Impairment of Host Liver Repopulation by Transplanted Hepatocytes in Aged Rats and the Release by Short-Term Growth Hormone Treatment.

PubMed

Stock, Peggy; Bielohuby, Maximilian; Staege, Martin S; Hsu, Mei-Ju; Bidlingmaier, Martin; Christ, Bruno

2017-03-01

Hepatocyte transplantation is an alternative to whole liver transplantation. Yet, efficient liver repopulation by transplanted hepatocytes is low in livers of old animals. This restraint might be because of the poor proliferative capacity of aged donor hepatocytes or the regenerative impairment of the recipient livers. The age-dependent liver repopulation by transplanted wild-type hepatocytes was investigated in juvenile and senescent rats deficient in dipeptidyl-peptidase IV. Repopulation was quantified by flow cytometry and histochemical estimation of dipeptidyl-peptidase IV enzyme activity of donor cells in the negative host liver. As a potential pathway involved, expression of cell cycle proteins was assessed. Irrespective of the age of the donor hepatocytes, large cell clusters appeared in juvenile, but only small clusters in senescent host livers. Because juvenile and senescent donor hepatocytes were likewise functional, host-derived factor(s) impaired senescent host liver repopulation. Growth hormone levels were significantly higher in juvenile than in senescent rats, suggesting that growth hormone might promote host liver repopulation. Indeed, short-term treatment with growth hormone augmented senescent host liver repopulation involving the growth hormone-mediated release of the transcriptional blockade of genes associated with cell cycle progression. Short-term growth hormone substitution might improve liver repopulation by transplanted hepatocytes, thus augmenting the therapeutic benefit of clinical hepatocyte transplantation in older patients. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

PubMed

Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

2017-06-01

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

Distinct bone marrow blood vessels differentially regulate haematopoiesis.

PubMed

Itkin, Tomer; Gur-Cohen, Shiri; Spencer, Joel A; Schajnovitz, Amir; Ramasamy, Saravana K; Kusumbe, Anjali P; Ledergor, Guy; Jung, Yookyung; Milo, Idan; Poulos, Michael G; Kalinkovich, Alexander; Ludin, Aya; Kollet, Orit; Shakhar, Guy; Butler, Jason M; Rafii, Shahin; Adams, Ralf H; Scadden, David T; Lin, Charles P; Lapidot, Tsvee

2016-04-21

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration.

PubMed

Massie, Isobel; Spaniol, Kristina; Barbian, Andreas; Geerling, Gerd; Metzger, Marco; Schrader, Stefan

2018-04-01

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (β-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further. Copyright © 2017 John Wiley & Sons, Ltd.

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro.

PubMed

Kim, Suna; Piao, Jiyuan; Son, Youngsook; Hong, Hyun Sook

2017-03-25

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. Copyright © 2017 Elsevier Inc. All rights reserved.

Decellularized aortic conduits: could their cryopreservation affect post-implantation outcomes? A morpho-functional study on porcine homografts.

PubMed

Gallo, Michele; Bonetti, Antonella; Poser, Helen; Naso, Filippo; Bottio, Tomaso; Bianco, Roberto; Paolin, Adolfo; Franci, Paolo; Busetto, Roberto; Frigo, Anna Chiara; Buratto, Edward; Spina, Michele; Marchini, Maurizio; Ortolani, Fulvia; Iop, Laura; Gerosa, Gino

2016-11-01

Decellularized porcine aortic valve conduits (AVCs) implanted in a Vietnamese Pig (VP) experimental animal model were matched against decellularized and then cryopreserved AVCs to assess the effect of cryopreservation on graft hemodynamic performance and propensity to in vivo repopulation by host's cells. VPs (n = 12) underwent right ventricular outflow tract substitution using AVC allografts and were studied for 15-month follow-up. VPs were randomized into two groups, receiving AVCs treated with decellularization alone (D; n = 6) or decellularization/cryopreservation (DC; n = 6), respectively. Serial echocardiography was carried out to follow up hemodynamic function. All explanted AVCs were processed for light and electron microscopy. No signs of dilatation, progressive stenosis, regurgitation, and macroscopic calcification were echocardiographically observed in both D and DC groups. Explanted D grafts exhibited near-normal features, whereas the presence of calcification, inflammatory infiltrates, and disarray of elastic lamellae occurred in some DC grafts. In the unaltered regions of AVCs from both groups, almost complete re-endothelialization was observed for both valve cusps and aorta walls. In addition, side-by-side repopulation by recipient's fibroblasts, myofibroblasts, and smooth muscle cells was paralleled by ongoing tissue remodeling, as revealed by the ultrastructural identification of typical canals of collagen fibrillogenesis and elastogenesis-related features. Incipient neo-vascularization and re-innervation of medial and adventitial tunicae of grafted aortic walls were also detected for both D and DC groups. Cryopreservation did not affect post-implantation AVC hemodynamic behavior and was topically propensive to cell repopulation and tissue renewal, although graft deterioration including calcification was present in several areas. Thus, these preliminary data provide essential information on feasibility of decellularization and cryopreservation coupling in the perspective of treatment optimization and subsequent clinical trials using similarly treated human allografts as innovative heart valve substitutes.

Anti-retroviral therapy fails to restore the severe Th-17: Tc-17 imbalance observed in peripheral blood during simian immunodeficiency virus infection.

PubMed

Kader, M; Bixler, S; Piatak, M; Lifson, J; Mattapallil, J J

2009-10-01

Human immuno deficiency virus and simian immunodeficiency virus infections are characterized by a severe loss of Th-17 cells (IL-17(+)CD4(+) T cells) that has been associated with disease progression and systemic dissemination of bacterial infections. Anti-retroviral therapy (ART) has led to repopulation of CD4(+) T cells in peripheral tissues with little sustainable repopulation in mucosal tissues. Given the central importance of Th-17 cells in mucosal homeostasis, it is not known if the failure of ART to permanently repopulate mucosal tissues is associated with a failure to restore Th-17 cells that are lost during infection. Dynamics of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood of SIV infected rhesus macaques were evaluated and compared to animals that were treated with ART. The frequency of Th-17 and Tc-17 cells was determined following infection and after therapy. Relative expression of IL-21, IL-23, and TGFbeta was determined using Taqman PCR. Treatment of SIV infected rhesus macaques with anti-retroviral therapy was associated with a substantial repopulation of mucosal homing alpha4(+)beta7(hi)CD4(+) T cells in peripheral blood. This repopulation, however, was not accompanied by a restoration of Th-17 responses. Interestingly, SIV infection was associated with an increase in Tc-17 responses (IL-17(+)CD8(+) T cells) suggesting to a skewing in the ratio of Th-17: Tc-17 cells from a predominantly Th-17 phenotype to a predominantly Tc-17 phenotype. Surprisingly, Tc-17 responses remained high during the course of therapy suggesting that ART failed to correct the imbalance in Th-17 : Tc-17 responses induced following SIV infection. ART was associated with substantial repopulation of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood with little or no rebound of Th-17 cells. On the other hand, repopulation of alpha4(+)beta7(hi) CD4(+) T cells was accompanied by persistence of high levels of Tc-17 cells in peripheral blood. The dysregulation of Th-17 and Tc-17 responses likely plays a role in disease progression.

A human bone marrow mesodermal-derived cell population with hemogenic potential.

PubMed

Mokhtari, Saloomeh; Colletti, Evan; Yin, Weihong; Sanada, Chad; Lamar, Zanetta; Simmons, Paul J; Walker, Steven; Bishop, Colin; Atala, Anthony; Zanjani, Esmail D; Porada, Christopher D; Almeida-Porada, Graça

2018-02-02

The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, co-expressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

Long-term erythropoietic repopulating ability of old, young, and fetal stem cells.

PubMed

Harrison, D E

1983-05-01

It is possible that erythropoietic stem cells do not age. This would mean that stem cells from old donors can function as well as those from young or fetal donors. The competitive repopulation assay has been used to test long-term stem cell function by directly comparing how well competing stem cells repopulate a recipient and produce differentiated cell types. C57BL/6J (B6) mice were used as donors, while recipients and competitors were WBB6F1 hybrids with genetically distinguishable hemoglobin. Lethally irradiated young WBB6F1 recipients were given a mixture of 2.5 X 10(6) cells from B6 old marrow, young marrow, or fetal liver donors; each recipient also received a standard dose of 1 X 10(6) marrow cells from a pool of young WBB6F1 competitors. Surprisingly, the old marrow cells competed the best in repopulating the recipients. This pattern was maintained even after recovery from sublethal irradiation, a treatment that severely stresses stem cells. This stress was demonstrated when sublethal irradiation caused a 20-fold decline in repopulating ability measured using hemoglobin markers, and a 3- to 7-fold decline using chromosome markers. Stem cells from old marrow competed better than young or fetal cells in similar experiments using immunologically crippled recipients or using unirradiated W/Wv recipients that are immunologically intact. In both types of recipients, the advantage of old marrow cells again persisted after recovery from sublethal irradiation. Other genotypes were tested, and marrow cells from old B6CBAF1 donors competed better than those from young donors of that genotype. However, marrow cells from young CBA donors completed better than those from old CBA donors. These results support the hypothesis that stem cells do not age, and suggest that regulatory changes with age promote rapid stem cell repopulation in B6 and B6CBAF1 mice, but inhibit it in CBA mice.

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells.

PubMed

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-06-09

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin-CD34+/-MPL+/- cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/-MPL+/- cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/- and CD34-MPL- cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/-MPL- cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/- SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34- SRCs generate CD34+CD38-CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34-MPL- SRCs reside at the apex of the human HSC hierarchy.

Monte Carlo radiotherapy simulations of accelerated repopulation and reoxygenation for hypoxic head and neck cancer

PubMed Central

Harriss-Phillips, W M; Bezak, E; Yeoh, E K

2011-01-01

Objective A temporal Monte Carlo tumour growth and radiotherapy effect model (HYP-RT) simulating hypoxia in head and neck cancer has been developed and used to analyse parameters influencing cell kill during conventionally fractionated radiotherapy. The model was designed to simulate individual cell division up to 108 cells, while incorporating radiobiological effects, including accelerated repopulation and reoxygenation during treatment. Method Reoxygenation of hypoxic tumours has been modelled using randomised increments of oxygen to tumour cells after each treatment fraction. The process of accelerated repopulation has been modelled by increasing the symmetrical stem cell division probability. Both phenomena were onset immediately or after a number of weeks of simulated treatment. Results The extra dose required to control (total cell kill) hypoxic vs oxic tumours was 15–25% (8–20 Gy for 5×2 Gy per week) depending on the timing of accelerated repopulation onset. Reoxygenation of hypoxic tumours resulted in resensitisation and reduction in total dose required by approximately 10%, depending on the time of onset. When modelled simultaneously, accelerated repopulation and reoxygenation affected cell kill in hypoxic tumours in a similar manner to when the phenomena were modelled individually; however, the degree was altered, with non-additive results. Simulation results were in good agreement with standard linear quadratic theory; however, differed for more complex comparisons where hypoxia, reoxygenation as well as accelerated repopulation effects were considered. Conclusion Simulations have quantitatively confirmed the need for patient individualisation in radiotherapy for hypoxic head and neck tumours, and have shown the benefits of modelling complex and dynamic processes using Monte Carlo methods. PMID:21933980

Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter

PubMed Central

Birey, Fikri

2015-01-01

Neuron–glial antigen 2-positive (NG2+) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2+ glial cell ablation model in mice, we examined the repopulation dynamics of NG2+ glial cells in the mature and aged mice gray matter. We found that some resident NG2+ glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2+ glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2+ glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2+ glial cell homeostasis that is distinct from its role in myelination. PMID:25926469

Liver repopulation by c-Met-positive stem/progenitor cells isolated from the developing rat liver.

PubMed

Suzuki, Atsushi; Zheng, Yun-wen; Fukao, Katashi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-01-01

Self-renewing stem cells responsible for tissue or organ development and regeneration have been recently described. To isolate such cells using flow cytometry, it should be required to find molecules expressing on their cell surfaces. We have previously reported that, on cells fulfilling the criteria for hepatic stem cells, the hepatocyte growth factor receptor c-Met is expressed specifically in the developing mouse liver. In this study, to determine whether c-Met is an essential marker for hepatic stem cells in other animal strains, we examined the potential for in vivo liver-repopulation in sorted fetal rat-derived c-Met+ cells using the retrorsine model. Using flow cytometry and monoclonal antibodies for c-Met and leukocyte common antigen CD45, fetal rat liver cells were fractionated according to the expression of these molecules. Then, cells in each cell subpopulation were sorted and transplanted into the retrorsine-treated adult rats with two-third hepatectomy. At 9 months post transplant, frequency of liver-repopulation was examined by qualitative and quantitative analyses. When we transplanted c-Met+ CD45- sorted cells, many donor-derived cells formed colonies that included mature hepatocytes expressing albumin and containing abundant glycogen in their cytoplasm. In contrast, c-Met- cells and CD45+ cells could not repopulate damaged recipient livers. High enrichment of liver-repopulating cells was conducted by sorting of c-Met+ cells from the developing rat liver. This result suggests that c-Met/HGF interaction plays a crucial role for stem cell growth, differentiation, and self-renewal in rat liver organogenesis. Since the c-Met is also expressed in the fetal mouse-derived hepatic stem cells, this molecule could be expected to be an essential marker for such cell population in the various animal strains, including human.

CD34 Antigen and the MPL Receptor Expression Defines a Novel Class of Human Cord Blood-Derived Primitive Hematopoietic Stem Cells

PubMed Central

Matsuoka, Yoshikazu; Takahashi, Masaya; Sumide, Keisuke; Kawamura, Hiroshi; Nakatsuka, Ryusuke; Fujioka, Tatsuya; Sonoda, Yoshiaki

2017-01-01

In the murine hematopoietic stem cell (HSC) compartment, thrombopoietin (THPO)/MPL (THPO receptor) signaling plays an important role in the maintenance of adult quiescent HSCs. However, the role of THPO/MPL signaling in the human primitive HSC compartment has not yet been elucidated. We have identified very primitive human cord blood (CB)-derived CD34– severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method. In this study, we investigated the roles of the MPL expression in the human primitive HSC compartment. The SRC activities of the highly purified CB-derived 18Lin–CD34+/–MPL+/– cells were analyzed using NOG mice. In the primary recipient mice, nearly all mice that received CD34+/–MPL+/– cells were repopulated with human CD45+ cells. Nearly all of these mice that received CD34+MPL+/– and CD34–MPL– cells showed a secondary repopulation. Interestingly, the secondary recipient mice that received CD34+/–MPL– cells showed a distinct tertiary repopulation. These results clearly indicate that the CD34+/– SRCs not expressing MPL sustain a long-term (LT) (>1 year) human cell repopulation in NOG mice. Moreover, CD34– SRCs generate CD34+CD38–CD90+ SRCs in vitro and in vivo. These findings provide a new concept that CD34–MPL– SRCs reside at the apex of the human HSC hierarchy. PMID:27938494

CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

PubMed

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

2015-01-01

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.

PubMed

Caralt, M; Uzarski, J S; Iacob, S; Obergfell, K P; Berg, N; Bijonowski, B M; Kiefer, K M; Ward, H H; Wandinger-Ness, A; Miller, W M; Zhang, Z J; Abecassis, M M; Wertheim, J A

2015-01-01

The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention--indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained 3 h of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

PubMed

Ng, Elizabeth S; Azzola, Lisa; Bruveris, Freya F; Calvanese, Vincenzo; Phipson, Belinda; Vlahos, Katerina; Hirst, Claire; Jokubaitis, Vanta J; Yu, Qing C; Maksimovic, Jovana; Liebscher, Simone; Januar, Vania; Zhang, Zhen; Williams, Brenda; Conscience, Aude; Durnall, Jennifer; Jackson, Steven; Costa, Magdaline; Elliott, David; Haylock, David N; Nilsson, Susan K; Saffery, Richard; Schenke-Layland, Katja; Oshlack, Alicia; Mikkola, Hanna K A; Stanley, Edouard G; Elefanty, Andrew G

2016-11-01

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter.

PubMed

Birey, Fikri; Aguirre, Adan

2015-04-29

Neuron-glial antigen 2-positive (NG2(+)) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2(+) glial cell ablation model in mice, we examined the repopulation dynamics of NG2(+) glial cells in the mature and aged mice gray matter. We found that some resident NG2(+) glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2(+) glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2(+) glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2(+) glial cell homeostasis that is distinct from its role in myelination. Copyright © 2015 the authors 0270-6474/15/356946-06$15.00/0.

Foamy virus–mediated gene transfer to canine repopulating cells

PubMed Central

Kiem, Hans-Peter; Allen, James; Trobridge, Grant; Olson, Erik; Keyser, Kirsten; Peterson, Laura; Russell, David W.

2007-01-01

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)–expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/μL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical. PMID:16968897

Proliferation of pulmonary endothelial cells: time-lapse cinematography of growth to confluence and restitution of monolayer after wounding.

PubMed

Ryan, U S; Absher, M; Olazabal, B M; Brown, L M; Ryan, J W

1982-01-01

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establishment of a monolayer from a low-density seed (7.5 X 10(4) cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 x 10(6) cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 x 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture 1, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture 1). Interdivision times (IDT) were longer and relatively constant in culture 1 until near confluence; none were less than 10 h, whereas in 2, 24% of the IDT's were less than or equal to 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro

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

Kim, Suna; Piao, Jiyuan; Son, Youngsook

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culturemore » are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. - Highlights: • Long-term culture of ADSCs leads to cell senescence. • Paracrine potential of ADSC decreases as passage number increases. • SP enhances the weakened proliferation capacity of ADSCs. • SP stimulates cytokine secretion from ADSC with impaired paracrine potential.« less

Exploiting the unique regenerative capacity of the liver to underpin cell and gene therapy strategies for genetic and acquired liver disease.

PubMed

Logan, Grant J; de Alencastro, Gustavo; Alexander, Ian E; Yeoh, George C

2014-11-01

The number of genetic or acquired diseases of the liver treatable by organ transplantation is ever-increasing as transplantation techniques improve placing additional demands on an already limited organ supply. While cell and gene therapies are distinctly different modalities, they offer a synergistic alternative to organ transplant due to distinct architectural and physiological properties of the liver. The hepatic blood supply and fenestrated endothelial system affords relatively facile accessibility for cell and/or gene delivery. More importantly, however, the remarkable capacity of hepatocytes to proliferate and repopulate the liver creates opportunities for new treatments based on emerging technologies. This review will summarise current understanding of liver regeneration, describe clinical and experimental cell and gene therapeutic modalities and discuss critical challenges to translate these new technologies to wider clinical utility. This article is part of a Directed Issue entitled: "Regenerative Medicine: the challenge of translation". Copyright © 2014 Elsevier Ltd. All rights reserved.

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold.

PubMed

Hassanein, Wessam; Uluer, Mehmet C; Langford, John; Woodall, Jhade D; Cimeno, Arielle; Dhru, Urmil; Werdesheim, Avraham; Harrison, Joshua; Rivera-Pratt, Carlos; Klepfer, Stephen; Khalifeh, Ali; Buckingham, Bryan; Brazio, Philip S; Parsell, Dawn; Klassen, Charlie; Drachenberg, Cinthia; Barth, Rolf N; LaMattina, John C

2017-01-02

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

Clinical-scale expansion of CD34+ cord blood cells amplifies committed progenitors and rapid scid repopulation cells.

PubMed

Casamayor-Genescà, Alba; Pla, Arnau; Oliver-Vila, Irene; Pujals-Fonts, Noèlia; Marín-Gallén, Sílvia; Caminal, Marta; Pujol-Autonell, Irma; Carrascal, Jorge; Vives-Pi, Marta; Garcia, Joan; Vives, Joaquim

2017-03-25

Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34 + population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34 + controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγ null mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×10 6 CD34 + cells committed to the granulocytic lineage and 3.9×10 9 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product. Copyright © 2016 Elsevier B.V. All rights reserved.

Tumour volume response, initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

PubMed Central

Stephens, T. C.; Peacock, J. H.

1977-01-01

The relationship between tumour volume response and cell kill in B16 melanoma following treatment in vivo with cyclophosphamide (CY) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated. Tumour volume response, expressed as growth delay, was estimated from measurements of tumour dimensions. Depression of in vitro colony-forming ability of cells from treated tumours was used as the measure of tumour cell kill. The relationship between these parameters was clearly different for the two agents studied. CY produced more growth delay (7.5 days) per decade of tumour cell kill than CCNU (2 to 3.5 days). The possibility that this was due to a technical artefact was rejected in favour of an alternative explanation that different rates of cellular repopulation in tumours treated with CY and CCNU might be responsible. Cellular repopulation was measured directly, by performing cell-survival assays at various times after treatment with doses of CY and CCNU which produced about 3 decades of cell kill. The rate of repopulation by clonogenic cells was much slower after treatment with CY than with CCNU, and this appears to account for the longer duration of the growth delay obtained with CY. PMID:921888

Inhibiting the Aurora B Kinase Potently Suppresses Repopulation During Fractionated Irradiation of Human Lung Cancer Cell Lines

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

Sak, Ali, E-mail: ali.sak@uni-due.de; Stuschke, Martin; Groneberg, Michael

2012-10-01

Purpose: The use of molecular-targeted agents during radiotherapy of non-small-cell lung cancer (NSCLC) is a promising strategy to inhibit repopulation, thereby improving therapeutic outcome. We assessed the combined effectiveness of inhibiting Aurora B kinase and irradiation on human NSCLC cell lines in vitro. Methods and Materials: NSCLC cell lines were exposed to concentrations of AZD1152-hydroxyquinazoline pyrazol anilide (AZD1152-HQPA) inhibiting colony formation by 50% (IC50{sub clone}) in combination with single dose irradiation or different fractionation schedules using multiple 2-Gy fractions per day up to total doses of 4-40 Gy. The total irradiation dose required to control growth of 50% of themore » plaque monolayers (TCD50) was determined. Apoptosis, G2/M progression, and polyploidization were also analyzed. Results: TCD50 values after single dose irradiation were similar for the H460 and H661 cell lines with 11.4 {+-} 0.2 Gy and 10.7 {+-} 0.3 Gy, respectively. Fractionated irradiation using 3 Multiplication-Sign 2 Gy/day, 2 Multiplication-Sign 2 Gy/day, and 1 Multiplication-Sign 2 Gy/day schedules significantly increased TCD50 values for both cell lines grown as plaque monolayers with increasing radiation treatment time. This could be explained by a repopulation effect per day that counteracts 75 {+-} 8% and 27 {+-} 6% of the effect of a 2-Gy fraction in H460 and H661 cells, respectively. AZD1152-HQPA treatment concomitant to radiotherapy significantly decreased the daily repopulation effect (H460: 28 {+-} 5%, H661: 10 {+-} 4% of a 2-Gy fraction per day). Treatment with IC50{sub clone} AZD1152-HPQA did not induce apoptosis, prolong radiation-induced G2 arrest, or delay cell cycle progression before the spindle check point. However, polyploidization was detected, especially in cell lines without functional p53. Conclusions: Inhibition of Aurora B kinase with low AZD1152-HQPA concentrations during irradiation of NSCLC cell lines affects repopulation during radiotherapy. Thus, concomitant Aurora B kinase inhibition and irradiation may be a promising strategy for fast repopulating tumors, which are difficult to cure by dose escalation based on conventional fractionation.« less

Repopulation Kinetics and the Linear-Quadratic Model

NASA Astrophysics Data System (ADS)

O'Rourke, S. F. C.; McAneney, H.; Starrett, C.; O'Sullivan, J. M.

2009-08-01

The standard Linear-Quadratic (LQ) survival model for radiotherapy is used to investigate different schedules of radiation treatment planning for advanced head and neck cancer. We explore how these treament protocols may be affected by different tumour repopulation kinetics between treatments. The laws for tumour cell repopulation include the logistic and Gompertz models and this extends the work of Wheldon et al. [1], which was concerned with the case of exponential repopulation between treatments. Treatment schedules investigated include standarized and accelerated fractionation. Calculations based on the present work show, that even with growth laws scaled to ensure that the repopulation kinetics for advanced head and neck cancer are comparable, considerable variation in the survival fraction to orders of magnitude emerged. Calculations show that application of the Gompertz model results in a significantly poorer prognosis for tumour eradication. Gaps in treatment also highlight the differences in the LQ model with the effect of repopulation kinetics included.

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

Bertoncello, I.; Hodgson, G.S.; Bradley, T.R.

A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells that preferentially home to the marrow of lethally irradiated mice to be enriched and separated from the majority of spleen colony-forming cells that are assayed 13 days after transplantation (CFU-S13). First, bone marrow cells are centrifuged in a discontinuous bovine serum albumin gradient. Low-density cells are harvested and labeled with the supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled cells are analyzed using a fluorescence-activated cell sorter, and cells are sorted on the basis of relative Rh123 fluorescence within a predetermined forward versus 90 degrees red lightmore » scatter window that has been optimized for the recovery and enrichment of cells with marrow repopulating ability (MRA). Cells with MRA were characterized by relatively low Rh123 fluorescence and could be separated from a fraction that fluoresced more intensely and contained the majority of CFU-S13 but low MRA. Cells with platelet repopulating ability cofractionate with MRA whereas cells with erythroid repopulating ability remain associated with CFU-S13.« less

Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow–derived cells

PubMed Central

Day, Yuan-Ji; Huang, Liping; McDuffie, Marcia J.; Rosin, Diane L.; Ye, Hong; Chen, Jiang-Fan; Schwarzschild, Michael A.; Fink, J. Stephen; Linden, Joel; Okusa, Mark D.

2003-01-01

Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow–derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP→WT, A2A-KO→WT, or WT→WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO→WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT→WT chimera. ATL146e reduced the induction of IL-6, IL-1β, IL-1ra, and TGF-α mRNA in WT→WT mice but not in A2A-KO→WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells. PMID:12975473

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

PubMed Central

Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

2009-01-01

SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation.

