume overload (ventricular septal defect). Currently, primary correction at a young age is the treatment of choice. To unravel the role of extracellular matrix in RVH, we examined myocardial expression of collagens and fibronectin in TF patients with primary correction (TF1, age 0.7 +/- 0.2 yr.), secondary surgery (TF2, age 36.9 +/- 4.6 yr), and in age-matched control patients. Sirius red staining quantified by video imaging showed significantly increased interstitial staining for collagens in both TF1 and TF2 groups as compared to respective controls. Fibronectin was expressed in extracellular spaces, perivascular regions, and in some cardiomyocytes. Quantitative analysis of fibronectin revealed increased expression in only TF1 group as compared to respective control. Our results indicate an increased amount of myocardial extracellular matrix deposition as a sign of fibrosis during RVH in patients with TF.

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo*

PubMed Central

Soulet, Fabienne; Kilarski, Witold W.; Roux-Dalvai, Florence; Herbert, John M. J.; Sacewicz, Izabela; Mouton-Barbosa, Emmanuelle; Bicknell, Roy; Lalor, Patricia; Monsarrat, Bernard; Bikfalvi, Andreas

2013-01-01

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments. PMID:23674615

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

PubMed Central

Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-01-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

PubMed

Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-08-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Compression stiffening of brain and its effect on mechanosensing by glioma cells

NASA Astrophysics Data System (ADS)

Pogoda, Katarzyna; Chin, LiKang; Georges, Penelope C.; Byfield, FitzRoy J.; Bucki, Robert; Kim, Richard; Weaver, Michael; Wells, Rebecca G.; Marcinkiewicz, Cezary; Janmey, Paul A.

2014-07-01

Many cell types, including neurons, astrocytes and other cells of the central nervous system, respond to changes in the extracellular matrix or substrate viscoelasticity, and increased tissue stiffness is a hallmark of several disease states, including fibrosis and some types of cancers. Whether the malignant tissue in brain, an organ that lacks the protein-based filamentous extracellular matrix of other organs, exhibits the same macroscopic stiffening characteristic of breast, colon, pancreatic and other tumors is not known. In this study we show that glioma cells, like normal astrocytes, respond strongly in vitro to substrate stiffness in the range of 100 to 2000 Pa, but that macroscopic (mm to cm) tissue samples isolated from human glioma tumors have elastic moduli in the order of 200 Pa that are indistinguishable from those of normal brain. However, both normal brain and glioma tissues increase their shear elastic moduli under modest uniaxial compression, and glioma tissue stiffens more strongly under compression than normal brain. These findings suggest that local tissue stiffness has the potential to alter glial cell function, and that stiffness changes in brain tumors might arise not from increased deposition or crosslinking of the collagen-rich extracellular matrix, but from pressure gradients that form within the tumors in vivo.

Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering.

PubMed

Pellegata, Alessandro F; Asnaghi, M Adelaide; Stefani, Ilaria; Maestroni, Anna; Maestroni, Silvia; Dominioni, Tommaso; Zonta, Sandro; Zerbini, Gianpaolo; Mantero, Sara

2013-01-01

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at -80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Young's modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.

Mef2c Regulates Transcription of the Extracellular Matrix Protein Cartilage Link Protein 1 in the Developing Murine Heart

PubMed Central

Phelps, Aimee L.; Ghatnekar, Angela V.; Barth, Jeremy L.; Norris, Russell A.; Wessels, Andy

2013-01-01

Cartilage Link Protein 1 (Crtl1) is an extracellular matrix (ECM) protein that stabilizes the interaction between hyaluronan and versican and is expressed in endocardial and endocardially-derived cells in the developing heart, including cells in the atrioventricular (AV) and outflow tract (OFT) cushions. Previous investigations into the transcriptional regulation of the Crtl1 gene have shown that Sox9 regulates Crtl1 expression in both cartilage and the AV valves. The cardiac transcription factor Mef2c is involved in the regulation of gene expression in cardiac and skeletal muscle cell lineages. In this study we have investigated the potential role of Mef2c in the regulation of ECM production in the endocardial and mesenchymal cell lineages of the developing heart. We demonstrate that the Crtl1 5′ flanking region contains two highly conserved Mef2 binding sites and that Mef2c is able to bind to these sites in vivo during cardiovascular development. Additionally, we show that Crtl1 transcription is dependent on Mef2c expression in fetal mitral valve interstitial cells (VICs). Combined, these findings highlight a new role for Mef2c in cardiac development and the regulation of cardiac extracellular matrix protein expression. PMID:23468913

Autoradiographic localization of specific (/sup 3/H)dexamethasone binding in fetal lung

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

Beer, D.G.; Butley, M.S.; Cunha, G.R.

1984-10-01

The cellular and subcellular localization of specific (/sup 3/H)dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of (/sup 3/H)dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Autoradiographs of (/sup 3/H)dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled.more » In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of (/sup 3/H)dexamethasone. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and (/sup 3/H)dexamethasone binding, a relationship is observed between extensive mesenchymal (/sup 3/H)dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.« less

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

PubMed Central

Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

2016-01-01

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

Mechanical Control of Myotendinous Junction Formation and Tendon Differentiation during Development.

PubMed

Valdivia, Mauricio; Vega-Macaya, Franco; Olguín, Patricio

2017-01-01

The development of the musculoskeletal system is a great model to study the interplay between chemical and mechanical inter-tissue signaling in cell adhesion, tissue morphogenesis and differentiation. In both vertebrates and invertebrates (e.g., Drosophila melanogaster ) the formation of muscle-tendon interaction generates mechanical forces which are required for myotendinous junction maturation and tissue differentiation. In addition, these forces must be withstood by muscles and tendons in order to prevent detachment from each other, deformation or even losing their integrity. Extracellular matrix remodeling at the myotendinous junction is key to resist mechanical load generated by muscle contraction. Recent evidences in vertebrates indicate that mechanical forces generated during junction formation regulate chemical signaling leading to extracellular matrix remodeling, however, the mechanotransduction mechanisms associated to this response remains elusive. In addition to extracellular matrix remodeling, the ability of Drosophila tendon-cells to bear mechanical load depends on rearrangement of tendon cell cytoskeleton, thus studying the molecular mechanisms involved in this process is critical to understand the contribution of mechanical forces to the development of the musculoskeletal system. Here, we review recent findings regarding the role of chemical and mechanical signaling in myotendinous junction formation and tendon differentiation, and discuss molecular mechanisms of mechanotransduction that may allow tendon cells to withstand mechanical load during development of the musculoskeletal system.

LOXL4 Is Induced by Transforming Growth Factor β1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling

PubMed Central

Busnadiego, Oscar; González-Santamaría, José; Lagares, David; Guinea-Viniegra, Juan; Pichol-Thievend, Cathy; Muller, Laurent

2013-01-01

Transforming growth factor β1 (TGF-β1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-β1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-β1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-β1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-β1, which involved the extracellular signal-regulated kinase (ERK)-dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-β1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis. PMID:23572561

Detection of abnormal extracellular matrix in the interstitium of regenerating renal tubules.

PubMed

Minuth, Will W; Denk, Lucia

2014-12-15

Stem/progenitor cells are promising candidates for the regeneration of parenchyma in acute and chronic renal failure. However, recent data exhibit that survival of stem/progenitor cells after implantation in diseased renal parenchyma is restricted. To elaborate basic parameters improving survival, cell seeding was simulated under advanced in vitro conditions. After isolation, renal stem/progenitor cells were mounted in a polyester interstitium for perfusion culture. During generation of tubules, chemically defined CO2 Independent Medium or Leibovitz's L-15 Medium was applied. Specimens were then fixed for transmission electron microscopy to analyze morphological features in generated tubules. Fixation in conventional glutaraldehyde (GA) solution shows development of tubules each exhibiting a polarized epithelium, an intact basal lamina and an inconspicuous interstitium. In contrast, special fixation of specimens in GA solution containing cupromeronic blue, ruthenium red or tannic acid unveils previously not visible extracellular matrix. Control experiments elucidate that a comparable extracellular matrix is not present in the interstitium of the matured kidney. Thus, generation of renal tubules in combination with advanced fixation of specimens for electron microscopy demonstrates that development of abnormal features in the newly developed interstitium has to be considered, when repair of renal parenchyma is performed by implantation of stem/progenitor cells.

Smooth muscle cell biglycan overexpression results in increased lipoprotein retention on extracellular matrix: implications for the retention of lipoproteins in atherosclerosis.

PubMed

O'Brien, Kevin D; Lewis, Katherine; Fischer, Jens W; Johnson, Pamela; Hwang, Jin-Yong; Knopp, Eleanor A; Kinsella, Michael G; Barrett, P Hugh R; Chait, Alan; Wight, Thomas N

2004-11-01

Lipoprotein retention on extracellular matrix (ECM) may play a central role in atherogenesis, and a specific extracellular matrix proteoglycan, biglycan, has been implicated in lipoprotein retention in human atherosclerosis. To test whether increased cellular biglycan expression results in increased retention of lipoproteins on ECM, rat aortic smooth muscle cells (SMCs) were transduced with a human biglycan cDNA-containing retroviral vector (LBSN) or with an empty retroviral vector (LXSN). To assess the importance of biglycan's glycosaminoglycan side chains in lipoprotein retention, ECM binding studies were also performed using RASMCs transduced with a retroviral vector encoding for a mutant, glycosaminoglycan-deficient biglycan (LBmutSN). Human biglycan mRNA and protein were confirmed in LBSN and LBmutSN RASMCs by Northern and Western blot analyses. HDL3+E binding to SMC ECM was increased significantly (as determined by 95% confidence intervals for binding curves) for LBSN as compared to either LXSN or LBmutSN cells; the increases for LBSN cell ECM were due primarily to an approximately 50% increase in binding sites (increased Bmax) versus LXSN cell ECM and of approximately 25% versus LBmutSN cell ECM. These results are consistent with the hypothesis that biglycan, through its glycosaminoglycan side chains, may mediate lipoprotein retention on atherosclerotic plaque ECM.

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

PubMed

Martins, Margarida; Uppuluri, Priya; Thomas, Derek P; Cleary, Ian A; Henriques, Mariana; Lopez-Ribot, José L; Oliveira, Rosário

2010-05-01

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

Extracellular matrix hydrogels from decellularized tissues: Structure and function.

PubMed

Saldin, Lindsey T; Cramer, Madeline C; Velankar, Sachin S; White, Lisa J; Badylak, Stephen F

2017-02-01

Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed. More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway.