PubMed

Kirby, S; Walton, W; Smithies, O

2000-06-15

In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM. (Blood. 2000;95:3710-3715)

High-efficiency Transduction of Rhesus Hematopoietic Repopulating Cells by a Modified HIV1-based Lentiviral Vector

PubMed Central

Uchida, Naoya; Hargrove, Phillip W.; Lap, Coen J.; Evans, Molly E.; Phang, Oswald; Bonifacino, Aylin C.; Krouse, Allen E.; Metzger, Mark E.; Nguyen, Anh-Dao; Hsieh, Matthew M.; Wolfsberg, Tyra G.; Donahue, Robert E.; Persons, Derek A.; Tisdale, John F.

2012-01-01

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34+ cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34+ cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. PMID:22871664

Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction.

PubMed

Bachy, M; Sherifi, I; Zadegan, F; Petite, H; Vialle, R; Hannouche, D

2016-04-01

Tissue engineering strategies include both cell-based and cell homing therapies. Ligamentous tissues are highly specialized and constitute vital components of the musculoskeletal system. Their damage causes significant morbidity and loss in function. The aim of this study is to analyze tendinous graft integration, cell repopulation and ligamentization by using GFP+/- allografts in GFP+/- transgenic New Zealand white (NZW) rabbits. Graft implantation was designed to closely mimic anterior cruciate ligament (ACL) repair surgery. Allografts were implanted in 8 NZW rabbits and assessed at 5 days, 3 weeks and 6 weeks through: (1) arthroCT imaging, (2) morphological analysis of the transplanted allograft, (3) histological analysis, (4) collagen type I immunochemistry, and (5) GFP cell tracking. Collagen remodeling was appreciated at 3 and 6 weeks. Graft repopulation with host cells, chondrocyte-like cells at the tendon-bone interface and graft corticalization in the bone tunnels were noticed at 3 weeks. By contrast we noticed a central necrosis aspect in the allografts intra-articularly at 6 weeks with a cell migration towards the graft edge near the synovium. Our study has served to gain a better understanding of tendinous allograft bone integration, ligamentization and allograft repopulation. We believe that both cell-based therapies and cell homing therapies are beneficial in ligament tissue engineering. Future studies may elucidate whether cell repopulation occurs with pre-differentiated or progenitor cells. We believe that both cell-based therapies and cell homing therapies are beneficial in ligament tissue engineering. Level V (animal study). Copyright © 2015 Elsevier Masson SAS. All rights reserved.

In vitro study of the adverse effect of nicotine and physical strain on human gingival fibroblasts as a model of the healing of wounds commonly found in the military.

PubMed

Dinos, Michael E; Borke, James L; Swiec, Gary D; McPherson, James C; Goodin, Jeremy L; Chuang, Augustine H

2015-03-01

Significant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model. HGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation. The application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase. Initial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well. Reprint & Copyright © 2015 Association of Military Surgeons of the U.S.

Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia

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

Maggio-Price, L.; Wolf, N.S.; Priestley, G.V.

1988-09-01

Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture ofmore » anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.« less

Unaltered repopulation properties of mouse hematopoietic stem cells transduced with lentiviral vectors

PubMed Central

Gonzalez-Murillo, Africa; Lozano, M. Luz; Montini, Eugenio; Bueren, Juan A.

2008-01-01

Recent studies of retroviral-mediated gene transfer have shown that retroviral integrations themselves may trigger nonmalignant clonal expansion of hematopoietic stem cells (HSCs) in transplant recipients. These observations suggested that previous conclusions of HSC dynamics based on gamma-retroviral gene marking should be confirmed with improved vectors having a more limited capacity to transactivate endogenous genes. Because of the low trans-activation activity of self-inactivating lentiviral vectors (LVs), we have investigated whether the LV marking of mouse HSCs induces a competitive repopulation advantage in recipients of serially transplants. As deduced from analyses conducted in primary and secondary recipients, we concluded that lentivirally transduced HSCs have no competitive repopulation advantages over untransduced HSCs. By linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis, we characterized LV-targeted genes in HSC clones that engrafted up to quaternary recipients. Although 9 clones harbored integrations close to defined retroviral insertion sites, none was characterized as a common integration site, and none was present in HSC clones repopulating quaternary recipients. Taken together, our results show unaltered repopulation properties of HSCs transduced with LVs, and confirm early studies suggesting the natural capacity of a few HSC clones to generate a monoclonal or oligoclonal hematopoiesis in transplant recipients. PMID:18684860

Influence of anaesthetics on tumour-cell kill and repopulation in B16 melanoma treated with melphalan.

PubMed Central

Peacock, J. H.; Stephens, T. C.

1978-01-01

The influence of anaesthetics on the in vivo response of B16 melanoma to melphalan was studied using an in vitro cell-survival assay. Three anaesthetics were used, Saffan (Althesin) Sagatal (Nembutal) and Hypnorm. When Saffan was administered to tumour-bearing animals before melphalan there was a significant increase in tumour-cell kill. This effect was not observed with Sagatal or Hypnorm. Maximum increase in tumour-cell kill was achieved when Saffan was administered about 1 h before melphalan, and was dependent on Saffan dose. Clonogenic tumour-cell repopulation after melphalan was rapid (TD - 1 day) and the rate was similar from 2 levels of cell kill. When Saffan was combined with melphalan the repopulation rate was the same as with melphalan alone, and the increased cell kill was reflected in increased growth delay. The in vitro response of B16 melanoma cells to melphalan was unaltered by pretreatment with, or simultaneous exposure to Saffan. The results suggest that the mechanism of the enhanced cell kill in vivo is probably due to an indirect systemic effect, rather than a direct effect on the tumour cells. PMID:743490

Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes

PubMed Central

Yovchev, Mladen I.; Xue, Yuhua; Shafritz, David A.; Locker, Joseph; Oertel, Michael

2013-01-01

Background & Aim Considerable progress has been made in developing anti-fibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Methods Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV+ F344 rats were transplanted into DPPIV− rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, RT-PCR, and immunohistochemistry. Results After chronic TAA administration, DPPIV− F344 rats exhibited progressive fibrosis, cirrhosis and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1+, AFP+, CD133+, Sox-9+, FoxJ1+) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of α-SMA, PDGFRβ, desmin, vimentin, TIMP1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than FLSPCs. Conclusions This study is a Proof of Principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit anti-fibrotic effects. PMID:23840008

B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

PubMed

Da Rosa, Larissa C; Boldison, Joanne; De Leenheer, Evy; Davies, Joanne; Wen, Li; Wong, F Susan

2018-06-01

Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4 + and CD8 + T cells. These CD8 + T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location

PubMed Central

Yovchev, Mladen; Jaber, Fadi L.; Lu, Zhonglei; Patel, Shachi; Locker, Joseph; Rogler, Leslie E.; Murray, John W.; Sudol, Marius; Dabeva, Mariana D.; Zhu, Liang; Shafritz, David A.

2016-01-01

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs. PMID:26763940

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

PubMed

Brammer, I; Zywietz, F; Beck-Bornholdt, H P; Jung, H

1992-05-01

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

Modeling marrow damage from response data: Evolution from radiation biology to benzene toxicity

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

Jones, T.D.; Morris, M.D.; Hasan, J.S.

1996-12-01

Consensus principles from radiation biology were used to describe a generic set of nonlinear, first-order differential equations for modeling toxicity-induced compensatory cell kinetics in terms of sublethal injury, repair, direct killing, killing of cells with unrepaired sublethal injury, and repopulation. This cellular model was linked to a probit model of hematopoietic mortality that describes death from infection and/or hemorrhage between 5 and 30 days. Mortality data from 27 experiments with 851 dose-response groups, in which doses were protracted by rate and/or fractionation, were used to simultaneously estimate all rate constants by maximum-likelihood methods. Data used represented 18,940 test animals: 12,827more » mice, 2925 rats, 1676 sheep, 829 swine, 479 dogs, and 204 burros. Although a long-term, repopulating hematopoietic stem cell is ancestral to all lineages needed to restore normal homeostasis, the dose-response data from the protracted irradiations indicate clearly that the particular lineage that is critical to hematopoietic recovery does not resemble stemlike cells with regard to radiosensitivity and repopulation rates. Instead, the weakest link in the chain of hematopoiesis was found to have an intrinsic radioresistance equal to or greater than stromal cells and to repopulate at the same rates. Model validation has been achieved by predicting the LD50 and/or fractional group mortality in 38 protracted-dose experiments (rats and mice) that were not used in the fitting of model coefficients. 29 refs., 5 figs., 5 tabs.« less

Mechanisms of radiation-induced normal tissue toxicity and implications for future clinical trials

PubMed Central

Jenrow, Kenneth A.; Brown, Stephen L.

2014-01-01

To summarize current knowledge regarding mechanisms of radiation-induced normal tissue injury and medical countermeasures available to reduce its severity. Advances in radiation delivery using megavoltage and intensity-modulated radiation therapy have permitted delivery of higher doses of radiation to well-defined tumor target tissues. Injury to critical normal tissues and organs, however, poses substantial risks in the curative treatment of cancers, especially when radiation is administered in combination with chemotherapy. The principal pathogenesis is initiated by depletion of tissue stem cells and progenitor cells and damage to vascular endothelial microvessels. Emerging concepts of radiation-induced normal tissue toxicity suggest that the recovery and repopulation of stromal stem cells remain chronically impaired by long-lived free radicals, reactive oxygen species, and pro-inflammatory cytokines/chemokines resulting in progressive damage after radiation exposure. Better understanding the mechanisms mediating interactions among excessive generation of reactive oxygen species, production of pro-inflammatory cytokines and activated macrophages, and role of bone marrow-derived progenitor and stem cells may provide novel insight on the pathogenesis of radiation-induced injury of tissues. Further understanding the molecular signaling pathways of cytokines and chemokines would reveal novel targets for protecting or mitigating radiation injury of tissues and organs. PMID:25324981

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

Revised radiobiological modelling of the contribution of synchronous chemotherapy to the rate of grades 3-4 mucositis in head and neck cancer.

PubMed

Meade, Sara; McConkey, Chris; Sanghera, Paul; Mehanna, Hisham; Hartley, Andrew

2013-12-01

Biological effective dose (BED) calculations modelled on reduced accelerated repopulation when synchronous chemotherapy is delivered significantly correlate with observed differences in local control in randomised trials of platinum-based chemoradiation. The purpose of this study was to examine whether a similar relationship existed in the context of grades 3-4 mucositis. Biological effective dose from radiotherapy and synchronous chemotherapy was calculated using three different models: AB using the additional BED attributable to chemotherapy and standard repopulation parameters; zero repopulation (ZRP) using zero correction for repopulation; and variable t(p) (Vt(p)) using a variable doubling time for mucosal stem cell repopulation. The correlation between the percentage change in biological effective dose between trial arms, and the observed percentage change in the rate of grades 3-4 mucositis was examined by using the Pearson product-moment correlation. With the AB model, there were no significant correlations with observed differences in rates of grades 3-4 mucositis. With either the ZRP or Vt(p) models, significant correlations were observed. A value of 5 days for the doubling time during repopulation (T(p)) was associated with the most significant correlation (P = 0.002). Models where the dose lost due to accelerated repopulation is reduced imply a therapeutic loss from the use of synchronous chemotherapy when only local control and the rate of acute grades 3-4 mucositis are considered. © 2013 The Royal Australian and New Zealand College of Radiologists.

Mitomycin C in combination with radiotherapy as a potent inhibitor of tumour cell repopulation in a human squamous cell carcinoma

PubMed Central

Budach, W; Paulsen, F; Welz, S; Classen, J; Scheithauer, H; Marini, P; Belka, C; Bamberg, M

2002-01-01

The potential of Mitomycin C in combination with fractionated irradiation to inhibit tumour cell repopulation of a fast growing squamous cell carcinoma after fractionated radiotherapy was investigated in vivo. A rapidly growing human squamous cell carcinoma (FaDudd) was used for the study. For experiments, NMRI (nu/nu) mice with subcutaneously growing tumours were randomly allocated to no treatment, Mitomycin C, fractionated irradiation (ambient: 11x4.5 Gy in 15 days), or fractionated irradiation combined with Mitomycin C. Graded top up doses (clamped blood flow: 0–57 Gy) were given at day 16, 23, 30 or 37. End point of the study was the time to local tumour progression. Data were examined by multiple regression analysis (Cox). Mitomycin C alone resulted in a median time to local tumour progression of 23 (95% confidence limits: 17–43) days, fractionated irradiation in 31 (25–35) days and combined Mitomycin C plus fractionated irradiation in 65 (58–73) days (P=0.02). Mitomycin C decreased the relative risk of local recurrence by 94% (P<<0.001) equivalent to 31.7 Gy top up dose. Repopulation accounted for 1.33 (0.95–1.72) Gy per day top up dose after fractionated irradiation alone and for 0.68 (0.13–1.22) Gy per day after fractionated irradiation+Mitomycin C (P=0.018). Mitomycin C significantly reduces the risk of local recurrence and inhibits tumour cell repopulation in combination with fractionated irradiation in vivo in the tested tumour model. British Journal of Cancer (2002) 86, 470–476. DOI: 10.1038/sj/bjc/6600081 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11875717

Recellularization of Rat Liver Scaffolds by Human Liver Stem Cells

PubMed Central

Navarro-Tableros, Victor; Herrera Sanchez, Maria Beatriz; Figliolini, Federico; Romagnoli, Renato; Tetta, Ciro

2015-01-01

In the present study, rat liver acellular scaffolds were used as biological support to guide the differentiation of human liver stem-like cells (HLSC) to hepatocytes. Once recellularized, the scaffolds were maintained for 21 days in different culture conditions to evaluate hepatocyte differentiation. HLSC lost the embryonic markers (alpha-fetoprotein, nestin, nanog, sox2, Musashi1, Oct 3/4, and pax2), increased the expression of albumin, and acquired the expression of lactate dehydrogenase and three subtypes of cytochrome P450. The presence of urea nitrogen in the culture medium confirmed their metabolic activity. In addition, cells attached to tubular remnant matrix structures expressed cytokeratin 19, CD31, and vimentin. The rat extracellular matrix (ECM) provides not only a favorable environment for differentiation of HLSC in functional hepatocytes (hepatocyte like) but also promoted the generation of some epithelial-like and endothelial-like cells. When fibroblast growth factor–epidermal growth factor or HLSC-derived conditioned medium was added to the perfusate, an improvement of survival rate was observed. The conditioned medium from HLSC potentiated also the metabolic activity of hepatocyte-like cells repopulating the acellular liver. In conclusion, HLSC have the potential, in association with the natural ECM, to generate in vitro a functional “humanized liver-like tissue.” PMID:25794768

Mammary stem cells have myoepithelial cell properties

PubMed Central

Prater, Michael D.; Petit, Valérie; Russell, I. Alasdair; Giraddi, Rajshekhar; Shehata, Mona; Menon, Suraj; Schulte, Reiner; Kalajzic, Ivo; Rath, Nicola; Olson, Michael F.; Metzger, Daniel; Faraldo, Marisa M.; Deugnier, Marie-Ange; Glukhova, Marina A.; Stingl, John

2014-01-01

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt acin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using 2 independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage tracing approach we follow the progeny of α-smooth muscle actin-expressing myoepithelial cells and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy. PMID:25173976

Increased Engraftment of Human Short Term Repopulating Hematopoietic Cells in NOD/SCID/IL2rγnull Mice by Lentiviral Expression of NUP98-HOXA10HD

PubMed Central

Zhao, Huifen; Humphries, Keith; Persons, Derek A.

2016-01-01

Techniques to expand human hematopoietic stem cells ex-vivo could be beneficial to the fields of clinical hematopoietic stem cell transplantation and gene therapy targeted at hematopoietic stem cells. NUP98-HOXA10HD is a relatively newly discovered fusion gene that in mouse transplant experiments has been shown to increase numbers of hematopoietic stem cells. We evaluated whether this fusion gene could be used to expand engrafting human primitive CD34+ cells in an immunodeficient mouse model. Gene transfer was achieved using a lentiviral based vector. The engraftment of mobilized peripheral blood human CD34+ cells grown in culture for one week after gene transfer was evaluated 3–4 months after transplant and found to be 2–3 fold higher in the NUP98-HOXA10HD groups as compared to controls. These data suggest an expansive effect at least at the short term human repopulating cell level. Further evaluation in long term repopulating models and investment in a NUP98-HOXA10HD protein seems worthy of consideration. Additionally, the results here provide strong impetus to utilize NUP98-HOXA10HD as a tool to search for underlying genes and pathways involved in hematopoietic stem cell expansion that can be enhanced and have an even more potent expansive effect. PMID:26761813

Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived chemotherapeutic microparticles

PubMed Central

Ma, Jingwei; Zhang, Yi; Tang, Ke; Zhang, Huafeng; Yin, Xiaonan; Li, Yong; Xu, Pingwei; Sun, Yanling; Ma, Ruihua; Ji, Tiantian; Chen, Junwei; Zhang, Shuang; Zhang, Tianzhen; Luo, Shunqun; Jin, Yang; Luo, Xiuli; Li, Chengyin; Gong, Hongwei; Long, Zhixiong; Lu, Jinzhi; Hu, Zhuowei; Cao, Xuetao; Wang, Ning; Yang, Xiangliang; Huang, Bo

2016-01-01

Developing novel approaches to reverse the drug resistance of tumor-repopulating cells (TRCs) or stem cell-like cancer cells is an urgent clinical need to improve outcomes of cancer patients. Here we show an innovative approach that reverses drug resistance of TRCs using tumor cell-derived microparticles (T-MPs) containing anti-tumor drugs. TRCs, by virtue of being more deformable than differentiated cancer cells, preferentially take up T-MPs that release anti-tumor drugs after entering cells, which in turn lead to death of TRCs. The underlying mechanisms include interfering with drug efflux and promoting nuclear entry of the drugs. Our findings demonstrate the importance of tumor cell softness in uptake of T-MPs and effectiveness of a novel approach in reversing drug resistance of TRCs with promising clinical applications. PMID:27167569

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates.

PubMed

Engram, Jessica C; Cervasi, Barbara; Borghans, Jose A M; Klatt, Nichole R; Gordon, Shari N; Chahroudi, Ann; Else, James G; Mittler, Robert S; Sodora, Donald L; de Boer, Rob J; Brenchley, Jason M; Silvestri, Guido; Paiardini, Mirko

2010-08-05

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4(+) or CD8(+) lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4(+) or CD8(+) T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4(+) and CD8(+) lymphocyte depletions were followed by a largely lineage-specific CD4(+) and CD8(+) T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4(+) T cells than RMs. In addition, in both species CD8(+) T-cell repopulation was faster than that of CD4(+) T cells, with CD8(+) T cells reconstituting a normal pool within 60 days and CD4(+) T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4(+) T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4(+) T-cell destruction is chronic.

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates

PubMed Central

Engram, Jessica C.; Cervasi, Barbara; Borghans, Jose A. M.; Klatt, Nichole R.; Gordon, Shari N.; Chahroudi, Ann; Else, James G.; Mittler, Robert S.; Sodora, Donald L.; de Boer, Rob J.; Brenchley, Jason M.; Silvestri, Guido

2010-01-01

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4+ or CD8+ lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4+ or CD8+ T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4+ and CD8+ lymphocyte depletions were followed by a largely lineage-specific CD4+ and CD8+ T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4+ T cells than RMs. In addition, in both species CD8+ T-cell repopulation was faster than that of CD4+ T cells, with CD8+ T cells reconstituting a normal pool within 60 days and CD4+ T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4+ T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4+ T-cell destruction is chronic. PMID:20484087

Second Cancers After Fractionated Radiotherapy: Stochastic Population Dynamics Effects

NASA Technical Reports Server (NTRS)

Sachs, Rainer K.; Shuryak, Igor; Brenner, David; Fakir, Hatim; Hahnfeldt, Philip

2007-01-01

When ionizing radiation is used in cancer therapy it can induce second cancers in nearby organs. Mainly due to longer patient survival times, these second cancers have become of increasing concern. Estimating the risk of solid second cancers involves modeling: because of long latency times, available data is usually for older, obsolescent treatment regimens. Moreover, modeling second cancers gives unique insights into human carcinogenesis, since the therapy involves administering well characterized doses of a well studied carcinogen, followed by long-term monitoring. In addition to putative radiation initiation that produces pre-malignant cells, inactivation (i.e. cell killing), and subsequent cell repopulation by proliferation can be important at the doses relevant to second cancer situations. A recent initiation/inactivation/proliferation (IIP) model characterized quantitatively the observed occurrence of second breast and lung cancers, using a deterministic cell population dynamics approach. To analyze ifradiation-initiated pre-malignant clones become extinct before full repopulation can occur, we here give a stochastic version of this I I model. Combining Monte Carlo simulations with standard solutions for time-inhomogeneous birth-death equations, we show that repeated cycles of inactivation and repopulation, as occur during fractionated radiation therapy, can lead to distributions of pre-malignant cells per patient with variance >> mean, even when pre-malignant clones are Poisson-distributed. Thus fewer patients would be affected, but with a higher probability, than a deterministic model, tracking average pre-malignant cell numbers, would predict. Our results are applied to data on breast cancers after radiotherapy for Hodgkin disease. The stochastic IIP analysis, unlike the deterministic one, indicates: a) initiated, pre-malignant cells can have a growth advantage during repopulation, not just during the longer tumor latency period that follows; b) weekend treatment gaps during radiotherapy, apart from decreasing the probability of eradicating the primary cancer, substantially increase the risk of later second cancers.

Large Animal Models for Foamy Virus Vector Gene Therapy

PubMed Central

Trobridge, Grant D.; Horn, Peter A.; Beard, Brian C.; Kiem, Hans-Peter

2012-01-01

Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit. PMID:23223198

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells

PubMed Central

Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson; Chute, John P

2013-01-01

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC counts in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34+CDCD38−Lin− cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration PMID:20305662

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.

PubMed

Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson J; Chute, John P

2010-04-01

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC numbers in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34(+)CDCD38(-)Lin(-) cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration.

The In Vivo PDGF Response During Remyelination in Mouse Spinal Cord Following Murine Hepatitis Virus Strain AS9-Induced Transient Demyelination

DTIC Science & Technology

1998-09-14

repopulation. These and other growth factors interacting with cell adhesion molecules andlor matrix molecules would be expected to mediate oligodendrocyte...oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system. J...repopulate and remyelinate demyelinated lesions. In vitro studies have shown that platelet- derived growth factor (PDGF) induces proliferation

Tumor-Repopulating Cells Induce PD-1 Expression in CD8+ T Cells by Transferring Kynurenine and AhR Activation.

PubMed

Liu, Yuying; Liang, Xiaoyu; Dong, Wenqian; Fang, Yi; Lv, Jiadi; Zhang, Tianzhen; Fiskesund, Roland; Xie, Jing; Liu, Jinyan; Yin, Xiaonan; Jin, Xun; Chen, Degao; Tang, Ke; Ma, Jingwei; Zhang, Huafeng; Yu, Jing; Yan, Jun; Liang, Huaping; Mo, Siqi; Cheng, Feiran; Zhou, Yabo; Zhang, Haizeng; Wang, Jing; Li, Jingnan; Chen, Yang; Cui, Bing; Hu, Zhuo-Wei; Cao, Xuetao; Xiao-Feng Qin, F; Huang, Bo

2018-03-12

Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating T cells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8 + T cells through a transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) pathway. Interferon-γ produced by CD8 + T cells stimulates release of high levels of Kyn produced by TRCs, which is transferred into adjacent CD8 + T cells via the transporters SLC7A8 and PAT4. Kyn induces and activates AhR and thereby upregulates PD-1 expression. This Kyn-AhR pathway is confirmed in both tumor-bearing mice and cancer patients and its blockade enhances antitumor adoptive T cell therapy efficacy. Thus, we uncovered a mechanism of PD-1 upregulation with potential tumor immunotherapeutic applications. Copyright © 2018 Elsevier Inc. All rights reserved.

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

PubMed

Yoon, D S; Kim, Y H; Jung, H S; Paik, S; Lee, J W

2011-10-01

This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2. © 2011 Blackwell Publishing Ltd.

HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation.

PubMed

Antonchuk, J; Sauvageau, G; Humphries, R K

2001-09-01

Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, we have shown that overexpression of HOXB4 in mouse bone marrow can greatly enhance the level of hematopoietic stem cell (HSC) regeneration achieved at late times (> 4 months) posttransplantation. The objective of this study was to resolve if HOXB4 increases the rate and/or duration of HSC regeneration, and also to see if this enhancement was associated with impaired production of end cells or would lead to competitive reconstitution of all compartments. Retroviral vectors were generated with the GFP reporter gene +/- HOXB4 to enable the isolation and direct tracking of transduced cells in culture or following transplantation. Stem cell recovery was measured by limit dilution assay for long-term competitive repopulating cells (CRU). HOXB4-overexpressing cells have enhanced growth in vitro, as demonstrated by their rapid dominance in mixed cultures and their shortened population doubling time. Furthermore, HOXB4-transduced cells have a marked competitive repopulating advantage in vivo in both primitive and mature compartments. CRU recovery in HOXB4 recipients was extremely rapid, reaching 25% of normal by 14 days posttransplant or some 80-fold greater than control transplant recipients, and attaining normal numbers by 12 weeks. Mice transplanted with even higher numbers of HOXB4-transduced CRU regenerated up to but not beyond the normal CRU levels. HOXB4 is a potent enhancer of primitive hematopoietic cell growth, likely by increasing self-renewal probability but without impairing homeostatic control of HSC population size or the rate of production and maintenance of mature end cells.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in CD34+CD133+CXCR4low fraction.

PubMed

Lapostolle, Véronique; Chevaleyre, Jean; Duchez, Pascale; Rodriguez, Laura; Vlaski-Lafarge, Marija; Sandvig, Ioanna; Brunet de la Grange, Philippe; Ivanovic, Zoran

2018-06-01

Feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, CD34, CD133, CD90, CD45RA, CD26 and CD9 expression was determined on sorted CD34+ cells according to CXCR4 (neg, low, bright) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained in only the CD133+CXCR4low cells. The failure of CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood ones, as well as to those issued from the bone marrow. This data represent the first phenotypic characterization of steady-state blood cells exhibiting short and long term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation. Copyright © 2018, Ferrata Storti Foundation.