PubMed

Barreca, Maria M; Spinello, Walter; Cavalieri, Vincenzo; Turturici, Giuseppina; Sconzo, Gabriella; Kaur, Punit; Tinnirello, Rosaria; Asea, Alexzander A A; Geraci, Fabiana

2017-07-01

Mouse mesoangioblasts are vessel-associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. Therefore, they represent a promising tool in the regeneration of injured tissues. Several studies have demonstrated that homing of mesoangioblasts into blood and injured tissues are mainly controlled by cytokines/chemokines and other inflammatory factors. However, little is known about the molecular mechanisms regulating their ability to traverse the extracellular matrix (ECM). Here, we demonstrate that membrane vesicles released by mesoangioblasts contain Hsp70, and that the released Hsp70 is able to interact by an autocrine mechanism with Toll-like receptor 4 (TLR4) and CD91 to stimulate migration. We further demonstrate that Hsp70 has a positive role in regulating matrix metalloproteinase 2 (MMP2) and MMP9 expression and that MMP2 has a more pronounced effect on cell migration, as compared to MMP9. In addition, the analysis of the intracellular pathways implicated in Hsp70 regulated signal transduction showed the involvement of both PI3K/AKT and NF-κB. Taken together, our findings present a paradigm shift in our understanding of the molecular mechanisms that regulate mesoangioblast stem cells ability to traverse the extracellular matrix (ECM). J. Cell. Physiol. 232: 1845-1861, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Extracellular matrix remodeling and matrix metalloproteinase inhibition in visceral adipose during weight cycling in mice.

PubMed

Caria, Cíntia Rabelo E Paiva; Gotardo, Érica Martins Ferreira; Santos, Paola Souza; Acedo, Simone Coghetto; de Morais, Thainá Rodrigues; Ribeiro, Marcelo Lima; Gambero, Alessandra

2017-10-15

Extracellular matrix (ECM) remodeling is necessary for a health adipose tissue (AT) expansion and also has a role during weight loss. We investigate the ECM alteration during weight cycling (WC) in mice and the role of matrix metalloproteinases (MMPs) was assessed using GM6001, an MMP inhibitor, during weight loss (WL). Obesity was induced in mice by a high-fat diet. Obese mice were subject to caloric restriction for WL followed by reintroduction to high-fat diet for weight regain (WR), resulting in a WC protocol. In addition, mice were treated with GM6001 during WL period and the effects were observed after WR. Activity and expression of MMPs was intense during WL. MMP inhibition during WL results in inflammation and collagen content reduction. MMP inhibition during WL period interferes with the period of subsequent expansion of AT resulting in improvements in local inflammation and systemic metabolic alterations induced by obesity. Our results suggest that MMPs inhibition could be an interesting target to improve adipose tissue inflammation during WL and to support weight cyclers. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

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

Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian

2011-01-01

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80%more » of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.« less

On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage.

PubMed

Dhote, Valentin; Skaalure, Stacey; Akalp, Umut; Roberts, Justine; Bryant, Stephanie J; Vernerey, Franck J

2013-03-01

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one's quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. Copyright © 2012 Elsevier Ltd. All rights reserved.

On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage

PubMed Central

Dhote, Valentin; Skaalure, Stacey; Akalp, Umut; Roberts, Justine; Bryant, Stephanie J.; Vernerey, Franck J.

2012-01-01

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one’s quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. PMID:23276516

Matrix metalloproteinase processing of signaling molecules to regulate inflammation.

PubMed

Butler, Georgina S; Overall, Christopher M

2013-10-01

Inflammation is a complex and highly regulated process that facilitates the clearance of pathogens and mediates tissue repair. Failure to resolve inflammation can lead to chronic inflammatory diseases such as periodontitis. Matrix metalloproteinases are generally thought to be detrimental in disease because degradation of extracellular matrix contributes to pathology. However, proteomic techniques (degradomics) are revealing that matrix metalloproteinases process a diverse array of substrates and therefore have a broad range of functions. Many matrix metalloproteinase substrates modulate inflammation and hence, by processing these proteins, matrix metalloproteinases can orchestrate the inflammatory response. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development and characterization of a snapshot Mueller matrix polarimeter for the determination of cervical cancer risk in the low resource setting

NASA Astrophysics Data System (ADS)

Ramella-Roman, Jessica C.; Gonzalez, Mariacarla; Chue-Sang, Joseph; Montejo, Karla; Krup, Karl; Srinivas, Vijaya; DeHoog, Edward; Madhivanan, Purnima

2018-04-01

Mueller Matrix polarimetry can provide useful information about the function and structure of the extracellular matrix. Mueller Matrix systems are sophisticated and costly optical tools that have been used primarily in the laboratory or in hospital settings. Here we introduce a low-cost snapshot Mueller Matrix polarimeter that that does not require external power, has no moving parts, and can acquire a full Mueller Matrix in less than 50 milliseconds. We utilized this technology in the study of cervical cancer in Mysore India, yet the system could be translated in multiple diagnostic applications.

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.

Quantification of the extracellular matrix of the Listeria monocytogenes biofilms of different phylogenic lineages with optimization of culture conditions.

PubMed

Combrouse, T; Sadovskaya, I; Faille, C; Kol, O; Guérardel, Y; Midelet-Bourdin, G

2013-04-01

The purpose of this study was to quantify the extracellular matrix of Listeria monocytogenes biofilm. A preliminary study was carried out to establish a relationship between phylogenetic lineage of 27 strains and their ability to form biofilm in various conditions. Biofilm formation on microtitre plates of 27 strains of L. monocytogenes belonging to lineages I or II was evaluated in different conditions [two temperatures (37 and 22°C) and two media (tryptone soy broth yeast extract medium (TSBYE) and MCDB 202 defined medium)] using crystal violet assay. Lineage II strains produced significantly more biofilm than lineage I strains. In microtitre plates assay, biofilm quantities were greater in MCDB 202 vs TSBYE medium [confirmed by scanning electron microscopy (SEM) analysis] and at 37 vs 22°C. Cultivable bacteria from biofilm population on Petri dishes were enumerated in greater quantities in TSBYE than in MCDB 202 medium. The SEM investigation established that L. monocytogenes biofilms produce extracellular matrix in both media at 37°C. The amount of exopolymers in the extracellular matrix and the pH values were significantly higher in TSBYE than in MCDB 202 medium. The exception was the ScottA strain that presented similar pH values and exopolymer contents in both media. Proteins were the most abundant exopolymer components, followed by DNA and polysaccharides. The interpretation of results of biofilm quantification was depending on the growth conditions, the viability of the bacteria and the analysis method. The quantities of proteins, DNA and polysaccharides were different according to the strains and the medium. This study screened the potential of a wide panel of L. monocytogenes strains to synthesize exopolymers in biofilm growing condition. The characterization of L. monocytogenes biofilm composition may help to develop new strategies to prevent the formation of biofilms and to remove the biofilms. © 2013 The Society for Applied Microbiology.

Influence of the amyloid dye Congo red on curli, cellulose, and the extracellular matrix in E. coli during growth and matrix purification.

PubMed

Reichhardt, Courtney; McCrate, Oscar A; Zhou, Xiaoxue; Lee, Jessica; Thongsomboon, Wiriya; Cegelski, Lynette

2016-11-01

Microbial biofilms are communities of cells characterized by a hallmark extracellular matrix (ECM) that confers functional attributes to the community, including enhanced cohesion, adherence to surfaces, and resistance to external stresses. Understanding the composition and properties of the biofilm ECM is crucial to understanding how it functions and protects cells. New methods to isolate and characterize ECM are emerging for different biofilm systems. Solid-state nuclear magnetic resonance was used to quantitatively track the isolation of the insoluble ECM from the uropathogenic Escherichia coli strain UTI89 and understand the role of Congo red in purification protocols. UTI89 assembles amyloid-integrated biofilms when grown on YESCA nutrient agar. The ECM contains curli amyloid fibers and a modified form of cellulose. Biofilms formed by UTI89 and other E. coli and Salmonella strains are often grown in the presence of Congo red to visually emphasize wrinkled agar morphologies and to score the production of ECM. Congo red is a hallmark amyloid-binding dye and binds to curli, yet also binds to cellulose. We found that growth in Congo red enabled more facile extraction of the ECM from UTI89 biofilms and facilitates isolation of cellulose from the curli mutant, UTI89ΔcsgA. Yet, Congo red has no influence on the isolation of curli from curli-producing cells that do not produce cellulose. Sodium dodecyl sulfate can remove Congo red from curli, but not from cellulose. Thus, Congo red binds strongly to cellulose and possibly weakens cellulose interactions with the cell surface, enabling more complete removal of the ECM. The use of Congo red as an extracellular matrix purification aid may be applied broadly to other organisms that assemble extracellular amyloid or cellulosic materials. Graphical abstract Solid-state NMR was used to quantitatively track the isolation of the insoluble amyloid-associated ECM from uropathogenic E. coli and understand the role of Congo red in purification protocols.

Spindle pole body component 25 homolog expressed by ECM stiffening is required for lung cancer cell proliferation.

PubMed

Jeong, Jangho; Keum, Seula; Kim, Daehwan; You, Eunae; Ko, Panseon; Lee, Jieun; Kim, Jaegu; Kim, Jung-Woong; Rhee, Sangmyung

2018-06-12

Accumulating evidence has shown that matrix stiffening in cancer tissue by the deposition of extracellular matrix (ECM) is closely related with severe tumor progression. However, much less is known about the genes affected by matrix stiffness and its signaling for cancer progression. In the current research, we investigated the differential gene expression of a non-small lung adenocarcinoma cell line, H1299, cultured under the conditions of soft (∼0.5 kPa) and stiff (∼40 kPa) matrices, mimicking the mechanical environments of normal and cancerous tissues, respectively. For integrated transcriptome analysis, the genes identified by ECM stiffening were compared with 8248 genes retrieved from The Cancer Genome Atlas Lung Adenocarcinoma (TCGA). In stiff matrix, 29 genes were significantly upregulated, while 75 genes were downregulated. The screening of hazard ratios for these genes using the Kaplan-Meier Plotter identified 8 genes most closely associated with cancer progression under the condition of matrix stiffening. Among these genes, spindle pole body component 25 homolog (SPC25) was one of the most up-regulated genes in stiff matrix and tumor tissue. Knockdown of SPC25 in H1299 cells using shRNA significantly inhibited cell proliferation with downregulation of the expression of checkpoint protein, Cyclin B1, under the condition of stiff matrix whereas the proliferation rate in soft matrix was not affected by SPC25 silencing. Thus, our findings provide novel key molecules for studying the relationship of extracellular matrix stiffening and cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

PubMed Central

Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature. PMID:21779348

The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli.

PubMed

Hufnagel, David A; Evans, Margery L; Greene, Sarah E; Pinkner, Jerome S; Hultgren, Scott J; Chapman, Matthew R

2016-12-15

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Quantitative image analysis for investigating cell-matrix interactions

NASA Astrophysics Data System (ADS)

Burkel, Brian; Notbohm, Jacob

2017-07-01

The extracellular matrix provides both chemical and physical cues that control cellular processes such as migration, division, differentiation, and cancer progression. Cells can mechanically alter the matrix by applying forces that result in matrix displacements, which in turn may localize to form dense bands along which cells may migrate. To quantify the displacements, we use confocal microscopy and fluorescent labeling to acquire high-contrast images of the fibrous material. Using a technique for quantitative image analysis called digital volume correlation, we then compute the matrix displacements. Our experimental technology offers a means to quantify matrix mechanics and cell-matrix interactions. We are now using these experimental tools to modulate mechanical properties of the matrix to study cell contraction and migration.

ERK-regulated αB-crystallin induction by matrix detachment inhibits anoikis and promotes lung metastasis in vivo.

PubMed

Malin, D; Strekalova, E; Petrovic, V; Rajanala, H; Sharma, B; Ugolkov, A; Gradishar, W J; Cryns, V L

2015-11-05

Evasion of extracellular matrix detachment-induced apoptosis ('anoikis') is a defining characteristic of metastatic tumor cells. The ability of metastatic carcinoma cells to survive matrix detachment and escape anoikis enables them to disseminate as viable circulating tumor cells and seed distant organs. Here we report that αB-crystallin, an antiapoptotic molecular chaperone implicated in the pathogenesis of diverse poor-prognosis solid tumors, is induced by matrix detachment and confers anoikis resistance. Specifically, we demonstrate that matrix detachment downregulates extracellular signal-regulated kinase (ERK) activity and increases αB-crystallin protein and messenger RNA (mRNA) levels. Moreover, we show that ERK inhibition in adherent cancer cells mimics matrix detachment by increasing αB-crystallin protein and mRNA levels, whereas constitutive ERK activation suppresses αB-crystallin induction during matrix detachment. These findings indicate that ERK inhibition is both necessary and sufficient for αB-crystallin induction by matrix detachment. To examine the functional consequences of αB-crystallin induction in anoikis, we stably silenced αB-crystallin in two different metastatic carcinoma cell lines. Strikingly, silencing αB-crystallin increased matrix detachment-induced caspase activation and apoptosis but did not affect cell viability of adherent cancer cells. In addition, silencing αB-crystallin in metastatic carcinoma cells reduced the number of viable circulating tumor cells and inhibited lung metastasis in two orthotopic models, but had little or no effect on primary tumor growth. Taken together, our findings point to αB-crystallin as a novel regulator of anoikis resistance that is induced by matrix detachment-mediated suppression of ERK signaling and promotes lung metastasis. Our results also suggest that αB-crystallin represents a promising molecular target for antimetastatic therapies.

From nano- to macro-scale: nanotechnology approaches for spatially controlled delivery of bioactive factors for bone and cartilage engineering.

PubMed

Santo, Vítor E; Gomes, Manuela E; Mano, João F; Reis, Rui L

2012-07-01

The field of biomaterials has advanced towards the molecular and nanoscale design of bioactive systems for tissue engineering, regenerative medicine and drug delivery. Spatial cues are displayed in the 3D extracellular matrix and can include signaling gradients, such as those observed during chemotaxis. Architectures range from the nanometer to the centimeter length scales as exemplified by extracellular matrix fibers, cells and macroscopic shapes. The main focus of this review is the application of a biomimetic approach by the combination of architectural cues, obtained through the application of micro- and nanofabrication techniques, with the ability to sequester and release growth factors and other bioactive agents in a spatiotemporal controlled manner for bone and cartilage engineering.

Design properties of hydrogel tissue-engineering scaffolds

PubMed Central

Zhu, Junmin; Marchant, Roger E

2011-01-01

This article summarizes the recent progress in the design and synthesis of hydrogels as tissue-engineering scaffolds. Hydrogels are attractive scaffolding materials owing to their highly swollen network structure, ability to encapsulate cells and bioactive molecules, and efficient mass transfer. Various polymers, including natural, synthetic and natural/synthetic hybrid polymers, have been used to make hydrogels via chemical or physical crosslinking. Recently, bioactive synthetic hydrogels have emerged as promising scaffolds because they can provide molecularly tailored biofunctions and adjustable mechanical properties, as well as an extracellular matrix-like microenvironment for cell growth and tissue formation. This article addresses various strategies that have been explored to design synthetic hydrogels with extracellular matrix-mimetic bioactive properties, such as cell adhesion, proteolytic degradation and growth factor-binding. PMID:22026626

Kindler surprise: mutations in a novel actin-associated protein cause Kindler syndrome.

PubMed

White, Sharon J; McLean, W H Irwin

2005-06-01

Kindler syndrome is an autosomal recessive genodermatosis characterized by acral blistering in neonates and diffuse, progressive poikiloderma in later life. Other clinical features include photosensitivity, premature skin ageing and severe periodontal disease. Two groups have recently shown that the molecular basis of Kindler syndrome is loss of a novel epidermal protein, kindlin-1, encoded by the gene KIND1. Two additional kindlin proteins, kindlin-2 and kindlin-3, have also been described. Kindlin-1 is considered to be a component in the linkage of the actin cytoskeleton to the extracellular matrix and as such is proposed to have both structural and cell-signalling functions. Kindler syndrome is therefore the first skin fragility syndrome due to disruption of the actin-extracellular matrix system.

Contractile forces originating from Cancer Diskiod regulated by geometrical ECM properties

NASA Astrophysics Data System (ADS)

Alobaidi, Amani; Sun, Bo

Cancer cell migration in three-dimensional extracellular matrix is a major cause of death for cancer patients. Although extensive studies have enlightened detailed mechanism of single cell 3D invasion and cell-ECM interaction, 3D collective cancer invasion is still poorly understood. To capture collective cancer invasion with more realistic, we developed a novel 3D invasion assay, Diskiod In Geometrically Micropatterned ECM (DIGME). DIGME allows us to independently controlling the shape the shape of tumor organoids, microstructure and spatial heterogeneity of the extracellular matrix all at the same time. Here we study the affect of contractile forces originating from different geometrical cancer diskiods. We show that cancer invasion could be regulated by geometrical ECM properties.

Surface modification of implants in long bone.

PubMed

Förster, Yvonne; Rentsch, Claudia; Schneiders, Wolfgang; Bernhardt, Ricardo; Simon, Jan C; Worch, Hartmut; Rammelt, Stefan

2012-01-01

Coatings of orthopedic implants are investigated to improve the osteoinductive and osteoconductive properties of the implant surfaces and thus to enhance periimplant bone formation. By applying coatings that mimic the extracellular matrix a favorable environment for osteoblasts, osteoclasts and their progenitor cells is provided to promote early and strong fixation of implants. It is known that the early bone ongrowth increases primary implant fixation and reduces the risk of implant failure. This review presents an overview of coating titanium and hydroxyapatite implants with components of the extracellular matrix like collagen type I, chondroitin sulfate and RGD peptide in different small and large animal models. The influence of these components on cells, the inflammation process, new bone formation and bone/implant contact is summarized.

Surface modification of implants in long bone

PubMed Central

Förster, Yvonne; Rentsch, Claudia; Schneiders, Wolfgang; Bernhardt, Ricardo; Simon, Jan C.; Worch, Hartmut; Rammelt, Stefan

2012-01-01

Coatings of orthopedic implants are investigated to improve the osteoinductive and osteoconductive properties of the implant surfaces and thus to enhance periimplant bone formation. By applying coatings that mimic the extracellular matrix a favorable environment for osteoblasts, osteoclasts and their progenitor cells is provided to promote early and strong fixation of implants. It is known that the early bone ongrowth increases primary implant fixation and reduces the risk of implant failure. This review presents an overview of coating titanium and hydroxyapatite implants with components of the extracellular matrix like collagen type I, chondroitin sulfate and RGD peptide in different small and large animal models. The influence of these components on cells, the inflammation process, new bone formation and bone/implant contact is summarized. PMID:23507866

The extracellular matrix: A dynamic niche in cancer progression

PubMed Central

Lu, Pengfei; Weaver, Valerie M.

2012-01-01

The local microenvironment, or niche, of a cancer cell plays important roles in cancer development. A major component of the niche is the extracellular matrix (ECM), a complex network of macromolecules with distinctive physical, biochemical, and biomechanical properties. Although tightly controlled during embryonic development and organ homeostasis, the ECM is commonly deregulated and becomes disorganized in diseases such as cancer. Abnormal ECM affects cancer progression by directly promoting cellular transformation and metastasis. Importantly, however, ECM anomalies also deregulate behavior of stromal cells, facilitate tumor-associated angiogenesis and inflammation, and thus lead to generation of a tumorigenic microenvironment. Understanding how ECM composition and topography are maintained and how their deregulation influences cancer progression may help develop new therapeutic interventions by targeting the tumor niche. PMID:22351925

Potential Roles of Protease Inhibitors in Cancer Progression.

PubMed

Yang, Peng; Li, Zhuo-Yu; Li, Han-Qing

2015-01-01

Proteases are important molecules that are involved in many key physiological processes. Protease signaling pathways are strictly controlled, and disorders in protease activity can result in pathological changes such as cardiovascular and inflammatory diseases, cancer and neurological disorders. Many proteases have been associated with increasing tumor metastasis in various human cancers, suggesting important functional roles in the metastatic process because of their ability to degrade the extracellular matrix barrier. Proteases are also capable of cleaving non-extracellular matrix molecules. Inhibitors of proteases to some extent can reduce invasion and metastasis of cancer cells, and slow down cancer progression. In this review, we focus on the role of a few proteases and their inhibitors in tumors as a basis for cancer prognostication and therapy.

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

PubMed Central

Ahmed, Ahmed Ashour; Mills, Anthony D.; Ibrahim, Ashraf E.K.; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E.; Iyer, N. Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D.; Earl, Helena M.; Laskey, Ronald A.; Caldas, Carlos; Brenton, James D.

2007-01-01

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability. PMID:18068629

Extracellular matrix motion and early morphogenesis.

PubMed

Loganathan, Rajprasad; Rongish, Brenda J; Smith, Christopher M; Filla, Michael B; Czirok, Andras; Bénazéraf, Bertrand; Little, Charles D

2016-06-15

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. © 2016. Published by The Company of Biologists Ltd.

Dental Enamel: Genes Define Biomechanics

PubMed Central

Rauth, Rick J.; Potter, Karen S.; Ngan, Amanda Y.-W.; Saad, Deema M.; Mehr, Rana; Luong, Vivian Q.; Schuetter, Verna L.; Miklus, Vetea G.; Chang, PeiPei; Paine, Michael L.; Lacruz, Rodrigo S.; Snead, Malcolm L.; White, Shane N.

2010-01-01

Regulated gene expression assembles an extracellular proteinaceous matrix to control biomineralization and the resultant biomechanical function of tooth enamel. The importance of the dominant enamel matrix protein, amelogenin (Amel); a minor transiently expressed protein, dentin sialoprotein (Dsp); an electrogenic sodium bicarbonate cotransporter (NBCe1); the timely removal of the proteinaceous matrix by a serine protease, Kallikrein-4 (Klk4); and the late-stage expression of Amelotin (Amtn) on enamel biomechanical function were demonstrated and measured using mouse models. PMID:20066874

Different matrix micro-environments in colon cancer and diverticular disease.