The Culture-Repopulating Ability Assays and Incubation in Low Oxygen: A Simple Way to Test Drugs on Leukaemia Stem or Progenitor Cells

PubMed Central

Cipolleschi, Maria Grazia; Rovida, Elisabetta; Sbarba, Persio Dello

2013-01-01

The Culture-Repopulating Ability (CRA) assays is a method to measure in vitro the bone marrow-repopulating potential of haematopoietic cells. The method was developed in our laboratory in the course of studies based on the use of growth factor-supplemented liquid cultures to study haematopoietic stem/progenitor cell resistance to, and selection at, low oxygen tensions in the incubation atmosphere. These studies led us to put forward the first hypothesis of the existence in vivo of haematopoietic stem cell niches where oxygen tension is physiologically lower than in other bone marrow areas. The CRA assays and incubation in low oxygen were later adapted to the study of leukaemias. Stabilized leukaemia cell lines, ensuring genetically homogeneous cells and enhancing repeatability of results, were found nevertheless phenotypically heterogeneous, comprising cell subsets exhibiting functional phenotypes of stem or progenitor cells. These subsets can be assayed separately, provided an experimental system capable to select one from another (such as different criteria for incubation in low oxygen) is established. On this basis, a two-step procedure was designed, including a primary culture of leukaemia cells in low oxygen for different times, where drug treatment is applied, followed by the transfer of residual cell population (CRA assay) to a drug-free secondary culture incubated at standard oxygen tension, where the expansion of population is allowed. The CRA assays, applied to cell lines first and then to primary cells, represent a simple and relatively rapid, yet accurate and reliable, method for the pre-screening of drugs potentially active on leukaemias which in our opinion could be adopted systematically before they are tested in vivo. PMID:23394087

Vascularized bone transplant chimerism mediated by vascular endothelial growth factor.

PubMed

Willems, Wouter F; Larsen, Mikko; Friedrich, Patricia F; Bishop, Allen T

2015-01-01

Vascular endothelial growth factor (VEGF) induces angiogenesis and osteogenesis in bone allotransplants. We aim to determine whether bone remodeling in VEGF-treated bone allotransplants results from repopulation with circulation-derived autogenous cells or survival of allogenic transplant-derived cells. Vascularized femoral bone transplants were transplanted from female Dark Agouti rats (DA;RT1(a) ) to male Piebald Viral Glaxo (PVG;RT1(c) ). Arteriovenous bundle implantation and short-term immunosuppression were used to maintain cellular viability. VEGF was encapsulated in biodegradable microspheres and delivered intramedullary in the experimental group (n = 22). In the control group (n = 22), no VEGF was delivered. Rats were sacrificed at 4 or 18 weeks. Laser capture microdissection of bone remodeling areas was performed at the inner and outer cortex. Sex-mismatched genes were quantified with reverse transcription-polymerase chain reaction to determine the amount of male cells to total cells, defined as the relative expression ratio (rER). At 4 weeks, rER was significantly higher at the inner cortex in VEGF-treated transplants as compared to untreated transplants (0.622 ± 0.225 vs. 0.362 ± 0.081, P = 0.043). At 4 weeks, the outer cortex in the control group had a significantly higher rER (P = 0.038), whereas in the VEGF group, the inner cortex had a higher rER (P = 0.015). Over time, in the outer cortex the rER significantly increased to 0.634 ± 0.106 at 18 weeks in VEGF-treated rats (P = 0.049). At 18 weeks, the rER was >0.5 at all cortical areas in both groups. These in vivo findings suggest a chemotactic effect of intramedullary applied VEGF on recipient-derived bone and could imply that more rapid angiogenesis of vascularized allotransplants can be established with microencapsulated VEGF. © 2014 Wiley Periodicals, Inc.

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

PubMed Central

Desponts, Caroline; Hazen, Amy L.; Paraiso, Kim H. T.; Kerr, William G.

2006-01-01

The SH2 domain–containing inositol 5′-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP–/– mice. Despite expansion of the compartment, SHIP–/– HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP–/– stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing. PMID:16467196

[Anti-mouse CD122 antibody promotes the hematopoietic repopulating capacity of cord blood CD34⁺ cells in NOD/SCID mice].

PubMed

Sheng, Men-Yao; Shi, Hui; Xing, Wen; Wang, Wen-Jun; Si, Xiao-Hui; Bai, Jie; Yuan, Wei-Ping; Zhou, Yuan; Yang, Feng-Chun

2014-12-01

The study was aimed to investigate the effect of anti-mouse CD122 antibody on the hematopoietic repopulating capacity of cord blood CD34⁺ cells in a humanized murine model-non obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After sublethal irradiation with γ-ray, NOD/SCID mice were intraperitoneally injected with 200 µg mouse isotype control antibody or anti-mouse CD122 antibody. Human cord blood CD34⁺ cells or phosphate-buffered saline (PBS) were injected via the tail vein at 6-8 hours later. Cohort of the mice injected with anti-mice CD122 antibody or control antibody alone were sacrificed at different time point (at week 2, 3, and 4 weeks) after the injection, and the percentage of NK cells in the peripheral blood was analyzed by flow cytometry. To evaluate the effect of anti-mouse CD122 antibody on the repopulating capacity of cord blood CD34⁺ cells in the recipient mice, phenotype analysis was performed in the bone marrow at 6 and 8 weeks after the transplantation. The results showed that the proportion of NK cells in the peripheral blood were (4.6 ± 0.6)% and (5.7 ± 1.7)% at week 2 and 3 after anti-CD122 antibody injection respectively,which decreased by 60%, compared with the mice injected with isotype control antibody. After 6 and 8 weeks of cord blood CD34⁺ cell transplantation,the percentage of human CD45⁺ in the bone marrow of the recipient mice treated with anti-mice CD122 antibody was (63.0 ± 12.2)% and (53.2 ± 16.3)%,respectively,which were dramatically higher than that in the mice treated with isotype control antibody (7.7 ± 3.6)% and (6.1 ± 2.4)%. Moreover,at 8 weeks after transplantation,human CD34⁺ cells appeared significantly in the recipients treated with anti-CD122 antibody. It is concluded that the anti-mouse CD122 antibody enhances the hematopoietic repopulating capacity of cord blood CD34⁺ cells in the NOD/SCID mice through decreasing the proportion of NK cells.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

PubMed

Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

2011-05-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

SPONTANEOUS REPOPULATION OF β-CATENIN NULL LIVERS WITH β-CATENIN POSITIVE HEPATOCYTES AFTER CHRONIC MURINE LIVER INJURY

PubMed Central

Thompson, Michael D.; Wickline, Emily D.; Bowen, William B.; Lu, Amy; Singh, Sucha; Misse, Amalea; Monga, Satdarshan P. S.

2011-01-01

Prolonged exposure of mice to diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in hepatobiliary injury, atypical ductular proliferation, oval cell appearance and limited fibrosis. Previously, we reported that short-term ingestion of DDC diet by hepatocyte-specific β-catenin conditional knockout (KO) mice, led to fewer A6-positive oval cells than wild-type (WT) littermates. To examine the role of β-catenin in chronic hepatic injury and repair, we exposed WT and KO mice to DDC for 80 and 150 days. Paradoxically, long-term DDC exposure led to significantly more A6-positive cells indicating greater atypical ductular proliferation in KO, which coincided with increased fibrosis and cholestasis. Surprisingly, at 80 and 150 days in KO, we observed a significant amelioration of hepatocyte injury. This coincided with extensive repopulation of β-catenin null livers with β-catenin-positive hepatocytes at 150 days, which was preceded by appearance of β-catenin-positive hepatocyte clusters at 80 days and a few β-catenin-positive hepatocytes at earlier times. Intriguingly, occasional β-catenin-positive hepatocytes that were negative for progenitor markers were also observed at baseline in the KO livers suggesting spontaneous escape from cre-mediated recombination. These cells with hepatocyte morphology expressed mature hepatocyte markers but lacked markers of hepatic progenitors. The gradual repopulation of KO livers with β-catenin-positive hepatocytes occurred only following DDC injury and coincided with a progressive loss of hepatic cre-recombinase expression. A few β-catenin-positive cholangiocytes were observed albeit only after long-term DDC-exposure and trailed the appearance of β-catenin-positive hepatocytes. In conclusion, in a chronic liver injury model, β-catenin-positive hepatocytes exhibit growth and survival advantages and repopulate KO livers eventually limiting hepatic injury and dysfunction despite increased fibrosis and intrahepatic cholestasis. PMID:21721031

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

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

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

Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology;more » CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.« less

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

PubMed Central

Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

2014-01-01

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation. PMID:24853430

Chemotherapeutic tumor microparticles combining low-dose irradiation reprogram tumor-promoting macrophages through a tumor-repopulating cell-curtailing pathway

PubMed Central

Sun, Yanling; Zheng, Zu'an; Zhang, Huafeng; Yu, Yuandong; Ma, Jingwei; Tang, Ke; Xu, Pingwei; Ji, Tiantian; Liang, Xiaoyu; Chen, Degao; Jin, Xun; Zhang, Tianzhen; Long, Zhixiong; Liu, Yuying; Huang, Bo

2017-01-01

ABSTRACT Stem cell-like tumor-repopulating cells (TRCs) have a critical role in establishing a tumor immunosuppressive microenvironment. However, means to enhance antitumor immunity by disrupting TRCs are absent. Our previous studies have shown that tumor cell-derived microparticles (T-MPs) preferentially abrogate TRCs by delivering antitumor drugs into nuclei of TRCs. Here, we show that low dose irradiation (LDI) enhances the effect of cisplatin-packaging T-MPs (Cis-MPs) on TRCs, leading to inhibiting tumor growth in different tumor models. This antitumor effect is not due to the direct killing of tumor cells but is T cell-dependent and relies on macrophages for their efficacy. The underlying mechanism is involved in therapeutic reprograming macrophages from tumor-promotion to tumor-inhibition by disrupting TRCs and curtailing their vicious education on macrophages. These findings provide a novel strategy to reset macrophage polarization and confer their function more like M1 than M2 types with highly promising potential clinical applications. PMID:28680743

Optimizing autologous cell grafts to improve stem cell gene therapy.

PubMed

Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia

2016-07-01

Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function.

PubMed

Romero-Moya, Damia; Bueno, Clara; Montes, Rosa; Navarro-Montero, Oscar; Iborra, Francisco J; López, Luis Carlos; Martin, Miguel; Menendez, Pablo

2013-07-01

The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34(+) hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34(+) cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34(+) cells with high (CD34(+) Mito(High)) versus low (CD34(+) Mito(Low)) mitochondrial mass. The CD34(+) Mito(Low) fraction contained 6-fold more CD34(+)CD38(-) primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno-deficient mice. In contrast, the CD34(+) Mito(High) fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34(+) Mito(Low) cells was significantly delayed as compared to that of CD34(+) Mito(High) cells. The eventual complete differentiation of CD34(+) Mito(Low) cells, which coincided with a robust expansion of the CD34(-) differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34(+) cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell differentiation and proliferation of lineage-committed CD34(-) cells.

Wnt5a Regulates Hematopoietic Stem Cell Proliferation and Repopulation Through the Ryk Receptor

PubMed Central

Povinelli, Benjamin J.; Nemeth, Michael J.

2017-01-01

Proper regulation of the balance between hematopoietic stem cell (HSC) proliferation, self-renewal, and differentiation is necessary to maintain hematopoiesis throughout life. The Wnt family of ligands has been implicated as critical regulators of these processes through a network of signaling pathways. Previously, we have demonstrated that the Wnt5a ligand can induce HSC quiescence through a noncanonical Wnt pathway, resulting in an increased ability to reconstitute hematopoiesis. In this study, we tested the hypothesis that the Ryk protein, a Wnt ligand receptor that can bind the Wnt5a ligand, regulated the response of HSCs to Wnt5a. We observed that inhibiting Ryk blocked the ability of Wnt5a to induce HSC quiescence and enhance short-term and long-term hematopoietic repopulation. We found that Wnt5a suppressed production of reactive oxygen species, a known inducer of HSC proliferation. The ability of Wnt5a to inhibit ROS production was also regulated by Ryk. From these data, we propose that Wnt5a regulates HSC quiescence and hematopoietic repopulation through the Ryk receptor and that this process is mediated by suppression of reactive oxygen species. PMID:23939973

Wnt5a regulates hematopoietic stem cell proliferation and repopulation through the Ryk receptor.

PubMed

Povinelli, Benjamin J; Nemeth, Michael J

2014-01-01

Proper regulation of the balance between hematopoietic stem cell (HSC) proliferation, self-renewal, and differentiation is necessary to maintain hematopoiesis throughout life. The Wnt family of ligands has been implicated as critical regulators of these processes through a network of signaling pathways. Previously, we have demonstrated that the Wnt5a ligand can induce HSC quiescence through a noncanonical Wnt pathway, resulting in an increased ability to reconstitute hematopoiesis. In this study, we tested the hypothesis that the Ryk protein, a Wnt ligand receptor that can bind the Wnt5a ligand, regulated the response of HSCs to Wnt5a. We observed that inhibiting Ryk blocked the ability of Wnt5a to induce HSC quiescence and enhance short-term and long-term hematopoietic repopulation. We found that Wnt5a suppressed production of reactive oxygen species, a known inducer of HSC proliferation. The ability of Wnt5a to inhibit ROS production was also regulated by Ryk. From these data, we propose that Wnt5a regulates HSC quiescence and hematopoietic repopulation through the Ryk receptor and that this process is mediated by suppression of reactive oxygen species. © 2013 AlphaMed Press.

Biology of high single doses of IORT: RBE, 5 R's, and other biological aspects.

PubMed

Herskind, Carsten; Ma, Lin; Liu, Qi; Zhang, Bo; Schneider, Frank; Veldwijk, Marlon R; Wenz, Frederik

2017-01-19

Intraoperative radiotherapy differs from conventional, fractionated radiotherapy in several aspects that may influence its biological effect. The radiation quality influences the relative biologic effectiveness (RBE), and the role of the five R's of radiotherapy (reassortment, repair, reoxygenation, repopulation, radiosensitivity) is different. Furthermore, putative special biological effects and the small volume receiving a high single dose may be important. The present review focuses on RBE, repair, and repopulation, and gives an overview of the other factors that potentially contribute to the efficacy. The increased RBE should be taken into account for low-energy X-rays while evidence of RBE < 1 for high-energy electrons at higher doses is presented. Various evidence supports a hypothesis that saturation of the primary DNA double-strand break (DSB) repair mechanisms leads to increasing use of an error-prone backup repair system leading to genomic instability that may contribute to inactivate tumour cells at high single doses. Furthermore, the elimination of repopulation of residual tumour cells in the tumour bed implies that some patients are likely to have very few residual tumour cells which may be cured even by low doses to the tumour bed. The highly localised dose distribution of IORT has the potential to inactivate tumour cells while sparing normal tissue by minimising the volume exposed to high doses. Whether special effects of high single doses also contribute to the efficacy will require further experimental and clinical studies.

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow.

PubMed

Suryawanshi, Gajendra W; Xu, Song; Xie, Yiming; Chou, Tom; Kim, Namshin; Chen, Irvin S Y; Kim, Sanggu

2017-06-14

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis.

PubMed

Yang, Mo; Li, Karen; Ng, Pak Cheung; Chuen, Carmen Ka Yee; Lau, Tze Kin; Cheng, Yuan Shan; Liu, Yuan Sheng; Li, Chi Kong; Yuen, Patrick Man Pan; James, Anthony Edward; Lee, Shuk Man; Fok, Tai Fai

2007-07-01

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.

Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps.

PubMed

Zhang, Qixu; Johnson, Joshua A; Dunne, Lina W; Chen, Youbai; Iyyanki, Tejaswi; Wu, Yewen; Chang, Edward I; Branch-Brooks, Cynthia D; Robb, Geoffrey L; Butler, Charles E

2016-04-15

Using a perfusion decellularization protocol, we developed a decellularized skin/adipose tissue flap (DSAF) comprising extracellular matrix (ECM) and intact vasculature. Our DSAF had a dominant vascular pedicle, microcirculatory vascularity, and a sensory nerve network and retained three-dimensional (3D) nanofibrous structures well. DSAF, which was composed of collagen and laminin with well-preserved growth factors (e.g., vascular endothelial growth factor, basic fibroblast growth factor), was successfully repopulated with human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs), which integrated with DSAF and formed 3D aggregates and vessel-like structures in vitro. We used microsurgery techniques to re-anastomose the recellularized DSAF into nude rats. In vivo, the engineered flap construct underwent neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant adipose tissue formation at 3months postoperatively. Our results indicate that DSAF co-cultured with hASCs and HUVECs is a promising platform for vascularized soft tissue flap engineering. This platform is not limited by the flap size, as the entire construct can be immediately perfused by the recellularized vascular network following simple re-integration into the host using conventional microsurgical techniques. Significant soft tissue loss resulting from traumatic injury or tumor resection often requires surgical reconstruction using autologous soft tissue flaps. However, the limited availability of qualitative autologous flaps as well as the donor site morbidity significantly limits this approach. Engineered soft tissue flap grafts may offer a clinically relevant alternative to the autologous flap tissue. In this study, we engineered vascularized soft tissue free flap by using skin/adipose flap extracellular matrix scaffold (DSAF) in combination with multiple types of human cells. Following vascular reanastomosis in the recipient site, the engineered products successful regenerated large-scale fat tissue in vivo. This approach may provide a translatable platform for composite soft tissue free flap engineering for microsurgical reconstruction. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

PubMed Central

Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta

2011-01-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264

Hepatic progenitor cells of biliary origin with liver repopulation capacity

PubMed Central

Boulter, Luke; Tsuchiya, Atsunori; Cole, Alicia M; Hay, Trevor; Guest, Rachel V; Wojtacha, Davina; Man, Tak Yung; Mackinnon, Alison; Ridgway, Rachel A; Kendall, Timothy; Williams, Michael J; Jamieson, Thomas; Raven, Alex; Hay, David C; Iredale, John P; Clarke, Alan R; Sansom, Owen J; Forbes, Stuart J

2015-01-01

Summary Hepatocytes and cholangiocytes self renew following liver injury. Following severe injury hepatocytes are increasingly senescent, whether Hepatic Progenitor Cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where Mdm2 is inducibly deleted in over 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease. PMID:26192438

In vitro investigation of head and neck cancer stem cell proportions and their changes following X-ray irradiation as a function of HPV status.

PubMed

Reid, Paul; Wilson, Puthenparampil; Li, Yanrui; Marcu, Loredana G; Staudacher, Alexander H; Brown, Michael P; Bezak, Eva

2017-01-01

Some head and neck squamous cell carcinomas (HNSCC) have a distinct aetiology, which depends on the presence of oncogenic human papilloma virus (HPV). Also, HNSCC contains cancer stem cells (CSCs) that have greater radioresistance and capacity to change replication dynamics in response to irradiation compared to non-clonogenic cells. Since there is limited data on CSCs in HNSCC as a function of HPV status, better understanding of their radiobiology may enable improved treatment outcome. Baseline and post-irradiation changes in CSC proportions were investigated by flow cytometry in a HPV-negative (UM-SCC-1) and a HPV-positive (UM-SCC-47) HNSCC cell line, using fluorescent staining with CD44/ALDH markers. CSC proportions in both irradiated and unirradiated cultures were compared for the two cell lines at various times post-irradiation. To assess repopulation of CSCs, untreated cultures were depleted of CD44+/ALDH+ cells and re-cultured for 3 weeks before flow cytometry analysis. CSC proportions in untreated cell lines were 0.57% (UM-SCC-1) and 2.87% (UM-SCC-47). Untreated cell lines depleted of CD44+/ALDH+ repopulated this phenotype to a mean of 0.15% (UM-SCC-1) and 6.76% (UM-SCC-47). All UM-SCC-47 generations showed elevated CSC proportions after irradiation, with the most significant increase at 2 days post-irradiation. The highest elevation in UM-SCC-1 CSCs was observed at 1 day post-irradiation in the 2nd generation and at 3 days after irradiation in the 3rd generation. When measured after 10 days, only the 3rd generation of UM-SCC-1 showed elevated CSCs. CSC proportions in both cell lines were elevated after exposure and varied with time post irradiation. UM-SCC-47 displayed significant plasticity in repopulating the CSC phenotype in depleted cultures, which was not seen in UM-SCC-1.

Transcatheter treatment of tricuspid regurgitation by caval valve implantation--experimental evaluation of decellularized tissue valves in central venous position.

PubMed

Lauten, Alexander; Laube, Adrian; Schubert, Harald; Bischoff, Sabine; Nietzsche, Sandor; Horstkötter, Kim; Poudel-Bochmann, Bhawana; Franz, Marcus; Lichtenberg, Artur; Figulla, Hans R; Akhyari, Payam

2015-01-01

Caval valve implantation has been suggested for transcatheter treatment of severe tricuspid regurgitation (TR). Combining the interventional technique with the promising surgical experience with decellularized valves, we sought to evaluate the functional and structural outcome of decellularized pericardial tissue valves (dTVs) in the low-pressure venous circulation in a chronic model of TR. Sixteen pericardial tissue valves were heterotopically implanted in the inferior and superior vena cava in a sheep model (54-98 kg; median 74.5 kg, n = 8) of severe TR. The devices were assembled using self-expanding nitinol stents and bovine pericardia decellularized by a detergent-based protocol (group dTV; n = 8). Glutaraldehyde-fixed pericardial tissue valves served as control (GaTV, n = 8). After 6 months, device function and structural maturation were analyzed using echocardiographic, histologic, immunohistologic, and electron microscopic approaches. After implantation, cardiac output increased significantly from 3.7 ± 1.1 l/min to 4.8 ± 1.1 l/min (P < 0.05) and competent valve function was verified by angiography. At 6 months, angiographic and echocardiographic evaluation revealed moderate to severe regurgitation in all GaTV. In contrast, five of the eight dTVs functioned well with only minor regurgitation. In these animals, autopsy revealed preserved valve structure with tender leaflets without signs of thrombosis or calcification. Conversely, GaTV showed severe degeneration with large calcification areas. Microscopic and histologic analysis confirmed endothelial repopulation in both valve types. However, additional interstitial reseeding was observed in decellularized valves. In the venous circulation in severe TR, decellularized valves show superior functional performance compared to Ga-fixed tissue valves. Macroscopic and microscopic analyses suggest preserved structural integrity and advanced endothelial and interstitial repopulation with evidence of less degradation in dTV. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Natural Killer Cell Immunotherapy Targeting Cancer Stem Cells

PubMed Central

Luna, Jesus I; Grossenbacher, Steven K.; Murphy, William J; Canter, Robert J

2017-01-01

Introduction Standard cytoreductive cancer therapy, such as chemotherapy and radiotherapy, are frequently resisted by a small portion of cancer cells with “stem-cell” like properties including quiescence and repopulation. Immunotherapy represents a breakthrough modality for improving oncologic outcomes in cancer patients. Since the success of immunotherapy is not contingent on target cell proliferation, it may also be uniquely suited to address the problem of resistance and repopulation exerted by cancer stem cells (CSCs). Areas covered Natural killer (NK) cells have long been known for their ability to reject allogeneic hematopoietic stem cells, and there are increasing data demonstrating that NK cells can selectively identify and lyse CSCs. In this report, we review the current knowledge of CSCs and NK cells and highlight recent studies that support the concept that NK cells are capable of targeting CSC in solid tumors, especially in the context of combination therapy simultaneously targeting non-CSCs and CSCs. Expert Opinion Unlike cytotoxic cancer treatments, NK cells are able to target and eliminate quiescent/non-proliferating cells such as CSCs, and these enigmatic cells are an important source of relapse and metastasis. NK targeting of CSCs represents a novel and potentially high impact method to capitalize on the intrinsic therapeutic potential of NK cells. PMID:27960589

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

Kleiner, J.B.; Amiel, D.; Harwood, F.L.

A rabbit model for anterior cruciate ligament (ACL) reconstruction using autogenous patellar tendon was utilized to study the early events of autograft cellular dynamics. Biochemical, autoradiographic, histological, and vascular injection techniques demonstrated that the native autograft cell population rapidly necroses. This repopulation occurs without a vascular contribution; cells entering the autograft are reliant upon synovial fluid nutrition.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

PubMed

Mao, Leilei; Li, Peiying; Zhu, Wen; Cai, Wei; Liu, Zongjian; Wang, Yanling; Luo, Wenli; Stetler, Ruth A; Leak, Rehana K; Yu, Weifeng; Gao, Yanqin; Chen, Jun; Chen, Gang; Hu, Xiaoming

2017-07-01

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic studies demonstrated that regulatory T cells completely abolished the tPA-induced elevation of MMP9 and CCL2 after stroke. Using MMP9 and CCL2 knockout mice, we discovered that both molecules partially contributed to the protective actions of regulatory T cells. In an in vitro endothelial cell-based model of the blood-brain barrier, we confirmed that regulatory T cells inhibited tPA-induced endothelial expression of CCL2 and preserved blood-brain barrier integrity after an ischaemic challenge. Lentivirus-mediated CCL2 knockdown in endothelial cells completely abolished the blood-brain barrier protective effect of regulatory T cells in vitro. Altogether, our studies suggest that regulatory T cell adoptive transfer may alleviate thrombolytic treatment-induced haemorrhage in stroke victims. Furthermore, regulatory T cell-afforded protection in the tPA-treated stroke model is mediated by two inhibitory mechanisms involving CCL2 and MMP9. Thus, regulatory T cell adoptive transfer may be useful as a cell-based therapy to improve the efficacy and safety of thrombolytic treatment for ischaemic stroke. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

PubMed

Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

2017-09-01

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP) - Oct4 + cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP + NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4 fl/fl ;Sox1 Cre (Oct4 CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP + dNSCs, in these Oct4 CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP + dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

Prostaglandin E2 enhances long-term repopulation but does not permanently alter inherent stem cell competitiveness.

PubMed

Hoggatt, Jonathan; Mohammad, Khalid S; Singh, Pratibha; Pelus, Louis M

2013-10-24

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for malignant and nonmalignant hematologic diseases and metabolic disorders. Although successful, hematopoietic transplantation can be hindered by inadequate stem cell number or poor engrafting efficiency. To overcome these deficits, we and others have previously reported the HSC-enhancing ability of a short-term exposure of prostaglandin E2 (PGE2); this strategy has now progressed to phase 1 clinical trials in double cord blood transplantation. To further analyze the short- and long-term effects of HSC exposure to PGE2, we followed the repopulation kinetics of PGE2-treated hematopoietic grafts through 5 serial transplantations and compared inherent long-term competitiveness in a HSC head-to-head secondary transplantation model. Treatment with PGE2 did not result in a long-term increase in HSC competitiveness, lineage bias, or enhanced proliferative potential, demonstrating that pulse exposure to PGE2 results in transient increases in HSC homing and engraftment potential.

HTR8/SVneo cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity, and expression of "stemness-" associated transcription factors.

PubMed

Weber, Maja; Knoefler, Ilka; Schleussner, Ekkehard; Markert, Udo R; Fitzgerald, Justine S

2013-01-01

JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Endothelial-regenerating cells: an expanding universe.

PubMed

Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

2010-03-01

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

PubMed

Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

2009-07-01

Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

Biological and physicochemical characterization of a serum- and xeno-free chemically defined cryopreservation procedure for adult human progenitor cells.

PubMed

Zeisberger, Steffen M; Schulz, Julia C; Mairhofer, Mario; Ponsaerts, Peter; Wouters, Guy; Doerr, Daniel; Katsen-Globa, Alisa; Ehrbar, Martin; Hescheler, Jurgen; Hoerstrup, Simon P; Zisch, Andreas H; Kolbus, Andrea; Zimmermann, Heiko

2011-01-01

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

PubMed Central

Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

2017-01-01

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

Repopulation of interacting tumor cells during fractionated radiotherapy: stochastic modeling of the tumor control probability.

PubMed

Fakir, Hatim; Hlatky, Lynn; Li, Huamin; Sachs, Rainer

2013-12-01

Optimal treatment planning for fractionated external beam radiation therapy requires inputs from radiobiology based on recent thinking about the "five Rs" (repopulation, radiosensitivity, reoxygenation, redistribution, and repair). The need is especially acute for the newer, often individualized, protocols made feasible by progress in image guided radiation therapy and dose conformity. Current stochastic tumor control probability (TCP) models incorporating tumor repopulation effects consider "stem-like cancer cells" (SLCC) to be independent, but the authors here propose that SLCC-SLCC interactions may be significant. The authors present a new stochastic TCP model for repopulating SLCC interacting within microenvironmental niches. Our approach is meant mainly for comparing similar protocols. It aims at practical generalizations of previous mathematical models. The authors consider protocols with complete sublethal damage repair between fractions. The authors use customized open-source software and recent mathematical approaches from stochastic process theory for calculating the time-dependent SLCC number and thereby estimating SLCC eradication probabilities. As specific numerical examples, the authors consider predicted TCP results for a 2 Gy per fraction, 60 Gy protocol compared to 64 Gy protocols involving early or late boosts in a limited volume to some fractions. In sample calculations with linear quadratic parameters α = 0.3 per Gy, α∕β = 10 Gy, boosting is predicted to raise TCP from a dismal 14.5% observed in some older protocols for advanced NSCLC to above 70%. This prediction is robust as regards: (a) the assumed values of parameters other than α and (b) the choice of models for intraniche SLCC-SLCC interactions. However, α = 0.03 per Gy leads to a prediction of almost no improvement when boosting. The predicted efficacy of moderate boosts depends sensitively on α. Presumably, the larger values of α are the ones appropriate for individualized treatment protocols, with the smaller values relevant only to protocols for a heterogeneous patient population. On that assumption, boosting is predicted to be highly effective. Front boosting, apart from practical advantages and a possible advantage as regards iatrogenic second cancers, also probably gives a slightly higher TCP than back boosting. If the total number of SLCC at the start of treatment can be measured even roughly, it will provide a highly sensitive way of discriminating between various models and parameter choices. Updated mathematical methods for calculating repopulation allow credible generalizations of earlier results.

Effective Hepatocyte Transplantation Using Rat Hepatocytes with Low Asialoglycoprotein Receptor Expression

PubMed Central

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-01-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 × 105 cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied ∼76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for ∼12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes. PMID:15277224

Effective hepatocyte transplantation using rat hepatocytes with low asialoglycoprotein receptor expression.

PubMed

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-08-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 x 10(5) cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied approximately 76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for approximately 12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes.

Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver

PubMed Central

Viswanathan, Preeti; Kapoor, Sorabh; Kumaran, Vinay; Joseph, Brigid; Gupta, Sanjeev

2014-01-01

Engraftment of transplanted cells is critical for liver-directed cell therapy but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells. To define whether TNF-α served roles in cell-transplantation-induced hepatic inflammation, we used TNF-α antagonist, etanercept, for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas etanercept prior to cell transplantation essentially normalized these responses. Moreover, ETN downregulated cell transplantation-induced intrahepatic release of secretory cytokines, such as high mobility group box 1. These effects of etanercept decreased cell transplantation-induced activation of neutrophils but not of Kupffer cells. Transplanted cell engraftment improved by several-fold in etanercept-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with etanercept before multiple cell transplantation sessions. Transplanted cell numbers did not change over time indicating absence of cell proliferation after etanercept alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after etanercept pretreatment significantly accelerated liver repopulation compared with control rats. We concluded that TNF-α played a major role in orchestrating cell transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. As TNF-α antagonism by etanercept decreased transplanted cell clearance, improved cell engraftment and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy. PMID:24844924

Therapeutic modulation of growth factors and cytokines in regenerative medicine.

PubMed

Ioannidou, Effie

2006-01-01

Regeneration that takes place in the human body is limited throughout life. Therefore, when organs are irreparably damaged, they are usually replaced with an artificial device or donor organ. The term "regenerative medicine" covers the restoration or replacement of cells, tissues, and organs. Stem cells play a major role in regenerative medicine by providing the way to repopulate organs damaged by disease. Stem cells have the ability to self renew and to regenerate cells of diverse lineages within the tissue in which they reside. Stem cells could originate from embryos or adult tissues. Growth factors are proteins that may act locally or systemically to affect the growth of cells in several ways. Various cell activities, including division, are influenced by growth factors. Cytokines are a family of low-molecular-weight proteins that are produced by numerous cell types and are responsible for regulating the immune response, inflammation, tissue remodeling and cellular differentiation. Target cells of growth factors and cytokines are mesenchymal, epithelial and endothelial cells. These molecules frequently have overlapping activities and can act in an autocrine or paracrine fashion. A complex network of growth factors and cytokines guides cellular differentiation and regeneration in all organs and tissues. The aim of this paper is to review the role of growth factors and cytokines in different organs or systems and explore their therapeutic application in regenerative medicine. The role of stem cells combined with growth factors and cytokines in the regeneration of vascular and hematopoietic, neural, skeletal, pancreatic, periodontal, and mucosal tissue is reviewed. There is evidence that supports the use of growth factors and cytokines in the treatment of neurological diseases, diabetes, cardiovascular disease, periodontal disease, cancer and its complication, oral mucositis. After solving the ethical issues and establishing clear and reasonable regulations, regenerative medicine through stem cell application combined with specific growth factors and cytokines will have great potential in curing a variety of human diseases.

Reduced Ang2 expression in aging endothelial cells

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

Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at; Ebenbauer, B.; Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of agingmore » before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.« less

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

PubMed

Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by inflammation triggered by monocyte adhesion and increased endothelial cell proliferation. These events are manifest in inflammatory diseases, such as atherosclerosis. Therefore, our results suggest that DBMSCs could be usefully employed as a therapeutic strategy for atherosclerosis.

Substrate effects on endothelial cell adherence rates.

PubMed

Scott, W J; Mann, P

1990-01-01

Endothelial cell attachment to a synthetic substrate was studied using an in vitro model system. Attachment rate was defined as the number of tritium-labeled endothelial cells attached to a synthetic substrate after 30 minutes. The surface of the synthetic substrate was chemically modified with either laminin or fibronectin. Labeled endothelial cells attached more rapidly to synthetic substrate, chemically modified with biomolecules, as compared with the untreated substrate controls. Unlabeled endothelial cells were grown to confluency on a second set of modified and untreated substrates. The cells were removed with 1% Triton, and the rate of re-endothelialization with tritium-labeled endothelial cells was determined. The rate was 11-13 times that of the same cells on untreated substrate. These data confirm that biomolecules increase the attachment rate of endothelial cells to synthetic substrate, and also suggest that endothelial cells may secrete a Triton-insoluble product (Sigma, St. Louis, MO) into subendothelial matrix that increases re-endothelialization.

An evaluation of image quality and accuracy of eye bank measurement of donor cornea endothelial cell density in the Specular Microscopy Ancillary Study.

PubMed

Lass, Jonathan H; Gal, Robin L; Ruedy, Katrina J; Benetz, Beth Ann; Beck, Roy W; Baratz, Keith H; Holland, Edward J; Kalajian, Andrea; Kollman, Craig; Manning, Francis J; Mannis, Mark J; McCoy, Kristen; Montoya, Monty; Stulting, Doyle; Xing, Dongyuan

2005-03-01

The Specular Microscopy Ancillary Study was designed to examine donor corneal endothelial specular image quality, compare the central endothelial cell density determined by eye banks with the endothelial cell density determined by a central specular microscopy reading center, and evaluate donor factors that may have an impact on specular image quality and endothelial cell density accuracy. Nonrandomized comparative trial. Endothelial specular images of donor corneas assigned in the Cornea Donor Study. Certified readers assessed donor image quality (analyzable from fair to excellent vs. unanalyzable) and determined the central endothelial cell density. Independent adjudication was performed if there was a difference in the quality of grading or if the endothelial cell density varied by > or =5.0% between readers. Average reading center-determined endothelial cell density was compared with the endothelial cell density determined by each eye bank. Evaluation of image quality and accuracy of endothelial cell density. Of 688 donor endothelial images submitted by 23 eye banks, 663 (96%) were analyzable (excellent, 40 [6%]; good, 302 [44%]; fair, 321 [47%]), and 25 (4%) were unanalyzable by reading center standards. In situ retrieval and greater epithelial exposure correlated with a higher image quality grading. The eye bank-determined endothelial cell density of 434 of the 663 (65%) analyzable images were within 10% of the endothelial cell density determined by the reading center, whereas 185 (28%) were more than 10% higher and 44 (7%) were more than 10% lower. Greater variation in endothelial cell density between the eye banks and the reading center was observed with shorter time of death to preservation, presence of an epithelial defect, folds in Descemet's membrane, lower image quality, and the use of fixed-frame or center method endothelial cell density analysis. Overall, donor endothelial specular image quality and accuracy of endothelial cell density determination were good. However, the data suggest that factors that may affect image quality and contribute to variation in interpretation of the endothelial cell density should be addressed, because the donor endothelial cell density is an important parameter for assessing long-term corneal graft survival.

VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering

PubMed Central

Nourse, Marilyn B.; Halpin, Daniel E.; Scatena, Marta; Mortisen, Derek J.; Tulloch, Nathaniel L.; Hauch, Kip D.; Torok-Storb, Beverly; Ratner, Buddy D.; Pabon, Lil; Murry, Charles E.

2010-01-01

Objective Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. Methods and Results To enhance endothelial cell differentiation above a baseline of ∼2% in embryoid body (EB) spontaneous differentiation, three alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10-14. Continuous VEGF treatment resulted in a four- to five-fold enrichment of CD31+ cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31+ cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFα, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. Conclusions VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. These enrichment methods increase endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants. PMID:19875721

Efficient extravasation of tumor-repopulating cells depends on cell deformability

PubMed Central

Chen, Junjian; Zhou, Wenwen; Jia, Qiong; Chen, Junwei; Zhang, Shuang; Yao, Wenting; Wei, Fuxiang; Zhang, Yuejin; Yang, Fang; Huang, Wei; Zhang, Yao; Zhang, Huafeng; Zhang, Yi; Huang, Bo; Zhang, Zhihong; Jia, Haibo; Wang, Ning

2016-01-01

Cancer metastasis is the most deadly stage in cancer progression. Despite significant efforts over the past decades, it remains elusive why only a very small fraction of cancer cells is able to generate micrometastasis and metastatic colonization. Recently we have shown that tumor-repopulating cells (TRCs), a highly tumorigenic subpopulation of mouse melanoma cells, can be selected by being cultured and grown in 3D soft fibrin gels. Here we show that when injected into the yolk of a 2 day-post-fertilization (dpf) embryo of Tg (fli1:EGFP or kdrl:mCherry) zebrafish, TRCs are much more efficient in surviving and growing at various secondary sites to generate micrometastasis and metastatic colonization than control melanoma cells that are grown on rigid plastic. The metastasis of TRCs is dependent on the presence of Sox2, a self-renewal gene, and silencing Sox2 leads to the inhibition of TRC metastasis. High-resolution of 3D confocal images of the TRCs at the secondary sites show that extravasation and formation of micrometastases by TRCs are more efficient than by the control cells. Remarkably, efficient extravasation of TRCs in vivo and transmigration in vitro are determined by TRC deformability, as a result of low Cdc42 and high Sox2. Our findings suggest that tumor cell deformability is a key factor in controlling extravasation dynamics during metastasis. PMID:26787224

Cancer Cells Regulate Biomechanical Properties of Human Microvascular Endothelial Cells*

PubMed Central

Mierke, Claudia Tanja

2011-01-01

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1high cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs. PMID:21940631

Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

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

Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi

2015-07-03

Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed,more » because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.« less

Isolation and Characterization of Rat Pituitary Endothelial Cells

PubMed Central

Chaturvedi, Kirti; Sarkar, Dipak K.

2010-01-01

Most previous studies that determined the effect of estradiol on angiogenesis used endothelial cells from nonpituitary sources. Because pituitary tumor tissue receives its blood supply via portal and arterial circulation, it is important to use pituitary-derived endothelial cells in studying pituitary angiogenesis. We have developed a magnetic separation technique to isolate endothelial cells from pituitary tissues and have characterized these cells in primary cultures. Endothelial cells of the pituitary showed the existence of endothelial cell marker, CD31, and of von Willebrand factor protein. These cells in cultures also showed immunore-activity of estrogen receptors alpha and beta. The angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, significantly increased proliferation and migration of the pituitary-derived endothelial cells in primary cultures. These results suggest that a magnetic separation technique can be used for enrichment of pituitary-derived endothelial cells for determination of cellular mechanisms governing the vascularization in the pituitary. PMID:17028416

Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

PubMed Central

Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

2015-01-01

ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

Activation of PPARbeta/delta induces endothelial cell proliferation and angiogenesis.

PubMed

Piqueras, Laura; Reynolds, Andrew R; Hodivala-Dilke, Kairbaan M; Alfranca, Arántzazu; Redondo, Juan M; Hatae, Toshihisa; Tanabe, Tadashi; Warner, Timothy D; Bishop-Bailey, David

2007-01-01

The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-beta/delta in endothelial cells remains unclear. Interestingly, the selective PPARbeta/delta ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPARbeta/delta in endothelial cell proliferation and angiogenesis. Western blot analysis indicated PPARbeta/delta was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPARbeta/delta target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPARbeta/delta. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation. PPARbeta/delta is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

PubMed

Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

2012-01-01

Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Signaling hierarchy regulating human endothelial cell development.

PubMed

Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

PubMed

Ratajczak, Philippe; Leboeuf, Christophe; Wang, Li; Brière, Josette; Loisel-Ferreira, Irmine; Thiéblemont, Catherine; Zhao, Wei-Li; Janin, Anne

2012-06-01

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Spontaneous circulation of myeloid-lymphoid-initiating cells and SCID-repopulating cells in sickle cell crisis.

PubMed

Lamming, Christopher E D; Augustin, Lance; Blackstad, Mark; Lund, Troy C; Hebbel, Robert P; Verfaillie, Catherine M

2003-03-01

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid-initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture-initiating cells, consistent with the notion that SRCs are more primitive than long-term culture-initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation.

Rapid isolation of choriocapillary endothelial cells by Lycopersicon esculentum-coated Dynabeads.

PubMed

Hoffmann, S; Spee, C; Murata, T; Cui, J Z; Ryan, S J; Hinton, D R

1998-10-01

In vitro studies of choroidal endothelial cells may be critical for understanding the pathogenesis of neovascularization in age-related macular degeneration, since endothelial cells from different sites are highly heterogeneous in their morphology and behavior. Isolation of choroidal endothelial cells is complicated and labor intensive because of the small size of the choroid and the difficulty of excluding contaminating cells. We describe a rapid, simplified method for the isolation of bovine choroidal endothelial cells using microdissection followed by the use of superparamagnetic beads (Dynabeads) coated with the endothelial cell-specific lectin Lycopersicon esculentum, which selectively binds to fucose residues on the endothelial cell surface. Cells bound to beads are isolated using a magnetic particle concentrator. Isolated cells grew to confluence in a monolayer with a cobblestone morphology and were shown to be endothelial cells by their greater than 95% immunoreactivity to von Willebrand factor and phagocytosis of dil-acetylated LDL. Isolated cells grew as tubes in three-dimensional cultures. This method markedly reduces the time needed for pure culture of cells and makes the in vitro study of choroidal endothelial cells practical and reproducible.

Reduced survival in patients with early-stage non-small-cell lung cancer is associated with high pleural endothelial progenitor cell levels.

PubMed

Pirro, Matteo; Cagini, Lucio; Mannarino, Massimo R; Andolfi, Marco; Potenza, Rossella; Paciullo, Francesco; Bianconi, Vanessa; Frangione, Maria Rosaria; Bagaglia, Francesco; Puma, Francesco; Mannarino, Elmo

2016-12-01

Endothelial progenitor cells are capable of contributing to neovascularization in tumours. In patients with either malignant or transudative pleural effusion, we tested the presence of pleural endothelial progenitor cells. We also measured the number of endothelial progenitor cells in post-surgery pleural drainage of either patients with early non-small-cell lung cancer or control patients with benign lung disease undergoing pulmonary resection. The prospective influence of post-surgery pleural-drainage endothelial progenitor cells on cancer recurrence/survival was investigated. Pleural endothelial progenitor cell levels were quantified by fluorescence-activated cell sorting analysis in pleural effusion of 15 patients with late-stage non-small-cell lung cancer with pleural involvement and in 15 control patients with congestive heart failure. Also, pleural-drainage endothelial progenitor cells were measured in pleural-drainage fluid 48 h after surgery in 64 patients with early-stage non-small-cell lung cancer and 20 benign lung disease patients undergoing pulmonary resection. Cancer recurrence and survival was evaluated in patients with high pleural-drainage endothelial progenitor cell levels. The number of pleural endothelial progenitor cells was higher in non-small-cell lung cancer pleural effusion than in transudative pleural effusion. Also, pleural-drainage endothelial progenitor cell levels were higher in patients with non-small-cell lung cancer than in patients with benign lung disease undergoing pulmonary resection (P < 0.05). Non-small-cell lung cancer patients with high pleural-drainage endothelial progenitor cell levels had a significantly 4.9 higher rate of cancer recurrence/death than patients with lower pleural-drainage endothelial progenitor cell levels, irrespective of confounders. Endothelial progenitor cells are present in the pleural effusion and are higher in patients with late-stage non-small-cell lung cancer with pleural involvement than in congestive heart failure patients. Endothelial progenitor cell levels are higher in the post-surgery pleural drainage of patients with non-small-cell lung cancer than in non-neoplastic pleural-drainage fluid. High pleural-drainage endothelial progenitor cell levels in patients undergoing pulmonary resection for early non-small-cell lung cancer predict an increased risk of cancer recurrence and death. © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Heme oxygenase-1 protects INF-gamma primed endothelial cells from Jurkat T-cell adhesion.

PubMed

Du, D; Chang, S; Chen, B; Zhou, H; Chen, Z K

2007-12-01

The heme oxygenase-1 (HO-1) system is associated with the rate-limiting step of conversion of heme, one of the most critical roles in cytoprotective mechanisms. Our study investigated its potential role in protection of endothelial cells from T cells. The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells. Indirect fluorescent staining was used to examine the expression of HO-1 protein. Then endothelial cells primed by INF-gamma were mixed in culture with Jurkat T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The number of adhesive Jurkat T cells was determined using FACS to evaluate the adhesion effect. After being cultured with endothelial cells, the cell cycle of Jurkat T cells was detected using FACS. Expression of HO-1 on endothelial cells conferred significant protection against Jurkat T-cell-mediated adhesion. The rate of Jurkat T-cell adhesions was reduced to 19.06%, in contrast with 31.42% in the control group (P<.05). After using ZnPP, an inhibitor of HO-1, the rate of Jurkat T-cell adhesion recovered to 29.08%. The binding activities between endothelial cells and Jurkat T cells was blocked by HO-1 expression. The proliferation of Jurkat T cells was inhibited after culture with endothelial cells, which had been transfected with HO-1, which blocked cell cycle entry of T cells. More than 60% of Jurkat T cells remained in G0/G1 compared with 40% among the control group. HO-1 directly protected endothelial cells primed by INF-gamma from Jurkat T cells and down-regulated the expression of HLA-DR on the surface of endothelial cells. These results indicated that transgenic expression of HO-1 may be useful to prevent lymphocytes from responding to endothelial cells.

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

PubMed

Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-11-01

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target.

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos

2012-08-01

Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

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

Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.

1987-05-01

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cellsmore » (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.« less

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

PubMed

Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

2018-04-17

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection

PubMed Central

Zhang, Zhi-Qiang; Notermans, Daan W.; Sedgewick, Gerald; Cavert, Winston; Wietgrefe, Stephen; Zupancic, Mary; Gebhard, Kristin; Henry, Keith; Boies, Lawrence; Chen, Zongming; Jenkins, Marc; Mills, Roger; McDade, Hugh; Goodwin, Carolyn; Schuwirth, Caspar M.; Danner, Sven A.; Haase, Ashley T.

1998-01-01

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1. PMID:9448301

Kinetics of CD4+ T cell repopulation of lymphoid tissues after treatment of HIV-1 infection.

PubMed

Zhang, Z Q; Notermans, D W; Sedgewick, G; Cavert, W; Wietgrefe, S; Zupancic, M; Gebhard, K; Henry, K; Boies, L; Chen, Z; Jenkins, M; Mills, R; McDade, H; Goodwin, C; Schuwirth, C M; Danner, S A; Haase, A T

1998-02-03

Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

PubMed

Hiramoto, Takafumi; Li, Li B; Funk, Sarah E; Hirata, Roli K; Russell, David W

2018-05-02

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4 + and CD8 + T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Are there Race-Dependent Endothelial Cell Responses to Exercise?

PubMed Central

Brown, Michael D.; Feairheller, Deborah L.

2013-01-01

African Americans have endothelial dysfunction which likely contributes to their high prevalence of hypertension. Endothelial cell responses to stimuli could play a role in the development of endothelial dysfunction and hypertension. High physiological levels of vascular laminar shear stress can profoundly alter endothelial cell phenotype. It is not known whether there are race-dependent endothelial cell responses to laminar shear stress. PMID:23262464

Platelet recruitment promotes keratocyte repopulation following corneal epithelial abrasion in the mouse

USDA-ARS?s Scientific Manuscript database

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and ne...

A different regional response by mouse oligodendrocyte progenitor cells (OPCs) to high-dose X-irradiation has consequences for repopulating OPC-depleted normal tissue.

PubMed

Irvine, Karen-Amanda; Blakemore, William F

2007-01-01

This study was designed to investigate whether the residual, dysfunctional oligodendrocyte progenitor cells (OPCs) observed following X-irradiation of the mouse spinal cord [D. M. Chari et al. (2003) Exp. Neurol., 198, 145-153], the presence of which prevented the endogenous repopulation of these areas from normal tissue, reflects a general response of OPCs in the mouse central nervous system (CNS) to X-irradiation. The brains of adult mice were exposed to 40 Gy of X-irradiation and the effect of X-irradiation on the OPCs was assessed up to 4 weeks post-irradiation using anti-NG2 antibodies. X-irradiation resulted in almost complete depletion of OPCs within the telencephalon (cortex, corpus callosum and hippocampus) by 7 days post-irradiation, which was followed by progressive repopulation of OPCs from non-irradiated areas of the cortex. By contrast, within the lower brain centres (the diencephalon and mesencephalon) OPC loss occurred much more slowly so that 26% of the OPCs still remained 4 weeks after X-irradiation. The consequence of this heterogeneous response to X-irradiation was that whereas transplanted and endogenous OPCs rapidly established themselves in the OPC-depleted telencephalon this did not occur in the areas where there was incomplete depletion of endogenous OPCs. Our findings confirm not only the requirement for almost complete OPC depletion in order to establish transplanted OPCs in normal tissue but also highlight a heterogeneity of progenitor populations in different areas of the mouse CNS.