PubMed

Klinge, U; Rosch, R; Junge, K; Krones, C J; Stumpf, M; Lynen-Jansen, P; Mertens, P R; Schumpelick, V

2007-05-01

The extracellular matrix and the interactive signalling between its components are thought to play a pivotal role for tumour development and metastasis formation. An altered matrix composition as potential underlying pathology for the development of colorectal cancer was hypothesized. In a retrospective study of patients with colon cancer, the extracellular matrix in tumour-free bowel specimen was investigated in comparison with non-infected bowel specimen from patients operated on for colonic diverticulosis. The following matrix parameters with known associations to tumour formation, cell proliferation, invasion and metastasis were analysed by immunohistochemistry and quantified by a scoring system: VEGF, TGF-beta, ESDN, CD117, c-erb-2, cyclin D1, p53, p27, COX-2, YB-1, collagen I/III, MMP-13, PAI and uPAR. Expression profiles and correlations were calculated. The comparison of the two groups revealed a significantly decreased immunostaining for CD117 and TGF-beta in the cancer group (8.5+/-2.6 vs 10.3+/-2,1 and 4.9+/-1.5 vs 8.1+/-3, respectively), whereas PAI scores were significantly higher than in patients with diverticular disease (8.1+/-1.6 vs 6.2+/-0.9). Overall correlation patterns of matrix parameters indicated pronounced differences between tumour-free tissue in cancer patients compared with patients with diverticular disease. Our results indicate distinct differences in the colonic tissue architecture between cancer patients and patients with diverticulitis that support the notion of an altered matrix composition predisposing to the development of colon cancer.

Use of Ovine-based Collagen Extracellular Matrix and Gentian Violet/Methylene Blue Antibacterial Foam Dressings to Help Improve Clinical Outcomes in Lower Extremity Wounds: A Retrospective Cohort Study.

PubMed

Lullove, Eric J

2017-04-01

Dressings that provide broad spectrum metalloprotease reduction along with inherent aspects of an extracellular matrix may contribute to improved wound healing outcomes and shorter treatment times. The author performed a retrospective case series analysis to determine the clinical outcomes of regular debridement with the use of ovine-based collagen extracellular matrix dressings and gentian violet/methylene blue polyurethane antibacterial foam dressings in treating 53 patients with 53 chronic lower extremity wounds (diabetic foot ulcers [DFUs], venous leg ulcers, and heel pressure ulcers). Patients were treated twice weekly in an outpatient clinic for the first 4 weeks and weekly thereafter until closure. Average body mass index (BMI) for the study population was 28.3, and the average patient age was 75.9 years. Mean percent wound surface area reduction at 4, 8, and 12 weeks was 38.5%, 73.3%, and 91.3%, respectively. Average time to closure for all wounds was 10.6 weeks (range, 5-24 weeks). All wounds were 100% reepithelialized by week 20 except 1 DFU that reepithelialized at week 24. The average cost of care for a single wound episode (from presentation to closure) was $2749.49. Results of this analysis showed that the healing of chronic wounds in this series could be achieved at a reasonable cost with regular debridement and a collagen matrix dressing regimen, even in patients of advanced age and above average BMI as well as in wounds that did not achieve > 40% wound surface area reduction at 4 weeks.

Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.

PubMed

Ponec, Maria; El Ghalbzouri, Abdoelwaheb; Dijkman, Remco; Kempenaar, Johanna; van der Pluijm, Gabri; Koolwijk, Pieter

2004-01-01

A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

Effects of treatment with Maraviroc a CCR5 inhibitor on a human hepatic stellate cell line.

PubMed

Coppola, Nicola; Perna, Angelica; Lucariello, Angela; Martini, Salvatore; Macera, Margherita; Carleo, Maria A; Guerra, Germano; Esposito, Vincenzo; De Luca, Antonio

2018-08-01

After an acute liver damage, tissue regeneration repairs lesions with degradation of deposed fibrotic material, while mechanisms of tissue restoration are persistently activated following several repeated injuries, inducing deposition of extracellular matrix. (ECM). Factors responsible for ECM remodeling have been identified in a pathway involving a family of zinc-dependent enzyme matrix metalloproteinases (MMPs), together with tissue inhibitor of metalloproteinases (TIMPs). Recent experimental models suggested a role of CCR5 receptor in the genesis of liver fibrosis. Drawing from these background we decided to evaluate the effects of the treatment with the CCR5 inhibitor Maraviroc on LX-2, a human hepatic stellate cell line (HSC). Treatment with Maraviroc resulted in a block in S phase of LX-2 cells with increased expression levels of cyclin D1 and p21 while the expression of p53 was reduced. Treatment with Maraviroc was also able to block the accumulation of fibrillar collagens and extracellular matrix proteins (ECM), as demonstrated by the decrease of specific markers as Collagen type I, α-SMA, and TGF-β1. In addition we observed a down regulation of both metalloproteins (MMP-2, MMP-9), used for the degradation of the extracellular matrix and their inhibitors (TIMP-1, TIMP-2). The identification of a compound that may modulate the dynamic of liver fibrosis could be crucial in all chronic liver diseases. Maraviroc could play an important role because, in addition to its own anti-HIV activity, it could reduce the release of pro-inflammatory citokynes implicated in liver fibrogenesis. © 2018 Wiley Periodicals, Inc.

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

PubMed Central

Li, Shu-Hong; Sun, Zhuo; Guo, Lily; Han, Mihan; Wood, Michael F G; Ghosh, Nirmalya; Alex Vitkin, I; Weisel, Richard D; Li, Ren-Ke

2012-01-01

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re-establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full-length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9-week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell-based gene therapy provides a new approach to cardiac regeneration. PMID:22435995

Combination of platelet-rich plasma within periodontal ligament stem cell sheets enhances cell differentiation and matrix production.

PubMed

Xu, Qiu; Li, Bei; Yuan, Lin; Dong, Zhiwei; Zhang, Hao; Wang, Han; Sun, Jin; Ge, Song; Jin, Yan

2017-03-01

The longstanding goal of periodontal therapy is to regenerate periodontal tissues. Although platelet-rich plasma (PRP) has been gaining increasing popularity for use in the orofacial region, whether PRP is useful for periodontal regeneration is still unknown. The purpose of this study was to determine whether a mixture of periodontal ligament stem cell (PDLSC) sheets and PRP promoted bone regeneration, one of the most important measurement indices of periodontal tissue regenerative capability in vitro and in vivo. In this study, we evaluated the effects of different doses of PRP on the differentiation of human PDLSCs. Then cell sheet formation, extracellular matrix deposition and osteogenic gene expression in response to different doses of PRP treatment during sheet grafting was investigated. Furthermore, we implanted PDLSC sheets treated with 1% PRP subcutaneously into immunocompromised mice to evaluate their bone-regenerative capability. The results revealed that 1% PRP significantly enhanced the osteogenic differentiation of PDLSCs. Based on the production of extracellular matrix proteins, the results of scanning electron microscopy and the expression of the osteogenic genes ALP, Runx2, Col-1 and OCN, the provision of 1% PRP for PDLSC sheets was the most effective PRP administration mode for cell sheet formation. The results of in vivo transplantation showed that 1% PRP-mediated PDLSC sheets exhibited better periodontal tissue regenerative capability than those obtained without PRP intervention. These data suggest that a suitable concentration of PRP stimulation may enhance extracellular matrix production and positively affect cell behaviour in PDLSC sheets. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

PubMed

Oostendorp, Corien; Meyer, Sarah; Sobrio, Monia; van Arendonk, Joyce; Reichmann, Ernst; Daamen, Willeke F; van Kuppevelt, Toin H

2017-05-01

Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

Inactivation of genes TEC1 and EFG1 in Candida albicans influences extracellular matrix composition and biofilm morphology.

PubMed

Panariello, Beatriz Helena Dias; Klein, Marlise I; Pavarina, Ana Claudia; Duarte, Simone

2017-01-01

Background : Infections caused by Candida spp. have been associated with formation of a biofilm, i.e. a complex microstructure of cells adhering to a surface and embedded within an extracellular matrix (ECM). Methods : The ECMs of a wild-type (WT, SN425) and two Candida albicans mutant strains, Δ/Δ tec1 (CJN2330) and Δ/Δ efg1 (CJN2302), were evaluated. Colony-forming units (cfu), total biomass (mg), water-soluble polysaccharides (WSPs), alkali-soluble polysaccharides (ASPs), proteins (insoluble part of biofilms and matrix proteins), and extracellular DNA (eDNA) were quantified. Variable-pressure scanning electron microscopy and confocal scanning laser microscopy were performed. The biovolume (μm 3 /μm 2 ) and maximum thickness (μm) of the biofilms were quantified using COMSTAT2. Results : ASP content was highest in WT (mean ± SD: 74.5 ± 22.0 µg), followed by Δ/Δ tec1 (44.0 ± 24.1 µg) and Δ/Δ efg1 (14.7 ± 5.0 µg). The protein correlated with ASPs ( r  = 0.666) and with matrix proteins ( r  = 0.670) in the WT strain. The population in Δ/Δ efg1 correlated with the protein ( r  = 0.734) and its biofilms exhibited the lowest biomass and biovolume, and maximum thickness. In Δ/Δ tec1, ASP correlated with eDNA ( r  = 0.678). Conclusion : ASP production may be linked to C. albicans cell filamentous morphology.

Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia.

PubMed

Alì, Greta; Borrelli, Nicla; Riccardo, Giannini; Proietti, Agnese; Pelliccioni, Serena; Niccoli, Cristina; Boldrini, Laura; Lucchi, Marco; Mussi, Alfredo; Fontanini, Gabriella

2013-11-01

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples (p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples (p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.

Nuclear localization of matrix metalloproteinases.

PubMed

Mannello, Ferdinando; Medda, Virginia

2012-03-01

Matrix metalloproteinases (MMPs) were originally identified as matrixin proteases that act in the extracellular matrix. Recent works have uncovered nontraditional roles for MMPs in the extracellular space as well as in the cytosol and nucleus. There is strong evidence that subspecialized and compartmentalized matrixins participate in many physiological and pathological cellular processes, in which they can act as both degradative and regulatory proteases. In this review, we discuss the transcriptional and translational control of matrixin expression, their regulation of intracellular sorting, and the structural basis of activation and inhibition. In particular, we highlight the emerging roles of various matrixin forms in diseases. The activity of matrix metalloproteinases is regulated at several levels, including enzyme activation, inhibition, complex formation and compartmentalization. Most MMPs are secreted and have their function in the extracellular environment. MMPs are also found inside cells, both in the nucleus, cytosol and organelles. The role of intracellular located MMPs is still poorly understood, although recent studies have unraveled some of their functions. The localization, activation and activity of MMPs are regulated by their interactions with other proteins, proteoglycan core proteins and / or their glycosaminoglycan chains, as well as other molecules. Complexes formed between MMPs and various molecules may also include interactions with noncatalytic sites. Such exosites are regions involved in substrate processing, localized outside the active site, and are potential binding sites of specific MMP inhibitors. Knowledge about regulation of MMP activity is essential for understanding various physiological processes and pathogenesis of diseases, as well as for the development of new MMP targeting drugs. Copyright © 2011 Elsevier GmbH. All rights reserved.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis

PubMed Central

Bryan, Chase D.; Chien, Chi-Bin; Kwan, Kristen M.