Fractionated External Beam Radiotherapy as a Suitable Preparative Regimen for Hepatocyte Transplantation After Partial Hepatectomy

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

Krause, Petra; Wolff, Hendrik A.; Rave-Frank, Margret

2011-07-15

Purpose: Hepatocyte transplantation is strongly considered to be a promising option to correct chronic liver failure through repopulation of the diseased organ. We already reported on extensive liver repopulation by hepatocytes transplanted into rats preconditioned with 25-Gy single dose selective external beam irradiation (IR). Herein, we tested lower radiation doses and fractionated protocols, which would be applicable in clinical use. Methods and Material: Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with partial liver external beam single dose IR at 25 Gy, 8 Gy, or 5 Gy, or fractionated IR at 5 x 5 Gy or 5 x 2 Gy.more » Four days after completion of IR, a partial hepatectomy (PH) was performed to resect the untreated liver section. Subsequently, 12 million wild-type (DPPIV{sup +}) hepatocytes were transplanted via the spleen into the recipient livers. The degree of donor cell integration and liver repopulation was studied 16 weeks after transplantation by means of immunofluorescence and DPPIV-luminescence assay. Results: Donor hepatocyte integration and liver repopulation were more effective in the irradiated livers following pretreatment with the IR doses 1 x 25 Gy and 5 x 5 Gy (formation of large DPPIV-positive cell clusters) than single-dose irradiation at 8 Gy or 5 Gy (DPPIV-positive clusters noticeably smaller and less frequent). Quantitative analysis of extracted DPPIV revealed signals exceeding the control level in all transplanted animals treated with IR and PH. Compared with the standard treatment of 1 x 25 Gy, fractionation with 5 x 5 Gy was equally efficacious, the Mann-Whitney U test disclosing no statistically significant difference (p = 0.146). The lower doses of 1 x 5 Gy, 1 x 8 Gy, and 5 x 2 Gy were significantly less effective with p < 0.05. Conclusion: This study suggests that fractionated radiotherapy in combination with PH is a conceivable pretreatment approach to prime the host liver for hepatocyte transplantation, thus bringing the experimental model a step closer to clinical application.« less

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

Aerts-Kaya, Fatima S.F.; Visser, Trudi P.; Arshad, Shazia

Purpose: 5-Androstene-3{beta},17{beta}-diol (5-AED) stimulates recovery of hematopoiesis after exposure to radiation. To elucidate its cellular targets, the effects of 5-AED alone and in combination with (pegylated) granulocyte colony-stimulating factor and thrombopoietin (TPO) on immature hematopoietic progenitor cells were evaluated following total body irradiation. Methods and Materials: BALB/c mice were exposed to radiation delivered as a single or as a fractionated dose, and recovery of bone marrow progenitors and peripheral blood parameters was assessed. Results: BALB/c mice treated with 5-AED displayed accelerated multilineage blood cell recovery and elevated bone marrow (BM) cellularity and numbers of progenitor cells. The spleen colony-forming unitmore » (CFU-S) assay, representing the life-saving short-term repopulating cells in BM of irradiated donor mice revealed that combined treatment with 5-AED plus TPO resulted in a 20.1-fold increase in CFU-S relative to that of placebo controls, and a 3.7 and 3.1-fold increase in comparison to 5-AED and TPO, whereas no effect was seen of Peg-G-CSF with or without 5-AED. Contrary to TPO, 5-AED also stimulated reconstitution of the more immature marrow repopulating (MRA) cells. Conclusions: 5-AED potently counteracts the hematopoietic effects of radiation-induced myelosuppression and promotes multilineage reconstitution by stimulating immature bone marrow cells in a pattern distinct from, but synergistic with TPO.« less

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

PubMed

Siapati, Elena K; Bigger, Brian W; Miskin, James; Chipchase, Daniel; Parsley, Kathryn L; Mitrophanous, Kyriacos; Themis, Mike; Thrasher, Adrian J; Bonnet, Dominique

2005-09-01

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

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

Mathieu, Celine; Fouchet, Pierre; Gauthier, Laurent R.

2006-04-01

Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelialmore » cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having 'side population' phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.« less

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability*

PubMed Central

Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C.; Veillette, André

2012-01-01

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability. PMID:23105101

Bacterial repopulation of drinking water pipe walls after chlorination.

PubMed

Mathieu, Laurence; Francius, Grégory; El Zein, Racha; Angel, Edith; Block, Jean-Claude

2016-09-01

The short-term kinetics of bacterial repopulation were evaluated after chlorination of high-density polyethylene (HDPE) colonized with drinking water biofilms and compared with bare HDPE surfaces. The effect of chlorination was partial as a residual biofilm persisted and was time-limited as repopulation occurred immediately after water resupply. The total number of bacteria reached the same levels on both the bare and chlorinated biofilm-fouled HDPE after a seven-day exposure to drinking water. Due to the presence of a residual biofilm, the hydrophobicity of chlorinated biofilm-fouled surface exhibited much lower adhesion forces (2.1 nN) compared to bare surfaces (8.9 nN). This could explain the rapid repopulation after chlorination, with a twofold faster bacterial accumulation rate on the bare HDPE surface. γ-Proteobacteria dominated the early stages of repopulation of both surfaces and a shift in the dominance occurred over the colonization time. Such observations define a timescale for cleaning frequency in industrial environments and guidelines for a rinsing procedure using drinking water.

MicroRNA-34a regulation of endothelial senescence

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

Ito, Takashi; Yagi, Shusuke; Yamakuchi, Munekazu, E-mail: munekazu_yamakuchi@urmc.rochester.edu

2010-08-06

Research highlights: {yields} MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. {yields} MiR-34a expression increases during endothelial cell senescence and in older mice. {yields} SIRT1 is a miR-34a target gene in endothelial cells. {yields} SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells. -- Abstract: Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelialmore » cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.« less

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases

PubMed Central

Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-01-01

Background Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Design and Methods Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Results Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-α) and also induced allogeneic naive CD4+ T cells to proliferate and to produce type 1 cytokines such as interferon-γ and tumor necrosis factor-α. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Conclusions Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target. PMID:19648164

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

PubMed

Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

2017-07-25

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue.

PubMed

Chari, D M; Huang, W L; Blakemore, W F

2003-09-15

We have attempted to extend a previously described rat model of focal oligodendrocyte progenitor cell (OPC) depletion, using 40 Gy X-irradiation (Chari and Blakemore [2002] Glia 37:307-313), to the adult mouse spinal cord, to examine the ability of OPCs present in adjacent normal areas to colonise areas of progenitor depletion. In contrast to rat, OPCs in the mouse spinal cord appeared to be a comparatively radiation-resistant population, as 30-35% of OPCs survived in X-irradiated tissue (whereas <1% of OPCs survive in X-irradiated rat spinal cord). The numbers of surviving OPCs remained constant with time indicating that this population was incapable of regenerating itself in response to OPC loss. Additionally, these OPCs did not contribute to remyelination of axons when demyelinating lesions were placed in X-irradiated tissue, suggesting that the surviving cells are functionally impaired. Importantly, the length of the OPC-depleted area did not diminish with time, as would be expected if progressive repopulation of OPC-depleted areas by OPCs from normal areas was occurring. Our findings therefore raise the possibility that the presence of a residual dysfunctional OPC population may inhibit colonisation of such areas by normal OPCs. Copyright 2003 Wiley-Liss, Inc.

Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration across Brain and Lung Microvascular Endothelium

PubMed Central

Fan, Jie; Fu, Bingmei M.

2015-01-01

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin Palbumin was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased Palbumin; more tumor cells adhered to and transmigrated through the endothelial monolayer with higher Palbumin; Palbumin and tumor transmigration were increased by vascular endothelial growth factor (VEGF), a representative of cytokines, and lipopolysaccharides (LPS), a typical systemic inflammatory factor, but reduced by adenosine 3′, 5′-cyclic monophosphate (cAMP). These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation. PMID:26603751

Rac regulates vascular endothelial growth factor stimulated motility.

PubMed

Soga, N; Connolly, J O; Chellaiah, M; Kawamura, J; Hruska, K A

2001-01-01

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood. Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF. These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.

Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

PubMed

Desai, Amar; Qing, Yulan; Gerson, Stanton L

2014-02-01

Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long-term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5' HR end resection. We use cultured Exo1(mut) fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1(mut) mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1(mut) mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1(mut) HSCs were pushed into cell cycle in vivo with 5-fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1-mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. © AlphaMed Press.

Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources.

PubMed

Reed, Daniel M; Foldes, Gabor; Kirkby, Nicholas S; Ahmetaj-Shala, Blerina; Mataragka, Stefania; Mohamed, Nura A; Francis, Catherine; Gara, Edit; Harding, Sian E; Mitchell, Jane A

2014-12-12

Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

Protection from radiation-induced damage to spermatogenesis by hormone treatment

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

Kurdoglu, B.; Wilson, G.; Parchuri, N.

1994-07-01

Infertility caused by killing of the spermatogonial stem cells occurs frequently in men treated for cancer with radiotherapy and chemotherapy. We investigated whether pretreatment of rats with testosterone plus estradiol, which reversibly inhibits the completion of spermatogenesis and protects spermatogonial stem cells from procarbazine-induced damage, would also protect these cells from radiation. Adult male LBNF rats were implanted for 6 weeks with capsules containing testosterone and estradiol and then irradiated with doses from 2.5-7.0 Gy. Controls were irradiated with 1.8-3.5 Gy. Implants were removed 1 day after irradiation, and all animals were killed 10 weeks later for assessment of stemmore » cell survival by counting repopulating tubules in histological sections and by sperm head counts. At doses of 2.5 and 3.5 Gy the repopulation indices and sperm head counts were significantly higher (P < 0.001) in the rats treated with testosterone and estradiol than in the controls. Protection factors calculated from the dose-response curves were in the range of 1.5-2.2. Elucidation of the mechanism of protection is essential to apply it to clinical situations. The fact that the spermatogonia are protected against radiation as well as procarbazine indicates that the mechanism does not involve drug delivery or metabolism. 32 refs., 3 figs.« less

Molecular expression in transfected corneal endothelial cells

NASA Astrophysics Data System (ADS)

Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

2017-10-01

To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

Endothelial Cell Implantation and Survival within Experimental Gliomas

NASA Astrophysics Data System (ADS)

Lal, Bachchu; Indurti, Ravi R.; Couraud, Pierre-Olivier; Goldstein, Gary W.; Laterra, John

1994-10-01

The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the β-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by β-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

PubMed Central

Mantovani, A.; Introna, M.; Dejana, E.

1995-01-01

Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

[The role of endothelial cells and endothelial precursor cells in angiogenesis].

PubMed

Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

2006-01-01

Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

Mesenchymal-endothelial-transition contributes to cardiac neovascularization

PubMed Central

Ubil, Eric; Duan, Jinzhu; Pillai, Indulekha C.L.; Rosa-Garrido, Manuel; Wu, Yong; Bargiacchi, Francesca; Lu, Yan; Stanbouly, Seta; Huang, Jie; Rojas, Mauricio; Vondriska, Thomas M.; Stefani, Enrico; Deb, Arjun

2014-01-01

Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal-transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial cell like phenotype after acute ischemic cardiac injury. Fibroblast derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast derived endothelial cells, reduces post infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal to endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial-transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair. PMID:25317562

Comparison of apoptosis in human primary pulmonary endothelial cells and a brain microvascular endothelial cell line co-cultured with Plasmodium falciparum field isolates.

PubMed

Essone, Jean Claude Biteghe Bi; N'Dilimabaka, Nadine; Ondzaga, Julien; Lekana-Douki, Jean Bernard; Mba, Dieudonné Nkoghe; Deloron, Philippe; Mazier, Dominique; Gay, Frédrérick; Touré Ndouo, Fousseyni S

2017-06-27

Plasmodium falciparum infection can progress unpredictably to severe forms including respiratory distress and cerebral malaria. The mechanisms underlying the variable natural course of malaria remain elusive. The cerebral microvascular endothelial cells-D3 and lung endothelial cells both from human were cultured separately and challenged with P. falciparum field isolates taken directly from malaria patients or 3D7 strain (in vitro maintained culture). The capacity of these P. falciparum isolates to induce endothelial cell apoptosis via cytoadherence or not was then assessed. Overall, 27 P. falciparum isolates were collected from patients with uncomplicated malaria (n = 25) or severe malaria (n = 2). About half the isolates (n = 17) were able to bind brain endothelial cells (12 isolates, 44%) or lung endothelial cells (17 isolates, 63%) or both (12 isolates, 44%). Sixteen (59%) of the 27 isolates were apoptogenic for brain and/or lung endothelial cells. The apoptosis stimulus could be cytoadherence, direct cell-cell contact without cytoadherence, or diffusible soluble factors. While some of the apoptogenic isolates used two stimuli (direct contact with or without cytoadherence, plus soluble factors) to induce apoptosis, others used only one. Among the 16 apoptogenic isolates, eight specifically targeted brain endothelial cells, one lung endothelial cells, and seven both. These results indicate that the brain microvascular cell line was more susceptible to apoptosis triggered by P. falciparum than the primary pulmonary endothelial cells and may have relevance to host-parasite interaction.

Cell-SELEX-Based Identification of a Human and Mouse Cross-Reactive Endothelial Cell-Internalizing Aptamer.

PubMed

Dua, Pooja; Kang, Sinae; Shin, Hye-Soo; Kim, Soyoun; Lee, Dong-Ki

2018-04-02

Increased interest and insights gained by researchers on the roles of endothelial cells in the pathophysiology of cancer, inflammatory, and cardiovascular diseases have led to the design of pharmacological interventions aimed at the endothelium lining in the diseased sites. Toward this end, we used established brain microvascular endothelial cell lines mouse (bEND3), human (hCMEC/D3), and Toggle Cell-SELEX to identify a species cross-reactive, endothelial cell-internalizing aptamer R11-3. This 2'F-modified RNA aptamer is specific for endothelial cells as no internalization was seen with cells of nonendothelial origin. R11-3 was truncated in size, and its potential in endothelial targeted therapeutics was established using VEGFR2 targeting long interfering RNA (liRNA) aptamer chimera. Due to its specificity for both mouse and human endothelial cells, we believe that this aptamer not only fits for development of endothelial targeted drug development for human diseases but is also suitable for preclinical evaluation in mice.

Spontaneous circulation of myeloid-lymphoid–initiating cells and SCID-repopulating cells in sickle cell crisis

PubMed Central

Lamming, Christopher E.D.; Augustin, Lance; Blackstad, Mark; Lund, Troy C.; Hebbel, Robert P.; Verfaillie, Catherine M.

2003-01-01

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid–initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture–initiating cells, consistent with the notion that SRCs are more primitive than long-term culture–initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation. PMID:12639987

Sources of Hematopoietic Stem and Progenitor Cells and Methods to Optimize Yields for Clinical Cell Therapy.

PubMed

Panch, Sandhya R; Szymanski, James; Savani, Bipin N; Stroncek, David F

2017-08-01

Bone marrow (BM) aspirates, mobilized peripheral blood, and umbilical cord blood (UCB) have developed as graft sources for hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation and other cellular therapeutics. Individualized techniques are necessary to enhance graft HSPC yields and cell quality from each graft source. BM aspirates yield adequate CD34 + cells but can result in relative delays in engraftment. Granulocyte colony-stimulating factor (G-CSF)-primed BM HSPCs may facilitate faster engraftment while minimizing graft-versus-host disease in certain patient subsets. The levels of circulating HSPCs are enhanced using mobilizing agents, such as G-CSF and/or plerixafor, which act via the stromal cell-derived factor 1/C-X-C chemokine receptor type 4 axis. Alternate niche pathway mediators, including very late antigen-4/vascular cell adhesion molecule-1, heparan sulfate proteoglycans, parathyroid hormone, and coagulation cascade intermediates, may offer promising alternatives for graft enhancement. UCB grafts have been expanded ex vivo with cytokines, notch-ligand, or mesenchymal stromal cells, and most studies demonstrated greater quantities of CD34 + cells ex vivo and improved short-term engraftment. No significant changes were observed in long-term repopulating potential or in patient survival. Early phase clinical trials using nicotinamide and StemReginin1 may offer improved short- and long-term repopulating ability. Breakthroughs in genome editing and stem cell reprogramming technologies may hasten the generation of pooled, third-party HSPC grafts. This review elucidates past, present, and potential future approaches to HSPC graft optimization. Published by Elsevier Inc.

Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.

PubMed

Ilin, Yelena; Choi, Ji Sun; Harley, Brendan A C; Kraft, Mary L

2015-11-17

A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely related short-term repopulating HSCs (ST-HSCs) and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four subpopulations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that elicited them.

Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

PubMed

Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

2015-07-01

Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

PubMed

Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2015-01-01

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis.

PubMed

Piwowarczyk, Katarzyna; Paw, Milena; Ryszawy, Damian; Rutkowska-Zapała, Magdalena; Madeja, Zbigniew; Siedlar, Maciej; Czyż, Jarosław

2017-06-01

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43 high (DU-145 and MAT-LyLu) and Cx43 low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43 high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43 high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43 low AT-2 cells, Cx43 low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43 low and Cx43 high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43 high prostate cancer and endothelial cells. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

PubMed Central

Kim, Mihwa; Ham, Ahrom; Kim, Katelyn Yu-Mi; Brown, Kevin M.; Lee, H. Thomas

2014-01-01

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation. PMID:24945528

Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

NASA Astrophysics Data System (ADS)

Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

1983-11-01

Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

microRNAs as Pharmacological Targets in Endothelial Cell Function and Dysfunction

PubMed Central

Chamorro-Jorganes, Aránzazu; Araldi, Elisa; Suárez, Yajaira

2013-01-01

Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative, migratory and morphogenic capacities of endothelial cells, as well as control of leukocyte trafficking. MicroRNAs (miRNAs) are short non-coding RNAs that have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level. This review summarizes the latest insights in the identification of endothelial-specific miRNAs and their targets, as well as their roles in controlling endothelial cell functions in both autocrine and paracrine manner. In addition, we discuss the therapeutic potential for the treatment of endothelial cell dysfunction and associated vascular pathophysiological conditions. PMID:23603154

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

PubMed Central

Oubaha, Malika; Lin, Michelle I.; Margaron, Yoran; Filion, Dominic; Price, Emily N.; Zon, Leonard I.; Côté, Jean-François

2012-01-01

Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis. PMID:22936663

Responses of brain and non-brain endothelial cells to meningitis-causing Escherichia coli K1.

PubMed

Paul-Satyaseela, Maneesh; Xie, Yi; Di Cello, Francescopaolo; Kim, Kwang Sik

2006-03-31

Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.

Evaluation of hematopoietic potential generated by transplantation of muscle-derived stem cells in mice.

PubMed

Farace, Francoise; Prestoz, Laetitita; Badaoui, Sabrina; Guillier, Martine; Haond, Celine; Opolon, Paule; Thomas, Jean-Leon; Zalc, Bernard; Vainchenker, William; Turhan, Ali G

2004-02-01

Muscle tissue of adult mice has been shown to contain stem cells with hematopoietic repopulation ability in vivo. To determine the functional characteristics of stem cells giving rise to this hematopoietic activity, we have performed hematopoietic reconstitution experiments by the use of muscle versus marrow transplantation in lethally irradiated mice and followed the fate of transplanted cells by Y-chimerism using PCR and fluorescence in situ hybridization (FISH) analysis. We report here that transplantation of murine muscle generate a major hematopoietic chimerism at the level of CFU-C, CFU-S, and terminally-differentiated cells in three generations of lethally irradiated mice followed up to 1 year after transplantation. This potential is totally abolished when muscle grafts were performed by the use of muscle from previously irradiated mice. As compared to marrow transplantation, muscle transplants were able to generate similar potencies to give rise to myeloid, T, B, and natural killer (NK) cells. Interestingly, marrow stem cells that have been generated in primary and then in secondary recipients were able to contribute efficiently to myofibers in the muscle tissue of tertiary recipients. Altogether, our data demonstrate that muscle-derived stem cells present a major hematopoietic repopulating ability with evidence of self-replication in vivo. They are radiation-sensitive and similar to marrow-derived stem cells in terms of their ability to generate multilineage hematopoiesis. Finally, our data demonstrate that muscle-derived hematopoietic stem cells do not lose their ability to contribute to myofiber generation after at least two rounds of serial transplantation, suggesting a potential that is probably equivalent to that generated by marrow transplantation.

The Methods and Mechanisms to Differentiate Endothelial-Like Cells and Smooth Muscle Cells from Mesenchymal Stem Cells for Vascularization in Vaginal Reconstruction.

PubMed

Zhang, Hua; Zhang, Jingkun; Huang, Xianghua; Li, Yanan

2018-06-01

Endothelial cells and smooth muscle cells (SMCs) are important aspects of vascularization in vaginal reconstruction. Research has confirmed that mesenchymal stem cells could differentiate into endothelial-like cells and SMCs. But the methods were more complicated and the mechanism was unknown. In the current study, we induced the bone mesenchymal stem cells (BMSCs) to differentiate into endothelial-like cells and SMCs in vitro by differentiation medium and investigated the effect of Wnt/β-catenin signaling on the differentiation process of BMSCs. Results showed that the hypoxic environment combined with VEGF and bFGF could induce increased expression of endothelial-like cells markers VEGFR1, VEGFR2, and vWF. The SMCs derived from BMSCs induced by TGF-β1 and PDGF-AB significantly expressed SMC markers SMMHC11 and α-SMA. The data also showed that activation of Wnt/β-catenin signaling could promote the differentiation of BMSCs into endothelial-like cells and SMCs. Thus, we established endothelial-like cells and SMCs in vitro by more simple methods, presented the important role of hypoxic environment on the differentiation of BMSCs into endothelial-like cells, and confirmed that the Wnt/β-catenin signaling pathway has a positive impact on the differentiation of BMSCs into endothelial-like cells and SMCs. This is important for vascular reconstruction.

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells.

PubMed

Liu, Zhenguo; Jiang, Yuehua; Hao, Hong; Gupta, Kalpna; Xu, Jian; Chu, Ling; McFalls, Edward; Zweier, Jay; Verfaillie, Catherine; Bache, Robert J

2007-09-01

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.

Occurrence of Salmonella serotype Typhimurium DT104 on a commercial swine farm before, during, and after depopulation and repopulation.

PubMed

Erdman, Matthew M; Harris, Isabel T; Torremorell, Montserrat; Wilt, Vincil M; Harris, D L Hank

2005-08-01

To determine whether depopulation-repopulation could be used to eradicate Salmonella serotype Typhimurium DT104 from a commercial swine farm in the midwestern United States. Observational study A commercial swine farm undergoing depopulation-repopulation to eliminate porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Pooled fecal samples, tissue samples, and serum samples were collected from pigs on the farm before and after depopulation-repopulation. When there were no pigs on the farm, environmental swab specimens were collected for bacterial culture. Serum was analyzed for anti-Salmonella antibodies with an indirect ELISA. Salmonella isolates obtained by bacterial culture of fecal, tissue, and environmental samples were characterized by means of serotyping, phage typing, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. 167 Salmonella isolates representing 9 serotypes were recovered from the farm. Results of PFGE and antimicrobial susceptibility testing suggested that S. Typhimurium DT104 strain was not eradicated from the farm. However, seroprevalence of anti-Salmonella antibodies and the percentage of pooled fecal samples positive for Salmonella spp were significantly decreased following repopulation. Results suggested that depopulation-repopulation in conjunction with stringent cleaning and disinfection, attention to biosecurity procedures, control of other diseases, and changes in feed management may reduce the occurrence of, but likely will not eliminate, Salmonella spp in commercial swine herds.

Rolling the Human Amnion to Engineer Laminated Vascular Tissues

PubMed Central

Amensag, Salma

2012-01-01

The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact. PMID:22616610

Keratocyte Apoptosis and Not Myofibroblast Differentiation Mark the Graft/Host Interface at Early Time-Points Post-DSAEK in a Cat Model

PubMed Central

Weis, Adam J.; Huxlin, Krystel R.; Callan, Christine L.; DeMagistris, Margaret A.; Hindman, Holly B.

2013-01-01

Purpose To evaluate myofibroblast differentiation as an etiology of haze at the graft-host interface in a cat model of Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK). Methods DSAEK was performed on 10 eyes of 5 adult domestic short-hair cats. In vivo corneal imaging with slit lamp, confocal, and optical coherence tomography (OCT) were performed twice weekly. Cats were sacrificed and corneas harvested 4 hours, and 2, 4, 6, and 9 days post-DSAEK. Corneal sections were stained with the TUNEL method and immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and fibronectin with DAPI counterstain. Results At all in vivo imaging time-points, corneal OCT revealed an increase in backscatter of light and confocal imaging revealed an acellular zone at the graft-host interface. At all post-mortem time-points, immunohistochemistry revealed a complete absence of α-SMA staining at the graft-host interface. At 4 hours, extracellular fibronectin staining was identified along the graft-host interface and both fibronectin and TUNEL assay were positive within adjacent cells extending into the host stroma. By day 2, fibronectin and TUNEL staining diminished and a distinct acellular zone was present in the region of previously TUNEL-positive cells. Conclusions OCT imaging consistently showed increased reflectivity at the graft-host interface in cat corneas in the days post-DSAEK. This was not associated with myofibroblast differentiation at the graft-host interface, but rather with apoptosis and the development of a subsequent acellular zone. The roles of extracellular matrix changes and keratocyte cell death and repopulation should be investigated further as potential contributors to the interface optical changes. PMID:24098706

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.

PubMed

Zilla, P; Fasol, R; Dudeck, U; Siedler, S; Preiss, P; Fischlein, T; Müller-Glauser, W; Baitella, G; Sanan, D; Odell, J

1990-08-01

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

Low-level laser therapy prevents endothelial cells from TNF-α/cycloheximide-induced apoptosis.

PubMed

Chu, Yu-Hsiu; Chen, Shu-Ya; Hsieh, Yueh-Ling; Teng, Yi-Hsien; Cheng, Yu-Jung

2018-02-01

Low-level laser therapy (LLLT), widely used in physiotherapy, has been known to enhance wound healing and stimulate cell proliferation, including fibroblast and endothelial cells. Applying LLLT can increase cell proliferation in many kinds of cells including fibroblasts and endothelial cells. However, the protective mechanisms of LLLT on endothelial apoptosis remain unclear. We hypothesized LLLT can protect endothelial cells from inflammation-induced apoptosis. Human endothelial cell line, EA.hy926 cells, and TNF-α/cycloheximide (TNF/CHX) were used to explore the protective effects of LLLT (660 nm) on inflammation-induced endothelial apoptosis. Cell viability, apoptosis, caspase-3/7/8/9 activity, MAPKs signaling, NF-κB activity, and inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression were measured. Our results showed that LLLT increased EA.hy926 cell proliferation, attenuated the TNF/CHX-induced apoptosis, and reduced the TNF/CHX-mediated caspase-3/7/8/9 activation. In addition, LLLT increased ERK MAPK phosphorylation and suppressed the TNF/CHX-increased p38 MAPK, JNK, IKK phosphorylation, NF-κB translocation, and iNOS expression. The caspases-3 cleavage and cell death were not increased in cells treating with ERK inhibitor U0126, which implicated that ERK is not to be responsible for the protective effects of LLLT. After treating with p38 mitogen-activated protein kinase (MAPK) activator, the protection of LLLT in cell apoptosis was no longer existed, showing that LLLT protected the endothelial cells by suppressing p38 MAPK signaling. Our results provide a new insight into the possible molecular mechanisms in which LLLT protects against inflammatory-induced endothelial dysfunction.