2016-01-01

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1UW1) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1UW1 mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis. PMID:27339294

Matrix directed adipogenesis and neurogenesis of mesenchymal stem cells derived from adipose tissue and bone marrow.

PubMed

Lee, Junmin; Abdeen, Amr A; Tang, Xin; Saif, Taher A; Kilian, Kristopher A

2016-09-15

Mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical and biochemical properties of the extracellular matrix. In this work we conduct a combinatorial study of matrix properties that influence adipogenesis and neurogenesis including: adhesion proteins, stiffness, and cell geometry, for mesenchymal stem cells derived from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs). We uncover distinct differences in integrin expression, the magnitude of traction stress, and lineage specification to adipocytes and neuron-like cells between cell sources. In the absence of media supplements, adipogenesis in AT-MSCs is not significantly influenced by matrix properties, while the converse is true in BM-MSCs. Both cell types show changes in the expression of neurogenesis markers as matrix cues are varied. When cultured on laminin conjugated microislands of the same adhesive area, BM-MSCs display elevated adipogenesis markers, while AT-MSCs display elevated neurogenesis markers; integrin analysis suggests neurogenesis in AT-MSCs is guided by adhesion through integrin αvβ3. Overall, the properties of the extracellular matrix guides MSC adhesion and lineage specification to different degrees and outcomes, in spite of their similarities in general characteristics. This work will help guide the selection of MSCs and matrix components for applications where high fidelity of differentiation outcome is desired. Mesenchymal stem cells (MSCs) are an attractive cell type for stem cell therapies; however, in order for these cells to be useful in medicine, we need to understand how they respond to the physical and chemical environments of tissue. Here, we explore how two promising sources of MSCs-those derived from bone marrow and from adipose tissue-respond to the compliance and composition of tissue using model extracellular matrices. Our results demonstrate a source-specific propensity to undergo adipogenesis and neurogenesis, and uncover a role for adhesion, and the degree of traction force exerted on the substrate in guiding these lineage outcomes. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Can injured adult CNS axons regenerate by recapitulating development?

PubMed

Hilton, Brett J; Bradke, Frank

2017-10-01

In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS. © 2017. Published by The Company of Biologists Ltd.

Cartilage extracellular matrix as a biomaterial for cartilage regeneration.

PubMed

Kiyotake, Emi A; Beck, Emily C; Detamore, Michael S

2016-11-01

The extracellular matrix (ECM) of various tissues possesses the model characteristics that biomaterials for tissue engineering strive to mimic; however, owing to the intricate hierarchical nature of the ECM, it has yet to be fully characterized and synthetically fabricated. Cartilage repair remains a challenge because the intrinsic properties that enable its durability and long-lasting function also impede regeneration. In the last decade, cartilage ECM has emerged as a promising biomaterial for regenerating cartilage, partly because of its potentially chondroinductive nature. As this research area of cartilage matrix-based biomaterials emerged, investigators facing similar challenges consequently developed convergent solutions in constructing robust and bioactive scaffolds. This review discusses the challenges, emerging trends, and future directions of cartilage ECM scaffolds, including a comparison between two different forms of cartilage matrix: decellularized cartilage (DCC) and devitalized cartilage (DVC). To overcome the low permeability of cartilage matrix, physical fragmentation greatly enhances decellularization, although the process itself may reduce the chondroinductivity of fabricated scaffolds. The less complex processing of a scaffold composed of DVC, which has not been decellularized, appears to have translational advantages and potential chondroinductive and mechanical advantages over DCC, without detrimental immunogenicity, to ultimately enhance cartilage repair in a clinically relevant way. © 2016 New York Academy of Sciences.

An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration.

PubMed

Kutys, Matthew L; Yamada, Kenneth M

2014-09-01

Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments and discovered a form of Cdc42-RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified βPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of βPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this βPix knockdown phenotype to loss of polarized Cdc42 but not Rac1 activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates βPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF-GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.

Hydrogels Derived from Central Nervous System Extracellular Matrix

PubMed Central

Medberry, Christopher J.; Crapo, Peter M.; Siu, Bernard F.; Carruthers, Christopher A.; Wolf, Matthew T.; Nagarkar, Shailesh P.; Agrawal, Vineet; Jones, Kristen E.; Kelly, Jeremy; Johnson, Scott A.; Velankar, Sachin S.; Watkins, Simon C.; Modo, Michel

2012-01-01

Biologic scaffolds composed of extracellular matrix (ECM) are commonly used repair devices in preclinical and clinical settings; however the use of these scaffolds for peripheral and central nervous system (CNS) repair has been limited. Biologic scaffolds developed from brain and spinal cord tissue have recently been described, yet the conformation of the harvested ECM limits therapeutic utility. An injectable CNS-ECM derived hydrogel capable of in vivo polymerization and conformation to irregular lesion geometries may aid in tissue reconstruction efforts following complex neurologic trauma. The objectives of the present study were to develop hydrogel forms of brain and spinal cord ECM and compare the resulting biochemical composition, mechanical properties, and neurotrophic potential of a brain derived cell line to a non-CNS-ECM hydrogel, urinary bladder matrix. Results showed distinct differences between compositions of brain ECM, spinal cord ECM, and urinary bladder matrix. The rheologic modulus of spinal cord ECM hydrogel was greater than that of brain ECM and urinary bladder matrix. All ECMs increased the number of cells expressing neurites, but only brain ECM increased neurite length, suggesting a possible tissue-specific effect. All hydrogels promoted three-dimensional uni- or bi-polar neurite outgrowth following 7 days in culture. These results suggest that CNS-ECM hydrogels may provide supportive scaffolding to promote in vivo axonal repair. PMID:23158935

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

PubMed

Palchesko, Rachelle N; Szymanski, John M; Sahu, Amrita; Feinberg, Adam W

2014-09-01

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo . Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment

PubMed Central

Palchesko, Rachelle N.; Szymanski, John M.; Sahu, Amrita; Feinberg, Adam W.

2014-01-01

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments. PMID:25530816

Stress Chaperone GRP-78 Functions in Mineralized Matrix Formation*

PubMed Central

Ravindran, Sriram; Gao, Qi; Ramachandran, Amsaveni; Blond, Sylvie; Predescu, Sanda A.; George, Anne

2011-01-01

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78. PMID:21239500

Matrix Metalloproteinases 2 and 9 Are Differentially Expressed in Patients with Indeterminate and Cardiac Clinical Forms of Chagas Disease

PubMed Central

Fares, Rafaelle Christine Gomes; Gomes, Juliana de Assis Silva; Garzoni, Luciana Ribeiro; Waghabi, Mariana Caldas; Saraiva, Roberto Magalhães; Medeiros, Nayara Ingrid; Oliveira-Prado, Roberta; Sangenis, Luiz Henrique Conde; Chambela, Mayara da Costa; de Araújo, Fernanda Fortes; Teixeira-Carvalho, Andréa; Damásio, Marcos Paulo; Valente, Vanessa Azevedo; Ferreira, Karine Silvestre; Sousa, Giovane Rodrigo; Rocha, Manoel Otávio da Costa

2013-01-01

Dilated chronic cardiomyopathy (DCC) from Chagas disease is associated with myocardial remodeling and interstitial fibrosis, resulting in extracellular matrix (ECM) changes. In this study, we characterized for the first time the serum matrix metalloproteinase 2 (MMP-2) and MMP-9 levels, as well as their main cell sources in peripheral blood mononuclear cells from patients presenting with the indeterminate (IND) or cardiac (CARD) clinical form of Chagas disease. Our results showed that serum levels of MMP-9 are associated with the severity of Chagas disease. The analysis of MMP production by T lymphocytes showed that CD8+ T cells are the main mononuclear leukocyte source of both MMP-2 and MMP-9 molecules. Using a new 3-dimensional model of fibrosis, we observed that sera from patients with Chagas disease induced an increase in the extracellular matrix components in cardiac spheroids. Furthermore, MMP-2 and MMP-9 showed different correlations with matrix proteins and inflammatory cytokines in patients with Chagas disease. Our results suggest that MMP-2 and MMP-9 show distinct activities in Chagas disease pathogenesis. While MMP-9 seems to be involved in the inflammation and cardiac remodeling of Chagas disease, MMP-2 does not correlate with inflammatory molecules. PMID:23856618

Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting

PubMed Central

Davis, George E.; Stratman, Amber N.; Sacharidou, Anastasia; Koh, Wonshill

2013-01-01

Many studies reveal a fundamental role for extracellular matrix-mediated signaling through integrins and Rho GTPases as well as matrix metalloproteinases (MMPs) in the molecular control of vascular tube morphogenesis in three-dimensional (3D) tissue environments. Recent work has defined an EC lumen signaling complex of proteins that controls these vascular morphogenic events. These findings reveal a signaling interdependence between Cdc42 and MT1-MMP to control the 3D matrix-specific process of EC tubulogenesis. The EC tube formation process results in the creation of a network of proteolytically-generated vascular guidance tunnels in 3D matrices that are utilized to remodel EC-lined tubes through EC motility and could facilitate processes such as flow-induced remodeling and arteriovenous EC sorting and differentiation. Within vascular guidance tunnels, key dynamic interactions occur between endothelial cells (ECs) and pericytes to affect vessel remodeling, diameter, and vascular basement membrane matrix assembly, a fundamental process necessary for endothelial tube maturation and stabilization. Thus, the EC lumen and tube formation mechanism coordinates the concomitant establishment of a network of vascular tubes within tunnel spaces to allow for flow responsiveness, EC-mural cell interactions, and vascular extracellular matrix assembly to control the development of the functional microcirculation. PMID:21482411

MEPE Localization in the Craniofacial Complex and Function in Tooth Dentin Formation.

PubMed

Gullard, Angela; Gluhak-Heinrich, Jelica; Papagerakis, Silvana; Sohn, Philip; Unterbrink, Aaron; Chen, Shuo; MacDougall, Mary

2016-04-01

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein found in dental and skeletal tissues. Although information regarding the role of MEPE in bone and disorders of phosphate metabolism is emerging, the role of MEPE in dental tissues remains unclear. We performed RNA in situ hybridization and immunohistochemistry analyses to delineate the expression pattern of MEPE during embryonic and postnatal development in craniofacial mineralizing tissues. Mepe RNA expression was seen within teeth from cap through root formation in association with odontoblasts and cellular cementoblasts. More intense expression was seen in the alveolar bone within the osteoblasts and osteocytes. MEPE immunohistochemistry showed biphasic dentin staining in incisors and more intense staining in alveolar bone matrix and in forming cartilage. Analysis of Mepe null mouse molars showed overall mineralized tooth volume and density of enamel and dentin comparable with that of wild-type samples. However, Mepe(-/-) molars exhibited increased thickness of predentin, dentin, and enamel over controls and decreased gene expression of Enam, Bsp, Dmp1, Dspp, and Opnby RT-PCR. In vitro Mepe overexpression in odontoblasts led to significant reductions in Dspp reporter activity. These data suggest MEPE may be instrumental in craniofacial and dental matrix maturation, potentially functioning in the maintenance of non-mineralized matrix. © 2016 The Histochemical Society.