Reduced Ang2 expression in aging endothelial cells.

PubMed

Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

NASA Astrophysics Data System (ADS)

Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

1990-08-01

Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

Periodontal Wound Healing Responses to Varying Oxygen Concentrations and Atmospheric Pressures.

DTIC Science & Technology

1986-05-01

Presumably, epithelial and gingival connective tissue exclusion allowed periodontal ligament cells to repopulate the wound and to regenerate a new...However, it seems clear that the periodontal ligament cells provide a major source of connective tissue attachment and regeneration (Nyman et al., 1982a...Connective Tissue Regeneration to Periodontally Diseased Teeth. J. Perio. Res. 15:1. Davis, J. C., Dunn, J. M., Gates, G. A. and Heimbach, R. D. 1979

Angiopoietin-1 protects the endothelial cells against advanced glycation end product injury by strengthening cell junctions and inhibiting cell apoptosis.

PubMed

Zhao, Jingling; Chen, Lei; Shu, Bin; Tang, Jinming; Zhang, Lijun; Xie, Julin; Liu, Xusheng; Xu, Yingbin; Qi, Shaohai

2015-08-01

Endothelial dysfunction is a major characteristic of diabetic vasculopathy. Protection of the vascular endothelium is an essential aspect of preventing and treating diabetic vascular complications. Although Angiopoietin-1 (Ang-1) is an important endothelial-specific protective factor, whether Ang-1 protects vascular cells undergoing advanced glycation end product (AGE) injury has not been investigated. The aim of the present study was to determine the potential effects of Ang-1 on endothelial cells after exposure to AGE. We show here that Ang-1 prevented AGE-induced vascular leakage by enhancing the adherens junctions between endothelial cells, and this process was mediated by the phosphorylation and membrane localization of VE-cadherin. Furthermore, Ang-1 also protected endothelial cells from AGE-induced death by regulating phosphatidylinositol 3-kinase (PI3K)/Akt-dependent Bad phosphorylation. Our findings suggest that the novel protective mechanisms of Ang-1 on endothelium are achieved by strengthening endothelial cell junctions and reducing endothelial cell death after AGE injury. © 2014 Wiley Periodicals, Inc.

System for tracking transplanted limbal epithelial stem cells in the treatment of corneal stem cell deficiency (Conference Presentation)

NASA Astrophysics Data System (ADS)

Boadi, Joseph; Matcher, Stephen; MacNeil, Sheila; Sangwan, Virender S.

2016-04-01

The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells are continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. In the event that the cornea is damaged and the limbal stem cell population is severely reduced, this condition known as Limbal Stem Cell Deficiency and can lead to blindness. There are numerous treatments but most have high long term failure rates. Most treatment methods include the transplantation of limbal stem cells into damaged limbus with hope of repopulating the region and regenerating at healthy corneal epithelium. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images. A bespoke OCT has been built to investigate the trajectories of these limbal stem cells after transplantation to see whether if they do repopulate the damaged limbus or not. In the experimentation magneto-labelling was used to track the limbal stem cells. For the magneto-labelling a mixture of limbal stem cells and cornea epithelium are cultured with super paramagnetic iron (Fe3O4) nanoparticles (20-30nm in size) for 24hours, to allow for uptake. The cells are then transplanted onto the denuded cornea. The transplanted cell mixture with the encapsulated magnetic nanoparticles is actuated with an external magnetic field 0.08T leading to a phase modulation on the signal. A Phase sensitive Magneto-motive OCT is used to locate the transplanted cells. The location of the cells with embed SPIOs were located both in 2D and 3D.

Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells.

PubMed

Mariappan, Devi; Winkler, Johannes; Chen, Shuhua; Schulz, Herbert; Hescheler, Jürgen; Sachinidis, Agapios

2009-02-01

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells. CD31(+) cells exhibit endothelial-like behavior by being able to incorporate DiI-labeled acetylated low-density lipoprotein as well as form tubular structures on matrigel. Quantitative and semi-quantitative PCR analysis further demonstrated the increased expression of endothelial transcripts. To ascertain the specific transcriptomic identity of the CD31(+) cells, large-scale microarray analysis was carried out. Comparative bioinformatic analysis reveals an enrichment of the gene ontology categories angiogenesis, blood vessel morphogenesis, vasculogenesis and blood coagulation in the CD31(+) cell population. Based on the transcriptomic signatures of the CD31(+) cells, we conclude that this ES cell-derived population contains endothelial-like cells expressing a mesodermal marker BMP2 and possess an angiogenic potential. The transcriptomic characterization of CD31(+) cells enables an in vitro functional genomic model to identify genes required for angiogenesis.

Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

PubMed

Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

2015-07-01

Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p <0.001). A colony of circulating endothelial progenitor cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p <0.001). The culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p <0.001). The circulating endothelial progenitor cell level correlated positively with the number of patient colonies (r = 0.762, p <0.001). Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p <0.001). Earlier emergence of circulating endothelial progenitor cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells

PubMed Central

Suschek, Christoph; Kolb, Hubert; Kolb-Bachofen, Victoria

1997-01-01

Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.In each of the different endothelial cells Mg-Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor NG-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT–PCR. PMID:9421302

Approaches to improve angiogenesis in tissue-engineered skin.

PubMed

Sahota, Parbinder S; Burn, J Lance; Brown, Nicola J; MacNeil, Sheila

2004-01-01

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

Evidence for naive T-cell repopulation despite thymus irradiation after autologous transplantation in adults with multiple myeloma: role of ex vivo CD34+ selection and age.

PubMed

Malphettes, Marion; Carcelain, Guislaine; Saint-Mezard, Pierre; Leblond, Véronique; Altes, Hester Korthals; Marolleau, Jean-Pierre; Debré, Patrice; Brouet, Jean-Claude; Fermand, Jean-Paul; Autran, Brigitte

2003-03-01

Immunodeficiency following autologous CD34+-purified peripheral blood stem cell (PBSC) transplantation could be related to T-cell depletion of the graft or impaired T-cell reconstitution due to thymus irradiation. Aiming to assess the role of irradiated thymus in T-cell repopulation, we studied 32 adults with multiple myeloma, randomly assigned to receive high-dose therapy including total body irradiation (TBI) followed by autologous transplantation with either unselected or CD34+-selected PBSCs. The median number of reinfused CD3+ cells was lower in the selected group (0.03 versus 14 x 10(6)/kg; P =.002). Lymphocyte subset counts were evaluated from month 3 to 24 after grafting. Naive CD4+ T cells were characterized both by phenotype and by quantification of T-cell receptor rearrangement excision circles (TRECs). The reconstitution of CD3+ and CD4+ T cells was significantly delayed in the CD34+-selected group, but eventually led to counts similar to those found in the unselected group after month 12. Mechanism of reconstitution differed, however, between both groups. Indeed, a marked increase in the naive CD62L+CD45RA+CD4+ subset was observed in the selected group, but not in the unselected group in which half of the CD45RA+CD4+ T cells appear to be CD62L-. Age was identified as an independent adverse factor for CD4+ and CD62L+CD45RA+CD4+ T-cell reconstitution. Our results provide evidence that infusing PBSCs depleted of T cells after TBI in adults delays T-cell reconstitution but accelerates thymic regeneration.

Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells.

PubMed Central

Vlodavsky, I; Fielding, P E; Fielding, C J; Gospodarowicz, D

1978-01-01

Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque. Images PMID:203937

Endothelial cell membrane vesicles in the study of organ preference of metastasis.

PubMed

Johnson, R C; Augustin-Voss, H G; Zhu, D Z; Pauli, B U

1991-01-01

Many malignancies exhibit distinct patterns of metastasis that appear to be mediated by receptor/ligand-like interactions between tumor cells and organ-specific vascular endothelium. In order to study endothelial cell surface molecules involved in the binding of metastatic cells, we developed a perfusion method to isolate outside-out membrane vesicles from the lumenal surface of rat lung microvascular endothelium. Lungs were perfused in situ for 4 h at 37 degrees C with a solution of 100 mM formaldehyde, 2 mM dithiothreitol in phosphate-buffered saline to induce endothelial cell vesiculation. Radioiodinated rat lung endothelial cell membrane vesicles bound lung-metastatic tumor cells (B16F10, R323OAC-MET) in significantly higher numbers than their low or nonmetastatic counterparts (B16F0, R323OAC-LR). In contrast, leg endothelial membrane vesicle showed no binding preference for either cell line. Neuraminidase treatment of vesicles abolished specificity of adhesion of lung-derived vesicles to lung metastatic tumor cells. These results demonstrate that in situ perfusion is an appropriate technique to obtain pure endothelial cell membrane vesicles containing functionally active adhesion molecules. The preferential binding of lung-derived endothelial cell membrane vesicles by lung metastatic tumor cells is evidence of the importance of endothelial cell adhesion molecules in the formation of metastases.

Cross talk between primary human renal tubular cells and endothelial cells in cocultures.

PubMed

Tasnim, Farah; Zink, Daniele

2012-04-15

Interactions between renal tubular epithelial cells and adjacent endothelial cells are essential for normal renal functions but also play important roles in renal disease and repair. Here, we investigated cocultures of human primary renal proximal tubular cells (HPTC) and human primary endothelial cells to address the cross talk between these cell types. HPTC showed improved proliferation, marker gene expression, and enzyme activity in cocultures. Also, the long-term maintenance of epithelia formed by HPTC was improved, which was due to the secretion of transforming growth factor-β1 and its antagonist α2-macroglobulin. HPTC induced endothelial cells to secrete increased amounts of these factors, which balanced each other functionally and only displayed in combination the observed positive effects. In addition, in the presence of HPTC endothelial cells expressed increased amounts of hepatocyte growth factor and vascular endothelial growth factor, which have well-characterized effects on renal tubular epithelial cells as well as on endothelial cells. Together, the results showed that HPTC stimulated endothelial cells to express a functionally balanced combination of various factors, which in turn improved the performance of HPTC. The results give new insights into the cross talk between renal epithelial and endothelial cells and suggest that cocultures could be also useful models for the analysis of cellular communication in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, will be helpful for improving the performance of this cell type in in vitro applications including in vitro toxicology and kidney tissue engineering.

Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair

PubMed Central

Olszewska-Pazdrak, Barbara; McVicar, Scott D.; Rayavara, Kempaiah; Moya, Stephanie M.; Kantara, Carla; Gammarano, Chris; Olszewska, Paulina; Fuller, Gerald M.; Sower, Laurie E.; Carney, Darrell H.

2016-01-01

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation. PMID:27388041

Identification and functional analysis of endothelial tip cell-enriched genes.

PubMed

del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef; Eichmann, Anne

2010-11-11

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Repopulation of calibrations with samples from the target site: effect of the size of the calibration.

NASA Astrophysics Data System (ADS)

Guerrero, C.; Zornoza, R.; Gómez, I.; Mataix-Solera, J.; Navarro-Pedreño, J.; Mataix-Beneyto, J.; García-Orenes, F.

2009-04-01

Near infrared (NIR) reflectance spectroscopy offers important advantages because is a non-destructive technique, the pre-treatments needed in samples are minimal, and the spectrum of the sample is obtained in less than 1 minute without the needs of chemical reagents. For these reasons, NIR is a fast and cost-effective method. Moreover, NIR allows the analysis of several constituents or parameters simultaneously from the same spectrum once it is obtained. For this, a needed steep is the development of soil spectral libraries (set of samples analysed and scanned) and calibrations (using multivariate techniques). The calibrations should contain the variability of the target site soils in which the calibration is to be used. Many times this premise is not easy to fulfil, especially in libraries recently developed. A classical way to solve this problem is through the repopulation of libraries and the subsequent recalibration of the models. In this work we studied the changes in the accuracy of the predictions as a consequence of the successive addition of samples to repopulation. In general, calibrations with high number of samples and high diversity are desired. But we hypothesized that calibrations with lower quantities of samples (lower size) will absorb more easily the spectral characteristics of the target site. Thus, we suspect that the size of the calibration (model) that will be repopulated could be important. For this reason we also studied this effect in the accuracy of predictions of the repopulated models. In this study we used those spectra of our library which contained data of soil Kjeldahl Nitrogen (NKj) content (near to 1500 samples). First, those spectra from the target site were removed from the spectral library. Then, different quantities of samples of the library were selected (representing the 5, 10, 25, 50, 75 and 100% of the total library). These samples were used to develop calibrations with different sizes (%) of samples. We used partial least squares regression, and leave-one-out cross validation as methods of calibration. Two methods were used to select the different quantities (size of models) of samples: (1) Based on Characteristics of Spectra (BCS), and (2) Based on NKj Values of Samples (BVS). Both methods tried to select representative samples. Each of the calibrations (containing the 5, 10, 25, 50, 75 or 100% of the total samples of the library) was repopulated with samples from the target site and then recalibrated (by leave-one-out cross validation). This procedure was sequential. In each step, 2 samples from the target site were added to the models, and then recalibrated. This process was repeated successively 10 times, being 20 the total number of samples added. A local model was also created with the 20 samples used for repopulation. The repopulated, non-repopulated and local calibrations were used to predict the NKj content in those samples from the target site not included in repopulations. For the measurement of the accuracy of the predictions, the r2, RMSEP and slopes were calculated comparing predicted with analysed NKj values. This scheme was repeated for each of the four target sites studied. In general, scarce differences can be found between results obtained with BCS and BVS models. We observed that the repopulation of models increased the r2 of the predictions in sites 1 and 3. The repopulation caused scarce changes of the r2 of the predictions in sites 2 and 4, maybe due to the high initial values (using non-repopulated models r2 >0.90). As consequence of repopulation, the RMSEP decreased in all the sites except in site 2, where a very low RMESP was obtained before the repopulation (0.4 g×kg-1). The slopes trended to approximate to 1, but this value was reached only in site 4 and after the repopulation with 20 samples. In sites 3 and 4, accurate predictions were obtained using the local models. Predictions obtained with models using similar size of samples (similar %) were averaged with the aim to describe the main patterns. The r2 of predictions obtained with models of higher size were not more accurate than those obtained with models of lower size. After repopulation, the RMSEP of predictions using models with lower sizes (5, 10 and 25% of samples of the library) were lower than RMSEP obtained with higher sizes (75 and 100%), indicating that small models can easily integrate the variability of the soils from the target site. The results suggest that calibrations of small size could be repopulated and "converted" in local calibrations. According to this, we can focus most of the efforts in the obtainment of highly accurate analytical values in a reduced set of samples (including some samples from the target sites). The patterns observed here are in opposition with the idea of global models. These results could encourage the expansion of this technique, because very large data based seems not to be needed. Future studies with very different samples will help to confirm the robustness of the patterns observed. Authors acknowledge to "Bancaja-UMH" for the financial support of the project "NIRPROS".

Endothelial cells in the eyes of an immunologist.

PubMed

Young, M Rita

2012-10-01

Endothelial cell activation in the process of tumor angiogenesis and in various aspects of vascular biology has been extensively studied. However, endothelial cells also function in other capacities, including in immune regulation. Compared to the more traditional immune regulatory populations (Th1, Th2, Treg, etc.), endothelial cells have received far less credit as being immune regulators. Their regulatory capacity is multifaceted. They are critical in both limiting and facilitating the trafficking of various immune cell populations, including T cells and dendritic cells, out of the vasculature and into tissue. They also can be induced to stimulate immune reactivity or to be immune inhibitory. In each of these parameters (trafficking, immune stimulation and immune inhibition), their role can be physiological, whereby they have an active role in maintaining health. Alternatively, their role can be pathological, whereby they contribute to disease. In theory, endothelial cells are in an ideal location to recruit cells that can mediate immune reactivity to tumor tissue. Furthermore, they can activate the immune cells as they transmigrate across the endothelium into the tumor. However, what is seen is the absence of these protective effects of endothelial cells and, instead, the endothelial cells succumb to the defense mechanisms of the tumor, resulting in their acquisition of a tumor-protective role. To understand the immune regulatory potential of endothelial cells in protecting the host versus the tumor, it is useful to better understand the other circumstances in which endothelial cells modulate immune reactivities. Which of the multitude of immune regulatory roles that endothelial cells can take on seems to rely on the type of stimulus that they are encountering. It also depends on the extent to which they can be manipulated by potential dangers to succumb and contribute toward attack on the host. This review will explore the physiological and pathological roles of endothelial cells as they regulate immune trafficking, immune stimulation and immune inhibition in a variety of conditions and will then apply this information to their role in the tumor environment. Strategies to harness the immune regulatory potential of endothelial cells are starting to emerge in the non-tumor setting. Results from such efforts are expected to be applicable to being able to skew endothelial cells from having a tumor-protective role to a host-protective role.

The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

PubMed Central

Zhang, Jing-Chuan; Doñate, Fernando; Qi, Xiaoping; Ziats, Nicholas P.; Juarez, Jose C.; Mazar, Andrew P.; Pang, Yuan-Ping; McCrae, Keith R.

2002-01-01

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin. PMID:12196635

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

PubMed

Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

2018-05-01

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

PubMed Central

James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin

2010-01-01

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865

Fast revascularization of the injured area is essential to support zebrafish heart regeneration

PubMed Central

Marín-Juez, Rubén; Marass, Michele; Gauvrit, Sebastien; Rossi, Andrea; Lai, Shih-Lei; Materna, Stefan C.; Black, Brian L.; Stainier, Didier Y. R.

2016-01-01

Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization. PMID:27647901

Eradication of Murine Norovirus from a Mouse Barrier Facility

PubMed Central

Kastenmayer, Robin J; Perdue, Kathy A; Elkins, William R

2008-01-01

Murine norovirus (MNV) is a common viral infection of mice in many research facilities. MNV infects hematopoietic cells and alters their cellular morphology. Because of MNV's probable effects on the systemic immune response of infected mice the decision was made to eradicate the virus from 2 rooms containing infected animals in our vivarium. Two different eradication methods were selected. One room, in which most of the indirectly exposed sentinels had antibodies to MNV, was depopulated and thoroughly cleaned prior to repopulation. In the other room, in which only 13% of the sentinels had positive MNV titers, selective testing was used, and MNV-positive animals were removed. Data from surveillance of the sentinel mice exposed to dirty bedding indicate that the test-and-removal method was ineffective in eliminating MNV from the room, whereas sentinel mice in the room that underwent depopulation and cleaning prior to repopulation have not shown any evidence of MNV since December 2006. PMID:18210995

Antiapoptotic and antigenotoxic effects of N-acetylcysteine in human cells of endothelial origin.

PubMed

Aluigi, M G; De Flora, S; D'Agostini, F; Albini, A; Fassina, G

2000-01-01

N-Acetylcysteine (NAC) is a drug bearing multiple preventive properties that can inhibit genotoxicity and carcinogenicity. NAC also inhibits invasion and metastasis of malignant cells, as well as tumor take. We recently demonstrated the effects of NAC on Kaposi's sarcoma cells supernatant-induced invasion in vitro and angiogenesis in vivo. Many anticancer agents act through cytotoxicity of rapidly proliferating cells and several antineoplastic drugs induce apoptosis of cancer cells. Since endothelial cells are the target for the inhibition of angiogenesis, we wanted to verify that NAC, while inhibiting tumor vascularization and endothelial cell invasion would not induce endothelial cell apoptosis. We tested the ability of NAC to modulate apoptosis and cytogenetic damage in vitro and to promote differentiation on a reconstituted basement membrane (matrigel) in two endothelial cell lines (EAhy926 and HUVE). Treatment with NAC protected endothelial cells from TGF-beta-induced apoptosis and paraquat-induced cytogenetic damage. Therefore, NAC acts as an antiangiogenic agent and, at the same time, appears to prevent apoptosis and oxygen-related genotoxicity in endothelial cells.

Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells

PubMed Central

Wang, Xiaoli; Zhang, Wei; Tripodi, Joseph; Lu, Min; Xu, Mingjiang; Najfeld, Vesna; Li, Yan

2010-01-01

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC), we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34+ cells. Treatment of PMF CD34+ cells with chromatin-modifying agents (CMAs) but not hydroxyurea, Janus kinase 2 (JAK2) inhibitors, or low doses of interferon-α led to the generation of greater numbers of CD34+ chemokine (C-X-C motif) receptor (CXCR)4+ cells, which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F+. Furthermore, sequential treatment of PMF CD34+ cells but not normal CD34+ cells with decitabine (5-aza-2′-deoxycytidine [5azaD]), followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA), or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F+ PMF CD34+ cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγnull mice, the percentage of JAK2V617F/JAK2total in human CD45+ marrow cells was dramatically reduced. These findings suggest that both PMF HPCs, short-term and long-term SCID repopulating cells (SRCs), are JAK2V617F+ and that JAK2V617F+ HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs, therefore, represents a possible effective means of treating PMF at the level of the malignant SRC. PMID:20858855

Characterization of slow-cycling cells in the mouse cochlear lateral wall

PubMed Central

Ogawa, Kaoru

2017-01-01

Cochlear spiral ligament fibrocytes (SLFs) play essential roles in the physiology of hearing including ion recycling and the generation of endocochlear potential. In adult animals, SLFs can repopulate after damages, yet little is known about the characteristics of proliferating cells that support SLFs’ self-renewal. Here we report in detail about the characteristics of cycling cells in the spiral ligament (SL). Fifteen P6 mice and six noise-exposed P28 mice were injected with 5-bromo-2′-deoxyuridine (BrdU) for 7 days and we chased BrdU retaining cells for as long as 60 days. Immunohistochemistry revealed that the BrdU positive IB4 (an endotherial marker) negative cells expressed an early SLF marker Pou3f4 but negative for cleaved-Caspase 3. Marker studies revealed that type 3 SLFs displayed significantly higher percentage of BrdU+ cells compared to other subtypes. Notably, the cells retained BrdU until P72, demonstrating they were dividing slowly. In the noise-damaged mice, in contrast to the loss of the other types, the number of type 3 SLFs did not altered and the BrdU incorporating- phosphorylated Histone H3 positive type 3 cells were increased from day 1 to 14 after noise exposure. Furthermore, the cells repopulating type 1 area, where the cells diminished profoundly after damage, were positive for the type 3 SLF markers. Collectively, in the latral wall of the cochlea, type 3 SLFs have the stem cell capacity and may contribute to the endogenous regeneration of lateral wall spiral ligament. Manipulating type 3 cells may be employed for potential regenerative therapies. PMID:28632772

Dark Endothelial Spots After Descemet Membrane Endothelial Keratoplasty May Appear as Recurrent Fuchs Dystrophy or Herald Graft Failure or Rejection.

PubMed

Zygoura, Vasiliki; Baydoun, Lamis; Monnereau, Claire; Satué, Maria; Oellerich, Silke; Melles, Gerrit R J

2017-12-01

To evaluate the clinical significance of dark spots in the donor endothelial cell layer as observed with specular microscopy, in patients who underwent Descemet membrane endothelial keratoplasty (DMEK) for Fuchs endothelial dystrophy (FED). Specular microscopy images of 83 consecutive eyes up to 7 years after DMEK were retrospectively reviewed in a masked fashion for the presence of dark spots and morphologic changes in the endothelial cell layer and processed for endothelial cell density (ECD) measurements. A normal endothelial cell layer was found in 52/83 eyes (62.7%) (group 0). In the remaining 31/83 eyes, various dark discolorations with or without altered endothelial cell morphology were categorized into 4 groups. Dark spots were classified as artifacts in 10/83 (12.0%) eyes (group I) and as "superimposed" dots in 10/83 (12.0%) eyes (group II), that is, optical irregularities slightly anterior to a healthy endothelial cell layer. In 11/83 (13.3%) eyes, endothelial stress was characterized by dark grayish discolorations and/or nuclear activation (group III). Most of the latter eyes also had a significant ECD decrease; 3 of these eyes later developed secondary graft failure, of which one was preceded by allograft rejection. None of the eyes showed recurrent guttae typical for FED (group IV). Dark endothelial spots after DMEK for FED may not represent a recurrent disease, but tissue irregularities just anterior to the graft. However, if associated with changes in endothelial cell morphology, nuclear activation and/or ECD decrease, dark discolorations may reflect "cellular stress" heralding secondary graft failure or (subclinical) allograft rejection.

COPD as an endothelial disorder: endothelial injury linking lesions in the lungs and other organs? (2017 Grover Conference Series)

PubMed Central

Polverino, Francesca; Celli, Bartolome R.

2018-01-01

Chronic obstructive pulmonary disease (COPD) is characterized by chronic expiratory airflow obstruction that is not fully reversible. COPD patients develop varying degrees of emphysema, small and large airway disease, and various co-morbidities. It has not been clear whether these co-morbidities share common underlying pathogenic processes with the pulmonary lesions. Early research into the pathogenesis of COPD focused on the contributions of injury to the extracellular matrix and pulmonary epithelial cells. More recently, cigarette smoke-induced endothelial dysfunction/injury have been linked to the pulmonary lesions in COPD (especially emphysema) and systemic co-morbidities including atherosclerosis, pulmonary hypertension, and chronic renal injury. Herein, we review the evidence linking endothelial injury to COPD, and the pathways underlying endothelial injury and the “vascular COPD phenotype” including: (1) direct toxic effects of cigarette smoke on endothelial cells; (2) generation of auto-antibodies directed against endothelial cells; (3) vascular inflammation; (4) increased oxidative stress levels in vessels inducing increases in lipid peroxidation and increased activation of the receptor for advanced glycation end-products (RAGE); (5) reduced activation of the anti-oxidant pathways in endothelial cells; (6) increased endothelial cell release of mediators with vasoconstrictor, pro-inflammatory, and remodeling activities (endothelin-1) and reduced endothelial cell expression of mediators that promote vasodilation and homeostasis of endothelial cells (nitric oxide synthase and prostacyclin); and (7) increased endoplasmic reticular stress and the unfolded protein response in endothelial cells. We also review the literature on studies of drugs that inhibit RAGE signaling in other diseases (angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), or vasodilators developed for idiopathic pulmonary arterial hypertension that have been tested on cell culture systems, animal models of COPD, and/or smokers and COPD patients. PMID:29468936

Endothelial-derived interleukin-6 induces cancer stem cell motility by generating a chemotactic gradient towards blood vessels.