Isolation and characterization of chicken bile matrix metalloproteinase.

PubMed

Packialakshmi, B; Liyanage, R; Rasaputra, K S; Lay, Jackson O; Rath, N C

2014-06-01

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets. Poultry Science Association Inc.

Confocal microscopy imaging of the biofilm matrix.

PubMed

Schlafer, Sebastian; Meyer, Rikke L

2017-07-01

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the concentration of solutes and the diffusive properties of the biofilm matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices.

PubMed

Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, Lorena

2017-10-20

The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.

Acellular fetal bovine dermal matrix in the treatment of nonhealing wounds in patients with complex comorbidities.

PubMed

Lullove, Eric

2012-01-01

In contrast to the narrow indications for living skin equivalents, extracellular matrix biomaterials are clinically used in a wide range of wound-healing applications. Given the breadth of possible uses, the goal of this study was to retrospectively compile and analyze the clinical application and effectiveness of an extracellular matrix biomaterial derived from fetal bovine dermis (PriMatrix; TEI Biosciences, Boston, Massachusetts) in patients treated by a single physician and monitored postsurgically in an outpatient wound care center. A retrospective medical record review was conducted of consecutive patients treated from January 2007 through January 2009 with meshed PriMatrix after sharp/surgical debridement and coverage with standard moist wound therapy dressings. Twenty-nine patients and 34 wounds were compiled. All of the wounds were unresponsive to conservative treatment owing to complications, including infection, exposed bone or tendon, and other comorbidities known to delay healing. Wounds included 11 diabetic ulcers, 8 venous stasis ulcers, 10 nonhealing traumatic wounds, and 5 other chronic wounds. Thirty of 34 wounds healed, with four patients lost to follow-up. Mean time to healing for diabetic foot ulcers was 105 days with an average of 2.6 PriMatrix applications. Mean time to healing for venous, traumatic, and other chronic wounds was 74 to 82 days with an average of 1.2 to 1.4 PriMatrix applications. In patients with comorbidities known to delay healing, the implantation of PriMatrix promoted the healing and, ultimately, full reepithelialization of otherwise unresponsive wounds of varied etiology, including those with complications of infection or exposed bone or tendon.

The effect of a collagen-elastin matrix on adhesion formation after flexor tendon repair in a rabbit model.

PubMed

Wichelhaus, Dagmar Alice; Beyersdoerfer, Sascha Tobias; Gierer, Philip; Vollmar, Brigitte; Mittlmeier, Th

2016-07-01

The outcome of flexor tendon surgery is negatively affected by the formation of adhesions which can occur during the healing of the tendon repair. In this experimental study, we sought to prevent adhesion formation by wrapping a collagen-elastin scaffold around the repaired tendon segment. In 28 rabbit hind legs, the flexor tendons of the third and fourth digits were cut and then repaired using a two-strand suture technique on the fourth digit and a four-strand technique on the third digit. Rabbits were randomly assigned to study and control groups. In the control group, the operation ended by closing the tendon sheath and the skin. In the study group, a collagen-elastin scaffold was wrapped around the repaired tendon segment in both digits. After 3 and 8 weeks, the tendons were harvested and processed histologically. The range of motion of the digits and the gap formation between the repaired tendon ends were measured. The formation of adhesions, infiltration of leucocytes and extracellular inflammatory response were quantified. At the time of tendon harvesting, all joints of the operated toes showed free range of motion. Four-strand core sutures lead to significantly less diastasis between the repaired tendon ends than two-strand core suture repairs. The collagen-elastin scaffold leads to greater gapping after 3 weeks compared to the controls treated without the matrix. Within the tendons treated with the collagen-elastin matrix, a significant boost of cellular and extracellular inflammation could be stated after 3 weeks which was reflected by a higher level of CAE positive cells and more formation of myofibroblasts in the αSMA stain in the study group. The inflammatory response subsided gradually and significantly until the late stage of the study. Both the cellular and extracellular inflammatory response was emphasized with the amount of material used for the repair. The use of a collagen-elastin matrix cannot be advised for the prevention of adhesion formation in flexor tendon surgery, because it enhances both cellular and extracellular inflammation. Four-strand core sutures lead to less gapping than two-strand core sutures, but at the same time, the cellular and extracellular inflammatory response is more pronounced.

A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

PubMed

Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E; Araujo-Hernandez, Sophia A; Harrison, Joe J; MacCoss, Michael J; Parsek, Matthew R

2018-04-10

Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. IMPORTANCE Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix-interacting protein. Copyright © 2018 Tseng et al.

Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

PubMed

Shami, Annelie; Gonçalves, Isabel; Hultgårdh-Nilsson, Anna

2014-10-01

The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.

Attenuation of endocrine-exocrine pancreatic communication in type 2 diabetes: pancreatic extracellular matrix ultrastructural abnormalities.

PubMed

Hayden, Melvin R; Patel, Kamlesh; Habibi, Javad; Gupta, Deepa; Tekwani, Seema S; Whaley-Connell, Adam; Sowers, James R

2008-01-01

Ultrastructural observations reveal a continuous interstitial matrix connection between the endocrine and exocrine pancreas, which is lost due to fibrosis in rodent models and humans with type 2 diabetes mellitus (T2DM). Widening of the islet-exocrine interface appears to result in loss of desmosomes and adherens junctions between islet and acinar cells and is associated with hypercellularity consisting of pericytes and inflammatory cells in T2DM pancreatic tissue. Organized fibrillar collagen was closely associated with pericytes, which are known to differentiate into myofibroblasts-pancreatic stellate cells. Of importance, some pericyte cellular processes traverse both the connecting islet-exocrine interface and the endoacinar interstitium of the exocrine pancreas. Loss of cellular paracrine communication and extracellular matrix remodeling fibrosis in young animal models and humans may result in a dysfunctional insulino-acinar-ductal-incretin gut hormone axis, resulting in pancreatic insufficiency and glucagon-like peptide deficiency, which are known to exist in prediabetes and overt T2DM in humans.

Attenuation of Endocrine-Exocrine Pancreatic Communication in Type 2 Diabetes: Pancreatic Extracellular Matrix Ultrastructural Abnormalities

PubMed Central

Hayden, Melvin R; Patel, Kamlesh; Habibi, Javad; Gupta, Deepa; Tekwani, Seema S.; Whaley-Connell, Adam; Sowers, James R.

2009-01-01

Ultrastructural observations reveal a continuous interstitial matrix connection between the endocrine and exocrine pancreas, which is lost due to fibrosis in rodent models and humans with type 2 diabetes mellitus (T2DM). Widening of the islet exocrine interface (IEI) appears to result in loss of desmosomes and adherens junctions between islet and acinar cells and is associated with hypercellularity consisting of pericytes and inflammatory cells in T2DM pancreatic tissue. Organized fibrillar collagen was closely associated with pericytes, which are known to differentiate into myofibroblasts – pancreatic stellate cells. Importantly, some pericyte cellular processes traverse both the connecting IEI and the endoacinar interstitium of the exocrine pancreas. Loss of cellular paracrine communication and extracellular matrix remodeling fibrosis in young animal models and humans may result in a dysfunctional insulino-acinar-ductal – incretin gut hormone axis resulting in pancreatic insufficiency and glucagon like peptide deficiency known to exist in prediabetes and overt T2DM in humans. PMID:19040593

Semitransparent bandages based on chitosan and extracellular matrix for photochemical tissue bonding.

PubMed

Frost, Samuel J; Mawad, Damia; Wuhrer, Richard; Myers, Simon; Lauto, Antonio

2018-01-22

Extracellular matrices (ECMs) are often used in reconstructive surgery to enhance tissue regeneration and remodeling. Sutures and staples are currently used to fix ECMs to tissue although they can be invasive devices. Other sutureless and less invasive techniques, such as photochemical tissue bonding, cannot be coupled to ECMs because of their intrinsic opacity to light. We succeeded in fabricating a biocompatible and adhesive device that is based on ovine forestomach matrix (OFM) and a chitosan adhesive. The natural opacity of the OFM has been overcome by adding the adhesive into the matrix that allows for the light to effectively penetrate through it. The OFM-chitosan device is semitransparent (attenuation length ~ 106 µm) and can be photoactivated by green light to bond to tissue. This device does not require sutures or staples and guarantees a bonding strength of ~ 23 kPa. A new semitransparent and biocompatible bandage has been successfully fabricated and characterized for sutureless tissue bonding.

Balance between apical membrane growth and luminal matrix resistance determines epithelial tubule shape.

PubMed

Dong, Bo; Hannezo, Edouard; Hayashi, Shigeo

2014-05-22

The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM) of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Methamphetamine and HIV-1 gp120 Effects on Lipopolysaccharide Stimulated Matrix Metalloproteinase-9 Production by Human Monocyte-Derived Macrophages

PubMed Central

Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E.; Schwartz, Stanley A.

2011-01-01

Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS. PMID:21425912

Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

PubMed

Choi, Bogyu; Kim, Soyon; Lin, Brian; Wu, Benjamin M; Lee, Min

2014-11-26

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

PubMed

Jennings, Laura K; Storek, Kelly M; Ledvina, Hannah E; Coulon, Charlène; Marmont, Lindsey S; Sadovskaya, Irina; Secor, Patrick R; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J; Howell, P Lynne; Parsek, Matthew R

2015-09-08

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix

PubMed Central

Jennings, Laura K.; Storek, Kelly M.; Ledvina, Hannah E.; Coulon, Charlène; Marmont, Lindsey S.; Sadovskaya, Irina; Secor, Patrick R.; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J.; Howell, P. Lynne; Parsek, Matthew R.