PubMed

Kim, Hong Sun; Chen, Yu-Chih; Nör, Felipe; Warner, Kristy A; Andrews, April; Wagner, Vivian P; Zhang, Zhaocheng; Zhang, Zhixiong; Martins, Manoela D; Pearson, Alexander T; Yoon, Euisik; Nör, Jacques E

2017-11-21

Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDH high CD44 high ) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells. Analysis of tissue microarrays generated from the invasive fronts of 77 HNSCC patients followed-up for up to 11 years revealed that high expression of IL-6 receptor (IL-6R) (p=0.0217) or co-receptor gp130 (p=0.0422) correlates with low HNSCC patient survival. We observed that endothelial cell-secreted factors induce epithelial to mesenchymal transition (EMT) and enhance invasive capacity of HNSCC cancer stem cells. Conditioned medium from CRISPR/Cas9-mediated IL-6 knockout primary human endothelial cells is less chemotactic for cancer stem cells in a microfluidics-based system than medium from control endothelial cells (p<0.05). Blockade of the IL-6 pathway with a humanized anti-IL-6R antibody (tocilizumab) inhibited endothelial cell-induced motility in vitro and decreased the fraction of cancer stem cells in vivo . Notably, xenograft HNSCC tumors vascularized with IL-6-knockout endothelial cells exhibited slower tumor growth and smaller cancer stem cell fraction. These findings demonstrate that endothelial cell-secreted IL-6 enhances the motility and survival of highly tumorigenic cancer stem cells, suggesting that endothelial cells can create a chemotactic gradient that enables the movement of carcinoma cells towards blood vessels.

Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo.

PubMed

Marchetti, Sandrine; Gimond, Clotilde; Iljin, Kristiina; Bourcier, Christine; Alitalo, Kari; Pouysségur, Jacques; Pagès, Gilles

2002-05-15

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation, reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study, we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. Using EGFP as a reporter gene, we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently, tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected, puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers, including CD31, CD34, VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1, two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally, we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together, these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies.

Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1

NASA Astrophysics Data System (ADS)

Koch, Alisa E.; Halloran, Margaret M.; Haskell, Catherine J.; Shah, Manisha R.; Polverini, Peter J.

1995-08-01

ENDOTHELIAL adhesion molecules facilitate the entry of leukocytes into inflamed tissues. This in turn promotes neovascularization, a process central to the progression of rheumatoid arthritis, tumour growth and wound repair1. Here we test the hypothesis that soluble endothelial adhesion molecules promote angiogenesis2á¤-4. Human recombinant soluble E-selectin and soluble vascular cell adhesion molecule-1 induced chemotaxis of human endothelial cells in vitro and were angiogenic in rat cornea. Soluble E-selectin acted on endothelial cells in part through a sialyl Lewis-X-dependent mechanism, while soluble vascular cell adhesion molecule-1 acted on endothelial cells in part through a very late antigen (VLA)-4 dependent mechanism. The chemotactic activity of rheumatoid synovial fluid for endothelial cells, and also its angiogenic activity, were blocked by antibodies to either soluble E-selectin or soluble vascular cell adhesion molecule-1. These results suggest a novel function for soluble endothelial adhesion molecules as mediators of angiogenesis.

Gestational diabetes mellitus alters maternal and neonatal circulating endothelial progenitor cell subsets.

PubMed

Acosta, Juan C; Haas, David M; Saha, Chandan K; Dimeglio, Linda A; Ingram, David A; Haneline, Laura S

2011-03-01

The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity. Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit-endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging. Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks' gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit-endothelial cells or colony-forming unit-endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects. The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities. Copyright © 2011 Mosby, Inc. All rights reserved.

Dermal Stem Cells Can Differentiate Down an Endothelial Lineage

PubMed Central

Bell, Emma; Richardson, Gavin D.; Jahoda, Colin A.; Gledhill, Karl; Phillips, Helen M.; Henderson, Deborah; Owens, W. Andrew

2012-01-01

In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle. PMID:22571645

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

PubMed

Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian Gabriel

2017-01-01

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Expression of an insulin-regulatable glucose carrier in muscle and fat endothelial cells

NASA Astrophysics Data System (ADS)

Vilaró, Senen; Palacín, Manuel; Pilch, Paul F.; Testar, Xavier; Zorzano, Antonio

1989-12-01

INSULIN rapidly stimulates glucose use in the major target tissues, muscle and fat, by modulating a tissue-specific glucose transporter isoform1-6. Access of glucose to the target tissue is restricted by endothelial cells which line the walls of nonfenestrated capillaries of fat and muscle7. Thus, we examined whether the capillary endothelial cells are actively involved in the modulation of glucose availability by these tissues. We report here the abundant expression of the muscle/fat glucose transporter isoform in endothelial cells, using an immunocytochemical analysis with a monoclonal antibody specific for this isoform1. This expression is restricted to endothelial cells from the major insulin target tissues, and it is not detected in brain and liver where insulin does not activate glucose transport. The expression of the muscle/fat transporter isoform in endothelial cells is significantly greater than in the neighbouring muscle and fat cells. Following administration of insulin to animals in vivo, there occurs a rapid increase in the number of muscle/fat transporters present in the lumenal plasma membrane of the capillary endothelial cells. These results document that insulin promotes the translocation of the muscle/fat glucose transporter in endothelial cells. It is therefore likely that endothelial cells play an important role in the regulation of glucose use by the major insulin target tissues in normal and diseased states.

Calcium supplementation prevents endothelial cell activation: possible relevance to preeclampsia.

PubMed

Chen, Qi; Tong, Mancy; Wu, Man; Stone, Peter R; Snowise, Saul; Chamley, Lawrence W

2013-09-01

Preeclampsia is a leading cause of maternal and fetal mortality and morbidity. A hallmark of preeclampsia is endothelial cell dysfunction/activation in response to 'toxins' from the placenta. Necrotic trophoblastic debris (NTD) is one possible placental toxin and other activators of endothelial cells include inflammatory cytokines. Calcium supplementation appears to protect 'at-risk' women from developing preeclampsia but how is unclear. Placental explants were cultured with interleukin-6 (IL-6) in varied concentrations of calcium. The resultant trophoblastic debris was exposed to endothelial cells. Endothelial cells were exposed to activators including NTD, IL-6, and preeclamptic sera in the presence of varied concentrations of calcium and activation monitored by quantifying cell surface markers by ELISA. Raising the levels of calcium did not prevent the IL-6-induced shedding of NTD from placental explants but did prevent the activation of endothelial cells in response to IL-6, preeclamptic sera, or NTD. Reducing the level of calcium directly induced the activation of endothelial cells. Inhibiting nitric oxide synthetase ablated the ability of high calcium levels to protect endothelial cell activation. The activity of endothelial cell nitric oxide synthetase was blocked with L-N-nitroarginine methyl ester. Our results demonstrate calcium levels do not affect the shedding of trophoblastic debris but are important to endothelial cell activation and supplemental calcium may reverse the activation of the endothelium in preeclamptic women. These results may in part explain the benefits of calcium supplementation in the reduction of risk for developing preeclampsia and provide in-vitro mechanistic support for the use of calcium supplementation in at-risk women.

Flavorings in Tobacco Products Induce Endothelial Cell Dysfunction.

PubMed

Fetterman, Jessica L; Weisbrod, Robert M; Feng, Bihua; Bastin, Reena; Tuttle, Shawn T; Holbrook, Monica; Baker, Gregory; Robertson, Rose Marie; Conklin, Daniel J; Bhatnagar, Aruni; Hamburg, Naomi M

2018-06-14

Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function. Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1-100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction. Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity. © 2018 American Heart Association, Inc.

Somatic GNAQ Mutation is Enriched in Brain Endothelial Cells in Sturge-Weber Syndrome.

PubMed

Huang, Lan; Couto, Javier A; Pinto, Anna; Alexandrescu, Sanda; Madsen, Joseph R; Greene, Arin K; Sahin, Mustafa; Bischoff, Joyce

2017-02-01

Sturge-Weber syndrome (SWS) is a rare congenital neurocutaneous disorder characterized by facial and extracraniofacial capillary malformations and capillary-venule malformations in the leptomeninges. A somatic mosaic mutation in GNAQ (c.548G>A; p.R183Q) was found in SWS brain and skin capillary malformations. Our laboratory showed endothelial cells in skin capillary malformations are enriched for the GNAQ mutation. The purpose of this study is to determine whether the GNAQ mutation is also enriched in endothelial cells in affected SWS brain. Two human SWS brain specimens were fractionated by fluorescence-activated cell sorting into hematopoietic (CD45), endothelial (CD31, VE-Cadherin, and vascular endothelial growth factor receptor 2), and perivascular (platelet-derived growth factor receptor beta) cells and cells negative for all markers. The sorted cell populations were analyzed for GNAQ p.R183Q mutation by droplet digital polymerase chain reaction. SWS patient-derived brain endothelial cells were selected by anti-CD31-coated magnetic beads and cultured in endothelial growth medium in vitro. The GNAQ p.R183Q mutation was present in brain endothelial cells in two SWS specimens, with mutant allelic frequencies of 34.7% and 24.0%. Cells negative for all markers also harbored the GNAQ mutation. The mutant allelic frequencies in these unidentified cells were 9.2% and 8.4%. SWS patient-derived brain endothelial cells with mutant allelic frequencies of 14.7% and 21% survived and proliferated in vitro. Our study provides evidence that GNAQ p.R183Q mutation is enriched in endothelial cells in SWS brain lesions and thereby reveals endothelial cells as a source of aberrant Gαq signaling. This will help to understand the pathophysiology of SWS, to discover biomarkers for predicting cerebral involvement, and to develop therapeutic targets to prevent neurological impairments in SWS. Copyright © 2016 Elsevier Inc. All rights reserved.

Loss of the endothelial glycocalyx is associated with increased E-selectin mediated adhesion of lung tumour cells to the brain microvascular endothelium.

PubMed

Rai, Srijana; Nejadhamzeeigilani, Zaynab; Gutowski, Nicholas J; Whatmore, Jacqueline L

2015-09-25

Arrest of metastasising lung cancer cells to the brain microvasculature maybe mediated by interactions between ligands on circulating tumour cells and endothelial E-selectin adhesion molecules; a process likely to be regulated by the endothelial glycocalyx. Using human cerebral microvascular endothelial cells and non-small cell lung cancer (NSCLC) cell lines, we describe how factors secreted by NSCLC cells i.e. cystatin C, cathepsin L, insulin-like growth factor-binding protein 7 (IGFBP7), vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α), damage the glycocalyx and enhance initial contacts between lung tumour and cerebral endothelial cells. Endothelial cells were treated with tumour secreted-proteins or lung tumour conditioned medium (CM). Surface levels of E-selectin were quantified by ELISA. Adhesion of A549 and SK-MES-1 cells was examined under flow conditions (1 dyne/cm(2)). Alterations in the endothelial glycocalyx were quantified by binding of fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC). A549 and SK-MES-1 CM and secreted-proteins significantly enhanced endothelial surface E-selectin levels after 30 min and 4 h and tumour cell adhesion after 30 min, 4 and 24 h. Both coincided with significant glycocalyx degradation; A549 and SK-MES-1 CM removing 55 ± 12 % and 58 ± 18.7 % of WGA-FITC binding, respectively. Inhibition of E-selectin binding by monoclonal anti-E-selectin antibody completely attenuated tumour cell adhesion. These data suggest that metastasising lung cancer cells facilitate their own adhesion to the brain endothelium by secreting factors that damage the endothelial glycocalyx, resulting in exposure of the previously shielded adhesion molecules and engagement of the E-selectin-mediated adhesion axis.

In Vitro Impact of Conditioned Medium From Demineralized Freeze-Dried Bone on Human Umbilical Endothelial Cells.

PubMed

Harnik, Branko; Miron, Richard J; Buser, Daniel; Gruber, Reinhard

2017-03-01

Angiogenesis is essential for the consolidation of bone allografts. The underlying molecular mechanism, however, remains unclear. Soluble factors released from demineralized freeze-dried bone target mesenchymal cells; however, their effect on endothelial cells has not been investigated so far. The aim of the present study was therefore to examine the effect of conditioned medium from demineralized freeze-dried bone on human umbilical endothelial cells in vitro. Conditioned medium was first prepared from demineralized freeze-dried bone following 24 hours incubation at room temperature to produce demineralized bone conditioned media. Thereafter, conditioned medium was used to stimulate human umbilical vein endothelial cells in vitro by determining the cell response based on viability, proliferation, expression of apoptotic genes, a Boyden chamber to determine cell migration, and the formation of branches. The authors report here that conditioned medium decreased viability and proliferation of endothelial cells. Neither of the apoptotic marker genes was significantly altered when endothelial cells were exposed to conditioned medium. The Boyden chamber revealed that endothelial cells migrate toward conditioned medium. Moreover, conditioned medium moderately stimulated the formation of branches. These findings support the concept that conditioned medium from demineralized freeze-dried bone targets endothelial cells by decreasing their proliferation and enhancing their motility under these in vitro conditions.

Effect of proinflammatory cytokines on PIGA- hematopoiesis.

PubMed

Kulkarni, Shashikant; Bessler, Monica

2003-09-01

Blood cells from patients with paroxysmal nocturnal hemoglobinuria lack glycosyl phosphatidylinositol (GPI)-linked proteins, due to a somatic mutation in the X-linked PIGA gene. It is believed that clonal expansion of PIGA- blood cells is due to a survival advantage in the hostile marrow environment of aplastic anemia. Here we investigated the effects of inhibitory cytokines in mice genetically engineered to have blood cells deficient in GPI-linked proteins. The effect of inhibitory cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], macrophage inflammatory protein-1 alpha [MIP-1alpha], and transforming growth factor-beta1 [TGF-beta1]) was investigated, using clonogenic assays, competitive repopulation, and in vivo induction of proinflammatory cytokines by double-stranded RNA. The expression of Fas on progenitor cells and its up-regulation by inhibitory cytokines were analyzed by flow cytometry. TNF-alpha, IFN-gamma, MIP-1alpha, and TGF-beta1 suppressed colony formation in a dose-dependent fashion that was similar for PIGA+ and PIGA- blood bone marrow cells. Competitive repopulation of bone marrow cells cultured in IFN-gamma and TNF-alpha resulted in a comparable ability of PIGA+ and PIGA- hematopoietic stem cells to reconstitute hematopoiesis. Fas expression was minimal on PIGA+ and PIGA- progenitor cells and was up-regulated to the same extent in response to IFN-gamma and TNF-alpha as assessed by Fas antibody-mediated apoptosis. Similarly, in vivo induction of proinflammatory cytokines by double-stranded RNA had no effect on the proportion of circulating PIGA- blood cells. These results indicate that PIGA+ and PIGA- hematopoietic progenitor cells respond similarly to inhibitory cytokines, suggesting that other factors are responsible for the clonal expansion of paroxysmal nocturnal hemoglobinuria cells.

Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

PubMed Central

Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

2015-01-01

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

Hyperglycemia-induced PATZ1 negatively modulates endothelial vasculogenesis via repression of FABP4 signaling.

PubMed

Chen, Ren-An; Sun, Xiao-Mian; Yan, Chang-You; Liu, Li; Hao, Miao-Wang; Liu, Qiang; Jiao, Xi-Ying; Liang, Ying-Min

2016-09-02

Vascular endothelial dysfunction, a central hallmark of diabetes, predisposes diabetic patients to numerous cardiovascular complications. The POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1), is an important transcriptional regulatory factor and regulates divergent pathways depending on the cellular context, but its role in endothelial cells remains poorly understood. Herein, we report for the first time that endothelial PATZ1 expression was abnormally upregulated in diabetic endothelial cells (ECs) regardless of diabetes classification. This stimulatory effect was further confirmed in the high glucose-treated human umbilical vein endothelial cells (HUVECs). From a functional standpoint, transgenic overexpression of PATZ1 in endothelial colony forming cells (ECFCs) blunted angiogenesis in vivo and rendered endothelial cells unresponsive to established angiogenic factors. Mechanistically, PATZ1 acted as a potent transcriptional corepressor of fatty acid-binding protein 4 (FABP4), an essential convergence point for angiogenic and metabolic signaling pathways in ECs. Taken together, endothelial PATZ1 thus potently inhibits endothelial function and angiogenesis via inhibition of FABP4 expression, and abnormal induction of endothelial PATZ1 may contribute to multiple aspects of vascular dysfunction in diabetes. Copyright © 2016. Published by Elsevier Inc.

Efficacy and safety of a clinically relevant foamy vector design in human hematopoietic repopulating cells.

PubMed

Everson, Elizabeth M; Hocum, Jonah D; Trobridge, Grant D

2018-06-23

Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and insulators can improve FV vector safety. However, in a previous analysis of insulator effects on FV vector safety, strong viral promoters were used to elicit genotoxic events. Here we developed and analyzed the efficacy and safety of a high-titer, clinically relevant FV vector driven by the housekeeping promoter elongation factor-1α and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). Human CD34 + cord blood cells were exposed to an enhanced green fluorescent protein expressing vector, FV-EGW-A1, at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. FV-EGW-A1 resulted in high-marking, multi-lineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. An FV vector with an elongation factor-1α promoter and an A1 insulator is a promising vector design for use in the clinic. This article is protected by copyright. All rights reserved.

Telomerase deficiency in bone marrow-derived cells attenuates angiotensin II-induced abdominal aortic aneurysm formation.

PubMed

Findeisen, Hannes M; Gizard, Florence; Zhao, Yue; Cohn, Dianne; Heywood, Elizabeth B; Jones, Karrie L; Lovett, David H; Howatt, Deborah A; Daugherty, Alan; Bruemmer, Dennis

2011-02-01

Abdominal aortic aneurysms (AAA) are an age-related vascular disease and an important cause of morbidity and mortality. In this study, we sought to determine whether the catalytic component of telomerase, telomerase reverse transcriptase (TERT), modulates angiotensin (Ang) II-induced AAA formation. Low-density lipoprotein receptor-deficient (LDLr-/-) mice were lethally irradiated and reconstituted with bone marrow-derived cells from TERT-deficient (TERT-/-) mice or littermate wild-type mice. Mice were placed on a diet enriched in cholesterol, and AAA formation was quantified after 4 weeks of Ang II infusion. Repopulation of LDLr-/- mice with TERT-/- bone marrow-derived cells attenuated Ang II-induced AAA formation. TERT-deficient recipient mice revealed modest telomere attrition in circulating leukocytes at the study end point without any overt effect of the donor genotype on white blood cell counts. In mice repopulated with TERT-/- bone marrow, aortic matrix metalloproteinase-2 (MMP-2) activity was reduced, and TERT-/- macrophages exhibited decreased expression and activity of MMP-2 in response to stimulation with Ang II. Finally, we demonstrated in transient transfection studies that TERT overexpression activates the MMP-2 promoter in macrophages. TERT deficiency in bone marrow-derived macrophages attenuates Ang II-induced AAA formation in LDLr-/- mice and decreases MMP-2 expression. These results point to a previously unrecognized role of TERT in the pathogenesis of AAA.

Vascular Gene Expression in Nonneoplastic and Malignant Brain

PubMed Central

Madden, Stephen L.; Cook, Brian P.; Nacht, Mariana; Weber, William D.; Callahan, Michelle R.; Jiang, Yide; Dufault, Michael R.; Zhang, Xiaoming; Zhang, Wen; Walter-Yohrling, Jennifer; Rouleau, Cecile; Akmaev, Viatcheslav R.; Wang, Clarence J.; Cao, Xiaohong; St. Martin, Thia B.; Roberts, Bruce L.; Teicher, Beverly A.; Klinger, Katherine W.; Stan, Radu-Virgil; Lucey, Brenden; Carson-Walter, Eleanor B.; Laterra, John; Walter, Kevin A.

2004-01-01

Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression. PMID:15277233

Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier.

PubMed

McGuire, Paul G; Rangasamy, Sampathkumar; Maestas, Joann; Das, Arup

2011-12-01

The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

PubMed

Kono, Ken; Hiruma, Hitomi; Kobayashi, Shingo; Sato, Yoji; Tanaka, Masaru; Sawada, Rumi; Niimi, Shingo

2016-01-01

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

Theobroma cacao increases cells viability and reduces IL-6 and sVCAM-1 level in endothelial cells induced by plasma from preeclamptic patients.

PubMed

Rahayu, Budi; Baktiyani, Siti Candra Windu; Nurdiana, Nurdiana

2016-01-01

This study aims to investigate whether an ethanolic extract of Theobroma cacao bean is able to increase cell viability and decrease IL-6 and sVCAM-1 in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluency, endothelial cells were divided into six groups, which included control (untreated), endothelial cells exposed to plasma from normal pregnancy, endothelial cells exposed to 2% plasma from preeclamptic patients (PP), endothelial cells exposed to PP in the presence of ethanolic extract of T. cacao (PP+TC) at the following three doses: 25, 50, and 100 ppm. The analysis was performed in silico using the Hex 8.0, LigPlus and LigandScout 3.1 software. Analysis on IL-6 and sVCAM-1 levels were done by enzyme linked immunosorbent assay (ELISA). We found that seven of them could bind to the protein NFκB (catechin, leucoanthocyanidin, niacin, phenylethylamine, theobromine, theophylline, and thiamin). This increase in IL-6 was significantly (P<0.05) attenuated by both the 50 and 100 ppm treatments of T. cacao extract. Plasma from PP significantly increased sVCAM-1 levels compared to untreated cells. This increase in sVCAM-1 was significantly attenuated by all doses of the extract. In conclusion, T. cacao extract prohibits the increase in IL-6 and sVCAM-1 in endothelial cells induced by plasma from preeclamptic patients. Therefore this may provide a herbal therapy for attenuating the endothelial dysfunction found in preeclampsia. Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

Quantifying effects of cyclic stretch on cell-collagen substrate adhesiveness of vascular endothelial cells.

PubMed

Omidvar, Ramin; Tafazzoli-Shadpour, Mohammad; Mahmoodi-Nobar, Farbod; Azadi, Shohreh; Khani, Mohammad-Mehdi

2018-05-01

Vascular endothelium is continuously subjected to mechanical stimulation in the form of shear forces due to blood flow as well as tensile forces as a consequence of blood pressure. Such stimuli influence endothelial behavior and regulate cell-tissue interaction for an optimized functionality. This study aimed to quantify influence of cyclic stretch on the adhesive property and stiffness of endothelial cells. The 10% cyclic stretch with frequency of 1 Hz was applied to a layer of endothelial cells cultured on a polydimethylsiloxane substrate. Cell-substrate adhesion of endothelial cells was examined by the novel approach of atomic force microscope-based single-cell force spectroscopy and cell stiffness was measured by atomic force microscopy. Furthermore, the adhesive molecular bonds were evaluated using modified Hertz contact theory. Our results show that overall adhesion of endothelial cells with substrate decreased after cyclic stretch while they became stiffer. Based on the experimental results and theoretical modeling, the decrease in the number of molecular bonds after cyclic stretch was quantified. In conclusion, in vitro cyclic stretch caused alterations in both adhesive capacity and elastic modulus of endothelial cells through mechanotransductive pathways as two major determinants of the function of these cells within the cardiovascular system.

Dual inhibition of mTORC1 and mTORC2 perturbs cytoskeletal organization and impairs endothelial cell elongation.

PubMed

Tsuji-Tamura, Kiyomi; Ogawa, Minetaro

2018-02-26

Elongation of endothelial cells is an important process in vascular formation and is expected to be a therapeutic target for inhibiting tumor angiogenesis. We have previously demonstrated that inhibition of mTORC1 and mTORC2 impaired endothelial cell elongation, although the mechanism has not been well defined. In this study, we analyzed the effects of the mTORC1-specific inhibitor everolimus and the mTORC1/mTORC2 dual inhibitor KU0063794 on the cytoskeletal organization and morphology of endothelial cell lines. While both inhibitors equally inhibited cell proliferation, KU0063794 specifically caused abnormal accumulation of F-actin and disordered distribution of microtubules, thereby markedly impairing endothelial cell elongation and tube formation. The effects of KU0063794 were phenocopied by paclitaxel treatment, suggesting that KU0063794 might impair endothelial cell morphology through over-stabilization of microtubules. Although mTORC1 is a key signaling molecule in cell proliferation and has been considered a target for preventing angiogenesis, mTORC1 inhibitors have not been sufficient to suppress angiogenesis. Our results suggest that mTORC1/mTORC2 dual inhibition is more effective for anti-angiogenic therapy, as it impairs not only endothelial cell proliferation, but also endothelial cell elongation. Copyright © 2018 Elsevier Inc. All rights reserved.

The impact of left ventricular stretching in model cultivations with neonatal cardiomyocytes in a whole-heart bioreactor.