2015-01-01

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components. PMID:26311845

Matrix metalloproteinase-9 involvement in the structural plasticity of dendritic spines

PubMed Central

Stawarski, Michal; Stefaniuk, Marzena; Wlodarczyk, Jakub

2014-01-01

Dendritic spines are the locus for excitatory synaptic transmission in the brain and thus play a major role in neuronal plasticity. The ability to alter synaptic connections includes volumetric changes in dendritic spines that are driven by scaffolds created by the extracellular matrix (ECM). Here, we review the effects of the proteolytic activity of ECM proteases in physiological and pathological structural plasticity. We use matrix metalloproteinase-9 (MMP-9) as an example of an ECM modifier that has recently emerged as a key molecule in regulating the morphology and dysmorphology of dendritic spines that underlie synaptic plasticity and neurological disorders, respectively. We summarize the influence of MMP-9 on the dynamic remodeling of the ECM via the cleavage of extracellular substrates. We discuss its role in the formation, modification, and maintenance of dendritic spines in learning and memory. Finally, we review research that implicates MMP-9 in aberrant synaptic plasticity and spine dysmorphology in neurological disorders, with a focus on morphological abnormalities of dendritic protrusions that are associated with epilepsy. PMID:25071472

Matrix polymer species have distinct effects on the mechanics of bacterial biofilms

NASA Astrophysics Data System (ADS)

Kovach, Kristin; Davis-Fields, Megan; Gordon, Vernita

2015-03-01

Biofilms are aggregates of microorganisms embedded in a self-produced extracellular polymer matrix. The matrix confers protection to these microorganisms against mechanical and chemical stresses that they may experience in their environment. The bacterium Pseudomonas aeruginosa is widely used as a model biofilm-forming organism because it is an opportunistic human pathogen common in hospital-acquired infections, in chronic wounds, and in cystic fibrosis lung disease. P. aeruginosa strain PA01 forms biofilms that are primarily structured by the extracellular polysaccharides Pel and Psl. Using bulk rheological measurements, we show that these polysaccharides each play a unique role in the mechanical robustness of the biofilm. Psl increases the elastic storage modulus while Pel increases the ductility of the biofilm. Increased expression of either Psl or Pel increases the yield stress by about the same amount. Identifying the mechanism(s) by which these polymers contribute to the mechanical toughness of the biofilm could allow new approaches to effective biofilm clearance, by revealing targets for disruption that would weaken the biofilm.

Design of biomimetic cellular scaffolds for co-culture system and their application

PubMed Central

Kook, Yun-Min; Jeong, Yoon; Lee, Kangwon; Koh, Won-Gun

2017-01-01

The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell–cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment. PMID:29081966

Design of biomimetic cellular scaffolds for co-culture system and their application.

PubMed

Kook, Yun-Min; Jeong, Yoon; Lee, Kangwon; Koh, Won-Gun

2017-01-01

The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell-cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment.

MMP inhibition as a potential method to augment the healing of skeletal muscle and tendon extracellular matrix

PubMed Central

Davis, Max E.; Gumucio, Jonathan P.; Sugg, Kristoffer B.; Bedi, Asheesh

2013-01-01

The extracellular matrix (ECM) of skeletal muscle and tendon is composed of different types of collagen molecules that play important roles in the transmission of forces throughout the body, and in the repair and regeneration of injured tissues. Fibroblasts are the primary cells in muscle and tendon that maintain, repair, and modify the ECM in response to mechanical loading, injury, and inactivity. Matrix metalloproteinases (MMPs) are enzymes that digest collagen and other structural molecules, which are synthesized and excreted by fibroblasts. MMPs are required for baseline ECM homeostasis, but disruption of MMP regulation due to injury or disease can alter the normal ECM architecture and prevent proper force transmission. Chronic injuries and diseases of muscles and tendons can be severely debilitating, and current therapeutic modalities to enhance healing are quite limited. This review will discuss the mechanobiology of MMPs, and the potential use of MMP inhibitors to improve the treatment of injured and diseased skeletal muscle and tendon tissue. PMID:23640595

Developing defined substrates for stem cell culture and differentiation.

PubMed

Hagbard, Louise; Cameron, Katherine; August, Paul; Penton, Christopher; Parmar, Malin; Hay, David C; Kallur, Therése

2018-07-05

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro , but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Authors.

Isolation and Purification of Versican and Analysis of Versican Proteolysis

PubMed Central

Foulcer, Simon J.; Day, Anthony J.; Apte, Suneel S.

2017-01-01

Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. It is crucial for several developmental processes in the embryo and there is increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and a versican fragment provided here, are likely to facilitate further progress on the biology of versican and its proteolysis. PMID:25325983

Isolation and purification of versican and analysis of versican proteolysis.

PubMed

Foulcer, Simon J; Day, Anthony J; Apte, Suneel S

2015-01-01

Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. It is crucial for several developmental processes in the embryo and there is increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and a versican fragment provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.

Development of biomimetic nanocomposites as bone extracellular matrix for human osteoblastic cells.

PubMed

Bhowmick, Arundhati; Mitra, Tapas; Gnanamani, Arumugam; Das, Manas; Kundu, Patit Paban

2016-05-05

Here, we have developed biomimetic nanocomposites containing chitosan, poly(vinyl alcohol) and nano-hydroxyapatite-zinc oxide as bone extracellular matrix for human osteoblastic cells and characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction. Scanning electron microscopy images revealed interconnected macroporous structures. Moreover, in this study, the problem related to fabricating a porous composite with good mechanical strength has been resolved by incorporating 5wt% of nano-hydroxyapatite-zinc oxide into chitosan-poly(vinyl alcohol) matrix; the present composite showed high tensile strength (20.25MPa) while maintaining appreciable porosity (65.25%). These values are similar to human cancellous bone. These nanocomposites also showed superior water uptake, antimicrobial and biodegradable properties than the previously reported results. Compatibility with human blood and pH was observed, indicating nontoxicity of these materials to the human body. Moreover, proliferation of osteoblastic MG-63 cells onto the nanocomposites was also observed without having any negative effect. Copyright © 2016 Elsevier Ltd. All rights reserved.

Domain organizations of modular extracellular matrix proteins and their evolution.

PubMed

Engel, J

1996-11-01

Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts.

PubMed

Nishizaki, Tomoyuki

2018-01-01

In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis. © 2018 The Author(s). Published by S. Karger AG, Basel.

High resolution three-dimensional reconstruction of fibrotic skeletal muscle extracellular matrix.

PubMed

Gillies, Allison R; Chapman, Mark A; Bushong, Eric A; Deerinck, Thomas J; Ellisman, Mark H; Lieber, Richard L

2017-02-15

Fibrosis occurs secondary to many skeletal muscle diseases and injuries, and can alter muscle function. It is unknown how collagen, the most abundant extracellular structural protein, alters its organization during fibrosis. Quantitative and qualitative high-magnification electron microscopy shows that collagen is organized into perimysial cables which increase in number in a model of fibrosis, and cables have unique interactions with collagen-producing cells. Fibrotic muscles are stiffer and have a higher concentration of collagen-producing cells. These results improve our understanding of the organization of fibrotic skeletal muscle extracellular matrix and identify novel structures that might be targeted by antifibrotic therapy. Skeletal muscle extracellular matrix (ECM) structure and organization are not well understood, yet the ECM plays an important role in normal tissue homeostasis and disease processes. Fibrosis is common to many muscle diseases and is typically quantified based on an increase in ECM collagen. Through the use of multiple imaging modalities and quantitative stereology, we describe the structure and composition of wild-type and fibrotic ECM, we show that collagen in the ECM is organized into large bundles of fibrils, or collagen cables, and the number of these cables (but not their size) increases in desmin knockout muscle (a fibrosis model). The increase in cable number is accompanied by increased muscle stiffness and an increase in the number of collagen producing cells. Unique interactions between ECM cells and collagen cables were also observed and reconstructed by serial block face scanning electron microscopy. These results demonstrate that the muscle ECM is more highly organized than previously reported. Therapeutic strategies for skeletal muscle fibrosis should consider the organization of the ECM to target the structures and cells contributing to fibrotic muscle function. © 2016 Rehabilitation Institute of Chicago. The Journal of Physiology © 2016 The Physiological Society.

High resolution three‐dimensional reconstruction of fibrotic skeletal muscle extracellular matrix

PubMed Central

Gillies, Allison R.; Chapman, Mark A.; Bushong, Eric A.; Deerinck, Thomas J.; Ellisman, Mark H.

2016-01-01

Key points Fibrosis occurs secondary to many skeletal muscle diseases and injuries, and can alter muscle function.It is unknown how collagen, the most abundant extracellular structural protein, alters its organization during fibrosis.Quantitative and qualitative high‐magnification electron microscopy shows that collagen is organized into perimysial cables which increase in number in a model of fibrosis, and cables have unique interactions with collagen‐producing cells.Fibrotic muscles are stiffer and have a higher concentration of collagen‐producing cells.These results improve our understanding of the organization of fibrotic skeletal muscle extracellular matrix and identify novel structures that might be targeted by antifibrotic therapy. Abstract Skeletal muscle extracellular matrix (ECM) structure and organization are not well understood, yet the ECM plays an important role in normal tissue homeostasis and disease processes. Fibrosis is common to many muscle diseases and is typically quantified based on an increase in ECM collagen. Through the use of multiple imaging modalities and quantitative stereology, we describe the structure and composition of wild‐type and fibrotic ECM, we show that collagen in the ECM is organized into large bundles of fibrils, or collagen cables, and the number of these cables (but not their size) increases in desmin knockout muscle (a fibrosis model). The increase in cable number is accompanied by increased muscle stiffness and an increase in the number of collagen producing cells. Unique interactions between ECM cells and collagen cables were also observed and reconstructed by serial block face scanning electron microscopy. These results demonstrate that the muscle ECM is more highly organized than previously reported. Therapeutic strategies for skeletal muscle fibrosis should consider the organization of the ECM to target the structures and cells contributing to fibrotic muscle function. PMID:27859324

Nonlinear laser scanning microscopy of human vocal folds.

PubMed

Miri, Amir K; Tripathy, Umakanta; Mongeau, Luc; Wiseman, Paul W

2012-02-01

The purpose of this work was to apply nonlinear laser scanning microscopy (NLSM) for visualizing the morphology of extracellular matrix proteins within human vocal folds. This technique may potentially assist clinicians in making rapid diagnoses of vocal fold tissue disease or damage. Microstructural characterization based on NLSM provides valuable information for better understanding molecular mechanisms and tissue structure. Experimental, ex vivo human vocal fold. A custom-built multimodal nonlinear laser scanning microscope was used to scan fibrillar proteins in three 4% formaldehyde-fixed cadaveric samples. Collagen and elastin, key extracellular matrix proteins in the vocal fold lamina propria, were imaged by two nonlinear microscopy modalities: second harmonic generation (SHG) and two-photon fluorescence (TPF), respectively. An experimental protocol was introduced to characterize the geometrical properties of the imaged fibrous proteins. NLSM revealed the biomorphology of the human vocal fold fibrous proteins. No photobleaching was observed for the incident laser power of ∼60 mW before the excitation objective. Types I and III fibrillar collagen were imaged without label in the tissue by intrinsic SHG. Imaging while rotating the incident laser light-polarization direction confirmed a helical shape for the collagen fibers. The amplitude, periodicity, and overall orientation were then computed for the helically distributed collagen network. The elastin network was simultaneously imaged via TPF and found to have a basket-like structure. In some regions, particularly close to the epithelium, colocalization of both extracellular matrix components were observed. A benchmark study is presented for quantitative real-time, ex vivo, NLSM imaging of the extracellular macromolecules in human vocal fold lamina propria. The results are promising for clinical applications. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

Teaching the extracellular matrix and introducing online databases within a multidisciplinary course with i-cell-MATRIX: A student-centered approach.

PubMed

Sousa, João Carlos; Costa, Manuel João; Palha, Joana Almeida

2010-03-01

The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure. Internet-available resources can bridge the division between the molecular details and ECM's biological properties and associated processes. This article presents an approach to teach the ECM developed for first year medical undergraduates who, working in teams: (i) Explore a specific molecular component of the matrix, (ii) identify a disease in which the component is implicated, (iii) investigate how the component's structure/function contributes to ECM' supramolecular organization in physiological and in pathological conditions, and (iv) share their findings with colleagues. The approach-designated i-cell-MATRIX-is focused on the contribution of individual components to the overall organization and biological functions of the ECM. i-cell-MATRIX is student centered and uses 5 hours of class time. Summary of results and take home message: A "1-minute paper" has been used to gather student feedback on the impact of i-cell-MATRIX. Qualitative analysis of student feedback gathered in three consecutive years revealed that students appreciate the approach's reliance on self-directed learning, the interactivity embedded and the demand for deeper insights on the ECM. Learning how to use internet biomedical resources is another positive outcome. Ninety percent of students recommend the activity for subsequent years. i-cell-MATRIX is adaptable by other medical schools which may be looking for an approach that achieves higher student engagement with the ECM. Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.

Extracellular matrix and α5β1 integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells

PubMed Central

Di Maggio, Nunzia; Martella, Elisa; Frismantiene, Agne; Resink, Therese J.; Schreiner, Simone; Lucarelli, Enrico; Jaquiery, Claude; Schaefer, Dirk J.; Martin, Ivan; Scherberich, Arnaud

2017-01-01

Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion. PMID:28290502

Mesenchymal stem cell proliferation and mineralization but not osteogenic differentiation are strongly affected by extracellular pH.

PubMed

Fliefel, Riham; Popov, Cvetan; Tröltzsch, Matthias; Kühnisch, Jan; Ehrenfeld, Michael; Otto, Sven

2016-06-01

Osteomyelitis is a serious complication in oral and maxillofacial surgery affecting bone healing. Bone remodeling is not only controlled by cellular components but also by ionic and molecular composition of the extracellular fluids in which calcium phosphate salts are precipitated in a pH dependent manner. To determine the effect of pH on self-renewal, osteogenic differentiation and matrix mineralization of mesenchymal stem cells (MSCs). We selected three different pH values; acidic (6.3, 6.7), physiological (7.0-8.0) and severe alkaline (8.5). MSCs were cultured at different pH ranges, cell viability measured by WST-1, apoptosis detected by JC-1, senescence was analyzed by β-galactosidase whereas mineralization was detected by Alizarin Red and osteogenic differentiation analyzed by Real-time PCR. Self-renewal was affected by pH as well as matrix mineralization in which pH other than physiologic inhibited the deposition of extracellular matrix but did not affect MSCs differentiation as osteoblast markers were upregulated. The expression of osteocalcin and alkaline phosphatase activity was upregulated whereas osteopontin was downregulated under acidic pH. pH affected MSCs self-renewal and mineralization without influencing osteogenic differentiation. Thus, future therapies, based on shifting acid-base balance toward the alkaline direction might be beneficial for prevention or treatment of osteomyelitis. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

PubMed Central

Koo, Bon-Hun; Kim, Yeon Hyang; Han, Jung Ho; Kim, Doo-Sik

2012-01-01

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca2+ ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys102 and the neighboring Cys102. Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2. PMID:22577146

Stabilization, Rolling, and Addition of Other Extracellular Matrix Proteins to Collagen Hydrogels Improve Regeneration in Chitosan Guides for Long Peripheral Nerve Gaps in Rats.

PubMed

Gonzalez-Perez, Francisco; Cobianchi, Stefano; Heimann, Claudia; Phillips, James B; Udina, Esther; Navarro, Xavier

2017-03-01

Autograft is still the gold standard technique for the repair of long peripheral nerve injuries. The addition of biologically active scaffolds into the lumen of conduits to mimic the endoneurium of peripheral nerves may increase the final outcome of artificial nerve devices. Furthermore, the control of the orientation of the collagen fibers may provide some longitudinal guidance architecture providing a higher level of mesoscale tissue structure. To evaluate the regenerative capabilities of chitosan conduits enriched with extracellular matrix-based scaffolds to bridge a critical gap of 15 mm in the rat sciatic nerve. The right sciatic nerve of female Wistar Hannover rats was repaired with chitosan tubes functionalized with extracellular matrix-based scaffolds fully hydrated or stabilized and rolled to bridge a 15 mm nerve gap. Recovery was evaluated by means of electrophysiology and algesimetry tests and histological analysis 4 months after injury. Stabilized constructs enhanced the success of regeneration compared with fully hydrated scaffolds. Moreover, fibronectin-enriched scaffolds increased muscle reinnervation and number of myelinated fibers compared with laminin-enriched constructs. A mixed combination of collagen and fibronectin may be a promising internal filler for neural conduits for the repair of peripheral nerve injuries, and their stabilization may increase the quality of regeneration over long gaps. Copyright © 2017 by the Congress of Neurological Surgeons

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.

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.

Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues.

PubMed

Kočí, Zuzana; Výborný, Karel; Dubišová, Jana; Vacková, Irena; Jäger, Aleš; Lunov, Oleg; Jiráková, Klára; Kubinová, Šárka

2017-06-01

Extracellular matrix (ECM) hydrogels prepared by tissue decellularization have been reported as natural injectable materials suitable for neural tissue repair. In this study, we prepared ECM hydrogel derived from human umbilical cord (UC) and evaluated its composition and mechanical and biological properties in comparison with the previously described ECM hydrogels derived from porcine urinary bladder (UB), brain, and spinal cord. The ECM hydrogels did not differ from each other in the concentration of collagen, while the highest content of glycosaminoglycans as well as the shortest gelation time was found for UC-ECM. The elastic modulus was then found to be the highest for UB-ECM. In spite of a different origin, topography, and composition, all ECM hydrogels similarly promoted the migration of human mesenchymal stem cells (MSCs) and differentiation of neural stem cells, as well as axonal outgrowth in vitro. However, only UC-ECM significantly improved proliferation of tissue-specific UC-derived MSCs when compared with the other ECMs. Injection of UC-ECM hydrogels into a photothrombotic cortical ischemic lesion in rats proved its in vivo gelation and infiltration with host macrophages. In summary, this study proposes UC-ECM hydrogel as an easily accessible biomaterial of human origin, which has the potential for neural as well as other soft tissue reconstruction.

Normal number of CGG repeats in the FMR-1 gene and abnormal incorporation of fibrillin into the extracellular matrix in Lujan Syndrome

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

Greenhaw, G.A.; Stone, C.; Milewicz, D.

1994-09-01

Lujan syndrome is an X-linked condition that includes mild-to-moderate mental retardation, poor social integration, normal secondary sexual development with normal testicular size, generalized hypotonia, hypernasal voice and dolichostenomelia. Major cardiac complications and lens dislocation have not been reported although severe myopia may occur. All reported cases have had negative cytogenetic screening for fra(X) syndrome but establishing this constellation of findings as a distinctive entity has been difficult. We report 4 males in two sibships with clinical findings consistent with Lujan syndrome, normal karyotypes, negative cytogenetic screening for fra(X) syndrome and a normal number of CGG repeats in the FMR-1 gene.more » Dermal fibroblasts explanted from one of the affected males were used to study fibrillin synthesis secretion and extracellular matrix incorporation into microfibrils. Cells from the affected individual showed normal synthesis and secretion of fibrillin when compared to control cells, but the fibrillin was not incorporated into the extracellular matrix. These results suggest the presence of a gene on the X chromosome which may play a role in microfibril assembly and when deficient may disrupt the incorporation of fibrillin into microfibrils. This may be important not only in normal body morphogenesis but also in the development/function of the brain. More affected individuals are needed to investigate these findings further.« less

Aortic Wall Extracellular Matrix Proteins Correlate with Syntax Score in Patients Undergoing Coronary Artery Bypass Surgery

PubMed Central

Chiong, Terri; Cheow, Esther S. H.; Woo, Chin C.; Lin, Xiao Y.; Khin, Lay W.; Lee, Chuen N.; Hartman, Mikael; Sze, Siu K.; Sorokin, Vitaly A.

2016-01-01

Aims: The SYNTAX score correlate with major cardiovascular events post-revascularization, although the histopathological basis is unclear. We aim to evaluate the association between syntax score and extracellular matrix histological characteristics of aortic punch tissue obtained during coronary artery bypass surgery (CABG). This analysis compares coronary artery bypass surgery patients with High and Low syntax score which were followed up for one year period. Methods and Results: Patients with High (score ≥ 33, (n=77)) and Low Syntax Scores (score ≤ 22, (n=71)) undergoing elective CABG were recruited prospectively. Baseline clinical characteristics and surgical risks were well matched. At 1 year, EMACCE (Sum of cardiovascular death, stroke, congestive cardiac failure, and limb, gut and myocardial ischemia) was significantly elevated in the High syntax group (P=0.022). Mass spectrometry (MS)-based quantitative iTRAQ proteomic results validated on independent cohort by immunohistochemistry (IHC) revealed that the High syntax group had significantly upraised Collagen I (P<0.0001) and Elastin (P<0.0001) content in ascending aortic wall. Conclusion: This study shows that aortic extracellular matrix (ECM) differ between High and Low syntax groups with up-regulation of Collagen I and Elastin level in High Syntax Score group. This identifies aortic punches collected during CABG as another biomarker source related with atherosclerosis severity and possible clinical outcome. PMID:27347220

In vivo engineering of the vocal fold extracellular matrix with injectable hyaluronic acid hydrogels: early effects on tissue repair and biomechanics in a rabbit model.

PubMed

Hansen, Jennifer K; Thibeault, Susan L; Walsh, Jennifer F; Shu, Xiao Zheng; Prestwich, Glenn D

2005-09-01

A prospective, controlled animal study was performed to determine whether the use of injectable, chemically modified hyaluronic acid (HA) derivatives at the time of intentional vocal fold resection might facilitate wound repair and preserve the unique viscoelastic properties of the vocal fold extracellular matrix. We performed bilateral vocal fold biopsies on 33 rabbits. Two groups of rabbits were unilaterally treated with 2 different HA derivatives--Carbylan-SX and HA-DTPH-PEGDA--at the time of resection. Saline was injected as a control into the contralateral fold. The animals were painlessly sacrificed 3 weeks after biopsy and injection. The outcomes measured included histologic fibrosis level, tissue HA level, and tissue viscosity and elasticity. The Carbylan-SX-treated vocal folds were found to have significantly less fibrosis than the saline-treated controls. The levels of HA in the treated vocal folds were not significantly different from those in the controls at 3 weeks as measured by enzyme-linked immunosorbent assay. The Carbylan-SX-treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. The HA-DTPH-PEGDA injections yielded significantly improved viscosity, but not elasticity. Prophylactic in vivo manipulation of the extracellular matrix with an injectable Carbylan-SX hydrogel appears to induce vocal fold tissue regeneration to yield optimal tissue composition and biomechanical properties favorable for phonation.