PubMed

Hülsmann, Jörn; Aubin, Hug; Wehrmann, Alexander; Lichtenberg, Artur; Akhyari, Payam

2017-05-01

Here, we investigate the impact of integrated three-dimensional (3D) left ventricular (LV) stretching on myocardial maturation in a whole-heart bioreactor setting. Therefore, decellularized rat hearts were selectively repopulated with rodent neonatal cardiomyocytes (5 · 10 6 cells per heart) and cultured over 5 days. Continuous medium perfusion was maintained through the coronary artery system in a customized whole-heart bioreactor system with or without integrated biomechanical stimulation of LV. 3D repopulation effectiveness and cellular vitality were evaluated by repetitive metabolic WST-1 assays and 3D confocal microscopy analysis through fluorescent staining, also assessing cellular organization. Moreover, specific myocardial vitality was verified by detecting spontaneous electrophysiological activity using a multielectrode assay. Western blot analysis of cardiac myosin heavychain (MHC) and quantitative RT-PCR for Connexin 43 was used to analyze cardiomyocyte maturation. Decellularized whole-heart constructs repopulated with neonatal cardiomyocytes (repopWHC) showed vital 3D cell populations throughout the repopulation sites within the LV with a significant increase in metabolic activity (326 ± 113% for stimulated constructs vs. 162 ± 32% for non-stimulated controls after 96 h of continuous cultivation as compared to their state 24 h after injection, directly prior to bioreactor cultivation). Further, bioreactor cultivation under integrated mechanical LV stimulation not only led to a higher degree of cellular organization and an increased MHC content, but also to a significant increase of Cx43 gene expression resulting in a regain of 60 ± 19% of native neonatal hearts expression level in contrast to 20 ± 9% for non-stimulated controls (P = 0.03). Therefore, our study suggests that the integration of LV stretching into whole-heart bioreactor cultivation may enhance cardiac maturation not only by promoting cellular organization but also through adaptive protein and gene expression with particular implications for the formation of the conductive apparatus. Further, this study emphasizes the importance of suitable bioprocessing strategies within sophisticated bioreactor systems as tools for customized stimulation and cultivation of tissue engineered tissues and organs. Biotechnol. Bioeng. 2017;114: 1107-1117. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation ▿

PubMed Central

Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

2011-01-01

Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

PubMed

Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

2017-08-01

Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese individuals.

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells

PubMed Central

Tabet, Fatiha; Vickers, Kasey C.; Cuesta Torres, Luisa F.; Wiese, Carrie B.; Shoucri, Bassem M.; Lambert, Gilles; Catherinet, Claire; Prado-Lourenco, Leonel; Levin, Michael G.; Thacker, Seth; Sethupathy, Praveen; Barter, Philip J.; Remaley, Alan T.; Rye, Kerry-Anne

2014-01-01

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223−/− mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL’s anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells. PMID:24576947

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

PubMed

Hailer, N P; Oppermann, E; Leckel, K; Cinatl, J; Markus, B H; Blaheta, R A

2000-07-15

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.

PubMed

Jakobsson, Lars; Franco, Claudio A; Bentley, Katie; Collins, Russell T; Ponsioen, Bas; Aspalter, Irene M; Rosewell, Ian; Busse, Marta; Thurston, Gavin; Medvinsky, Alexander; Schulte-Merker, Stefan; Gerhardt, Holger

2010-10-01

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

Protective effects of sulphonated formononetin in a rat model of cerebral ischemia and reperfusion injury.

PubMed

Zhu, Haibo; Zou, Libo; Tian, Jingwei; Lin, Fei; He, Jie; Hou, Jian

2014-03-01

Sodium formononetin-3'-sulphonate is a derivative of the plant isoflavone formononetin. The present study aimed to investigate the neuroprotective and angiogenesis effects of sodium formononetin-3'-sulphonate in vivo and in vitro. Treatment with sodium formononetin-3'-sulphonate (3, 7.5, 15, and 30 mg/kg, intravenous injection) could protect the brain from ischemia and reperfusion injury by improving neurological function, suppressing cell apoptosis, and increasing expression levels of vascular endothelial growth factor and platelet endothelial cell adhesion molecule 1 by middle cerebral artery occlusion. Treatment with sodium formononetin-3'-sulphonate (10 and 20 µg/mL) significantly increased cell migration, tube formation, and vascular endothelial growth factor and platelet endothelial cell adhesion molecule levels in human umbilical vein endothelial cells. Our results suggest that sodium formononetin-3'-sulphonate provides significant neuroprotective effects against cerebral ischemia and reperfusion injury in rats, and improves cerebrovascular angiogenesis in human umbilical vein endothelial cells. The protective mechanisms of sodium formononetin-3'-sulphonate may be attributed to the suppression of cell apoptosis and improved cerebrovascular angiogenesis by promoting vascular endothelial growth factor and platelet endothelial cell adhesion molecule expression. Georg Thieme Verlag KG Stuttgart · New York.

iRhom2 regulates CSF1R cell surface expression and non-steady state myelopoiesis in mice.

PubMed

Qing, Xiaoping; Rogers, Lindsay; Mortha, Arthur; Lavin, Yonit; Redecha, Patricia; Issuree, Priya D; Maretzky, Thorsten; Merad, Miriam; McIlwain, David; Mak, Tak W; Overall, Christopher M; Blobel, Carl P; Salmon, Jane E

2016-12-01

CSF1R (colony stimulating factor 1 receptor) is the main receptor for CSF1 and has crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by ADAM17 (A disintegrin and metalloprotease 17). Here, we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2-/- mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2-/- BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild-type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2-/- Lin - SCA-1 + c-Kit + (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

iRhom2 regulates cell surface expression of CSF1R and non-steady state myelopoiesis in mice

PubMed Central

Qing, Xiaoping; Lavin, Yonit; Redecha, Patricia; Issuree, Priya D.; Maretzky, Thorsten; Merad, Miriam; McIlwain, David; Mak, Tak W.; Overall, Christopher M.

2016-01-01

The colony stimulating factor 1 receptor (CSF1R) functions as the major receptor for macrophage colony stimulating factor (CSF1) with crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by A disintegrin and metalloprotease 17 (ADAM17). Here we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2−/− mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2−/− BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2−/− Lin−SCA-1+c-Kit+ (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs. PMID:27601030

Epigallocatechin 3-gallate inhibits 7-ketocholesterol-induced monocyte-endothelial cell adhesion.

PubMed

Yamagata, Kazuo; Tanaka, Noriko; Suzuki, Koichi

2013-07-01

7-Ketocholesterol (7KC) induces monocytic adhesion to endothelial cells, and induces arteriosclerosis while high-density lipoprotein (HDL) inhibits monocytic adhesion to the endothelium. Epigallocatechin 3-gallate (EGCG) was found to have a protective effect against arteriosclerosis. Therefore, the purpose of this study was to examine the possible HDL-like mechanisms of EGCG in endothelial cells by investigating whether EGCG inhibits 7KC-induced monocyte-endothelial cell adhesion by activating HDL-dependent signal transduction pathways. 7KC and/or EGCG were added to human endothelial cells (ISO-HAS), and the adhesion of pro-monocytic U937 cells was examined. The expression of genes associated with HDL effects such as Ca(2+)/calmodulin-dependent kinase II (CaMKKII), liver kinase B (LKD1), PSD-95/Dlg/ZO-1 kinase 1 (PDZK1), phosphatidylinositol 3-kinase (PI3K), intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-1), and endothelial nitric oxide synthase (eNOS) was examined by RT-PCR, and ICAM-1 protein expression was evaluated by western blot (WB). Production of reactive oxygen species (ROS) was examined with H2DCFDA. 7KC significantly induced adhesion of U937 cells to human endothelial cells while significantly increasing gene expressions of ICAM-1 and MCP-1 and decreasing eNOS and CaMKKII gene expressions. EGCG inhibited 7KC-induced monocytic adhesion to endothelial cells, and induced expression of eNOS and several genes involved in the CaMKKII pathway. Stimulation of endothelial cells with EGCG produced intracellular ROS, whereas treatment with N-acetylcysteine (NAC) blocked EGCG-induced expression of eNOS and CaMKKII. These results suggest that inhibition of monocyte-endothelial cell adhesion by EGCG is associated with CaMKKII pathway activation by ROS. Inhibition of 7KC-induced monocyte-endothelial cell adhesion induced by EGCG may function similarly to HDL. Copyright © 2013 Elsevier Inc. All rights reserved.

Apigenin and naringenin ameliorate PKCβII-associated endothelial dysfunction via regulating ROS/caspase-3 and NO pathway in endothelial cells exposed to high glucose.

PubMed

Qin, Weiwei; Ren, Bei; Wang, Shanshan; Liang, Shujun; He, Baiqiu; Shi, Xiaoji; Wang, Liying; Liang, Jingyu; Wu, Feihua

2016-10-01

Endothelial dysfunction is a key event in the progression of atherosclerosis with diabetes. Increasing cell apoptosis may lead to endothelial dysfunction. Apigenin and naringenin are two kinds of widely used flavones. In the present study, we investigated whether and how apigenin and naringenin reduced endothelial dysfunction induced by high glucose in endothelial cells. We showed that apigenin and naringenin protected against endothelial dysfunction via inhibiting phosphorylation of protein kinase C βII (PKCβII) expression and downstream reactive oxygen species (ROS) production in endothelial cells exposed to high glucose. Furthermore, we demonstrated that apigenin and naringenin reduced high glucose-increased apoptosis, Bax expression, caspase-3 activity and phosphorylation of NF-κB in endothelial cells. Moreover, apigenin and naringenin effectively restored high glucose-reduced Bcl-2 expression and Akt phosphorylation. Importantly, apigenin and naringenin significantly increased NO production in endothelial cells subjected to high glucose challenge. Consistently, high glucose stimulation impaired acetylcholine (ACh)-mediated vasodilation in the rat aorta, apigenin and naringenin treatment restored the impaired endothelium-dependent vasodilation via dramatically increasing eNOS activity and nitric oxide (NO) level. Taken together, our results manifest that apigenin and naringenin can ameliorate endothelial dysfunction via regulating ROS/caspase-3 and NO pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

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

Lin, Ming-Chung; Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan; Chen, Chia-Ling

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-likemore » cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase in peritoneal vascular permeability.« less

Hypotonic shock stimulates ascorbate release from coronary artery endothelial cells by a Ca2+ -independent pathway.

PubMed

Davis, Kim A; Samson, Sue E; Wilson, John X; Grover, Ashok K

2006-10-24

In endothelial cells, anion channels open upon osmotic swelling during shear stress and hypotonic shock. Therefore, we examined the effects of hypotonic shock on release of the antioxidant anion ascorbate from pig coronary artery endothelial cells. Hypotonic shock potentiated ascorbate release from freshly isolated or cultured pig coronary artery endothelial cells; subsequently cultured endothelial cells were used. The hypotonic shock-induced increase in Asc release was rapid, depended on the degree of hypotonic shock, and not due to membrane leakiness. Stimulating P2Y2 like receptors in endothelial cells with ATP causes ascorbate release via a Ca2+ -mediated pathway. Hypotonic shock-induced release differed from the Ca2+-mediated Asc release because: (a) the increase in release with hypotonic shock was additive to that with ATP or A23187 (Ca2+ -ionophore), (b) apyrase, suramin or removing extracellular Ca2+ did not affect the hypotonic shock-stimulated release, (c) anion channel blockers inhibited the release by the two pathways differently, and (d) hypotonic shock increased the ascorbate release from endothelial cells and cultured smooth muscle cells whereas the Ca2+ -mediated ascorbate release occurred only in endothelial cells. Accumulation of ascorbate by endothelial cells was examined at extracellular ascorbate concentrations of 10 (Na+ -ascorbate symporter not saturated) and 5000 microM (Na+ -ascorbate symporter saturated). Hypotonic shock and A23187 decreased ascorbate accumulation at 10 microM ascorbate but increased it at 5000 microM. The effects of the two treatments were additive and also differed from each other with substitution of gluconate for extracellular chloride. Thus, ascorbate release from endothelial cells can be potentiated by two distinct pathways - hypotonic shock mediated and ATP/Ca2+ stimulated.

Apicobasal polarity of brain endothelial cells

PubMed Central

Worzfeld, Thomas

2015-01-01

Normal brain homeostasis depends on the integrity of the blood–brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood–brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. PMID:26661193

N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

PubMed Central

Madathil, Bernadette K.; Anil Kumar, Pallickaveedu RajanAsari; Kumary, Thrikkovil Variyath

2014-01-01

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty. PMID:25003113

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

PubMed

Joo, Jihoon E; Hiden, Ursula; Lassance, Luciana; Gordon, Lavinia; Martino, David J; Desoye, Gernot; Saffery, Richard

2013-07-15

The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase. Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes

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

Pakala, Rajbabu; Watanabe, Takuya; Benedict, Claude R

2002-06-01

Introduction: Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. Methods: Endothelial cell proliferation is determined by the amount of [{sup 3}H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels inmore » the conditioned medium were determined by ELISA. Results: Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. Conclusion: Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors.« less

Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

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

Song, Kai; Zhu, Fei; Zhang, Han-zhong

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oralmore » squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black-Right-Pointing-Pointer VCAM-1/VLA-4 mediated TNF-{alpha}-enhanced cell fusions.« less

[Endothelial keratoplasty: Descemet stripping (DSEK) using TAN EndoGlide™ device: case series].

PubMed

Pazos, Henrique Santiago Baltar; Pazos, Paula Fernanda Morais Ramalho Baltar; Nogueira Filho, Pedro Antônio; Grisolia, Ana Beatriz Diniz; Silva, André Berger Emiliano; Gomes, José Álvaro Pereira

2011-01-01

To report the results of Descemet stripping endothelial keratoplasty (DSEK) using the TAN EndoGlideTM device to facilitate the insertion of the endothelial membrane. Prospective clinical study that included nine patients presenting corneal edema secondary to endothelial dysfunction. Best corrected visual acuity, refraction, central corneal thickness, endothelial cell density and complications were analyzed after a six-month follow-up. There was a significant improvement in the corneal edema and visual acuity in 7 patients (77.78%). The best corrected visual acuity ranged between 20/40 and 20/200. The average density of endothelial cells in six months varied between 1,305 cells/mm² and 2,346 cells/mm² with an average loss of 33.14% cells. Detachment of part of the graft was observed in one eye (11.11%) and primary failure of the endothelial transplantation occurred in 2 eyes (22.22%). The device TAN EndoGlideTM facilitates the introduction of the graft in Descemet stripping endothelial keratoplasty.

Pericyte Derived Sphinogosine 1-Phosphate Induces the Expression of Adhesion Proteins and Modulates the Retinal Endothelial Cell Barrier

PubMed Central

McGuire, P.G.; Rangasamy, S.; Maestas, J.; Das, A.

2011-01-01

Objective The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Methods and Results Human retinal microvascular endothelial cells were co-cultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate (S1P). S1P aids in maintenance of microvascular stability by up-regulating the expression of N-cadherin and VE-cadherin, and down-regulating the expression of angiopoietin 2. Conclusion Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of S1P. Alteration of pericyte-derived S1P production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability. PMID:21940944

Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5.

PubMed

Kim, Young; Abplanalp, William A; Jung, Andrew D; Schuster, Rebecca M; Lentsch, Alex B; Gulbins, Erich; Caldwell, Charles C; Pritts, Timothy A

2018-03-01

Microparticles are submicron vesicles shed from aging erythrocytes as a characteristic feature of the red blood cell (RBC) storage lesion. Exposure of pulmonary endothelial cells to RBC-derived microparticles promotes an inflammatory response, but the mechanisms underlying microparticle-induced endothelial cell activation are poorly understood. In the present study, cultured murine lung endothelial cells (MLECs) were treated with microparticles isolated from aged murine packed RBCs or vehicle. Microparticle-treated cells demonstrated increased expression of the adhesion molecules ICAM and E-selectin, as well as the cytokine, IL-6. To identify mechanisms that mediate these effects of microparticles on MLECs, cells were treated with microparticles covalently bound to carboxyfluorescein succinimidyl ester (CFSE) and cellular uptake of microparticles was quantified via flow cytometry. Compared with controls, there was a greater proportion of CFSE-positive MLECs from 15 min up to 24 h, suggesting endocytosis of the microparticles by endothelial cells. Colocalization of microparticles with lysosomes was observed via immunofluorescence, indicating endocytosis and endolysosomal trafficking. This process was inhibited by endocytosis inhibitors. SiRNA knockdown of Rab5 signaling protein in endothelial cells resulted in impaired microparticle uptake as compared with nonsense siRNA-treated cells, as well as an attenuation of the inflammatory response to microparticle treatment. Taken together, these data suggest that endocytosis of RBC-derived microparticles by lung endothelial cells results in endothelial cell activation. This response seems to be mediated, in part, by the Rab5 signaling protein.

Corneal endothelial cell density after femtosecond thin-flap LASIK and PRK for myopia: a contralateral eye study.

PubMed

Smith, Ryan T; Waring, George O; Durrie, Daniel S; Stahl, Jason E; Thomas, Priscilla

2009-12-01

To compare the effect of femtosecond thinflap LASIK and photorefractive keratectomy (PRK) on postoperative endothelial cell density. In a prospective, randomized, contralateral, single-center clinical trial, 25 patients (mean age: 30+/-5 years [range: 21 to 38 years]) underwent PRK in one eye and thin-flap LASIK in the fellow eye for the correction of myopia using a wavefront-guided platform. The central corneal endothelial cell density was measured using the NIDEK Confoscan 4 preoperatively, and at 1 and 3 months postoperatively. Changes in endothelial cell density were analyzed over time between the two refractive techniques. In PRK, the average preoperative endothelial cell density was 3011+/-329 cells/mm(2), which decreased to 2951+/-327 cells/mm(2) at 1 month (P=.5736) and 2982+/-365 cells/mm(2) at 3 months (P=.6513). In thinflap LASIK, the average preoperative endothelial cell density was 2995+/-325 cells/mm(2), which decreased to 2977+/-358 cells/mm(2) at 1 month (P=.5756) and 2931+/-369 cells/mm(2) at 3 months (P=.4106). No statistically significant difference was found between the two groups at 1 (P=.7404) or 3 (P=.3208) months postoperatively. No statistically significant change was noted in endothelial cell density following either PRK or thin-flap LASIK for the treatment of myopia. Furthermore, no statistically significant difference was found between the two groups out to 3 months postoperatively, indicating that thin-flap LASIK is as safe as PRK with regards to endothelial health.

Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration: potential role of soluble pro-angiogenic factors.

PubMed

Aguilera, Valeria; Briceño, Luis; Contreras, Hector; Lamperti, Liliana; Sepúlveda, Esperanza; Díaz-Perez, Francisca; León, Marcelo; Veas, Carlos; Maura, Rafael; Toledo, Jorge Roberto; Fernández, Paulina; Covarrubias, Ambart; Zuñiga, Felipe Andrés; Radojkovic, Claudia; Escudero, Carlos; Aguayo, Claudio

2014-01-01

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo. Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

The endothelial sample size analysis in corneal specular microscopy clinical examinations.

PubMed

Abib, Fernando C; Holzchuh, Ricardo; Schaefer, Artur; Schaefer, Tania; Godois, Ronialci

2012-05-01

To evaluate endothelial cell sample size and statistical error in corneal specular microscopy (CSM) examinations. One hundred twenty examinations were conducted with 4 types of corneal specular microscopes: 30 with each BioOptics, CSO, Konan, and Topcon corneal specular microscopes. All endothelial image data were analyzed by respective instrument software and also by the Cells Analyzer software with a method developed in our lab. A reliability degree (RD) of 95% and a relative error (RE) of 0.05 were used as cut-off values to analyze images of the counted endothelial cells called samples. The sample size mean was the number of cells evaluated on the images obtained with each device. Only examinations with RE < 0.05 were considered statistically correct and suitable for comparisons with future examinations. The Cells Analyzer software was used to calculate the RE and customized sample size for all examinations. Bio-Optics: sample size, 97 ± 22 cells; RE, 6.52 ± 0.86; only 10% of the examinations had sufficient endothelial cell quantity (RE < 0.05); customized sample size, 162 ± 34 cells. CSO: sample size, 110 ± 20 cells; RE, 5.98 ± 0.98; only 16.6% of the examinations had sufficient endothelial cell quantity (RE < 0.05); customized sample size, 157 ± 45 cells. Konan: sample size, 80 ± 27 cells; RE, 10.6 ± 3.67; none of the examinations had sufficient endothelial cell quantity (RE > 0.05); customized sample size, 336 ± 131 cells. Topcon: sample size, 87 ± 17 cells; RE, 10.1 ± 2.52; none of the examinations had sufficient endothelial cell quantity (RE > 0.05); customized sample size, 382 ± 159 cells. A very high number of CSM examinations had sample errors based on Cells Analyzer software. The endothelial sample size (examinations) needs to include more cells to be reliable and reproducible. The Cells Analyzer tutorial routine will be useful for CSM examination reliability and reproducibility.

Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

PubMed

Abeysinghe, Hima C S; Bokhari, Laita; Quigley, Anita; Choolani, Mahesh; Chan, Jerry; Dusting, Gregory J; Crook, Jeremy M; Kobayashi, Nao R; Roulston, Carli L

2015-09-29

Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules.

PubMed

Zhou, Xueping; He, Pingnian

2011-11-01

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

Contribution of endothelial cells to organogenesis: a modern reappraisal of an old Aristotelian concept

PubMed Central

Crivellato, E; Nico, B; Ribatti, D

2007-01-01

It is well established that many tissue-derived factors are involved in blood vessel formation, but evidence is now emerging that endothelial cells themselves represent a crucial source of instructive signals to non-vascular tissue cells during organ development. Thus, endothelial cell signalling is currently believed to promote fundamental cues for cell fate specification, embryo patterning, organ differentiation and postnatal tissue remodelling. This review article summarizes some of the recent advances in our understanding of the role of endothelial cells as effector cells in organ formation. PMID:17683480

Corneal endothelium: developmental strategies for regeneration

PubMed Central

Zavala, J; López Jaime, G R; Rodríguez Barrientos, C A; Valdez-Garcia, J

2013-01-01

The main treatment available for restoration of the corneal endothelium is keratoplasty. This procedure is faced with several difficulties, including the shortage of donor tissue, post-surgical complications associated with the use of drugs to prevent immune rejection, and a significant increase in the occurrence of glaucoma. Recently, surgical procedures such as Descemet's stripping endothelial keratoplasty have focused on the transplant of corneal endothelium, yielding better visual results but still facing the need for donor tissue. The emergent strategies in the field of cell biology and tissue cultivation of corneal endothelial cells aim at the production of transplantable endothelial cell sheets. Cell therapy focuses on the culture of corneal endothelial cells retrieved from the donor, in the donor's cornea, followed by transplantation into the recipient. Recently, research has focused on overcoming the challenge of harvesting human corneal endothelial cells and the generation of new biomembranes to be used as cell scaffolds in surgical procedures. The use of corneal endothelial precursors from the peripheral cornea has also demonstrated to be effective and represents a valuable tool for reducing the risk of rejection in allogeneic transplants. Several animal model reports also support the use of adult stem cells as therapy for corneal diseases. Current results represent important progresses in the development of new strategies based on alternative sources of tissue for the treatment of corneal endotheliopathies. Different databases were used to search literature: PubMed, Google Books, MD Consult, Google Scholar, Gene Cards, and NCBI Books. The main search terms used were: ‘cornea AND embryology AND transcription factors', ‘human endothelial keratoplasty AND risk factors', ‘(cornea OR corneal) AND (endothelium OR endothelial) AND cell culture', ‘mesenchymal stem cells AND cell therapy', ‘mesenchymal stem cells AND cornea', and ‘stem cells AND (cornea OR corneal) AND (endothelial OR endothelium)'. PMID:23470788

Nesting of colon and ovarian cancer cells in the endothelial niche is associated with alterations in glycan and lipid metabolism.

PubMed

Halama, Anna; Guerrouahen, Bella S; Pasquier, Jennifer; Satheesh, Noothan J; Suhre, Karsten; Rafii, Arash

2017-01-04

The metabolic phenotype of a cancer cell is determined by its genetic makeup and microenvironment, which dynamically modulates the tumor landscape. The endothelial cells provide both a promoting and protective microenvironment - a niche for cancer cells. Although metabolic alterations associated with cancer and its progression have been fairly defined, there is a significant gap in our understanding of cancer metabolism in context of its microenvironment. We deployed an in vitro co-culture system based on direct contact of cancer cells with endothelial cells (E4 + EC), mimicking the tumor microenvironment. Metabolism of colon (HTC15 and HTC116) and ovarian (OVCAR3 and SKOV3) cancer cell lines was profiled with non-targeted metabolic approaches at different time points in the first 48 hours after co-culture was established. We found significant, coherent and non-cell line specific changes in fatty acids, glycerophospholipids and carbohydrates over time, induced by endothelial cell contact. The metabolic patterns pinpoint alterations in hexosamine biosynthetic pathway, glycosylation and lipid metabolism as crucial for cancer - endothelial cells interaction. We demonstrated that "Warburg effect" is not modulated in the initial stage of nesting of cancer cell in the endothelial niche. Our study provides novel insight into cancer cell metabolism in the context of the endothelial microenvironment.

Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor

PubMed Central

Bierings, Ruben; Meems, Henriet; Mul, Frederik P. J.; Geerts, Dirk; Vlaar, Alexander P. J.; Voorberg, Jan; Hordijk, Peter L.

2017-01-01

Adhesion of erythrocytes to endothelial cells lining the vascular wall can cause vaso-occlusive events that impair blood flow which in turn may result in ischemia and tissue damage. Adhesion of erythrocytes to vascular endothelial cells has been described in multiple hemolytic disorders, especially in sickle cell disease, but the adhesion of normal erythrocytes to endothelial cells has hardly been described. It was shown that calcium-loaded erythrocytes can adhere to endothelial cells. Because sickle erythrocyte adhesion to ECs can be enhanced by ultra-large von Willebrand factor multimers, we investigated whether calcium loading of erythrocytes could promote binding to endothelial cells via ultra-large von Willebrand factor multimers. We used (immunofluorescent) live-cell imaging of washed erythrocytes perfused over primary endothelial cells at venular flow rate. Using this approach, we show that calcium-loaded erythrocytes strongly adhere to histamine-stimulated primary human endothelial cells. This adhesion is mediated by ultra-large von Willebrand factor multimers. Von Willebrand factor knockdown or ADAMTS13 cleavage abolished the binding of erythrocytes to activated endothelial cells under flow. Platelet depletion did not interfere with erythrocyte binding to von Willebrand factor. Our results reveal platelet-independent adhesion of calcium-loaded erythrocytes to endothelium-derived von Willebrand factor. Erythrocyte adhesion to von Willebrand factor may be particularly relevant for venous thrombosis, which is characterized by the formation of erythrocyte-rich thrombi. PMID:28249049

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay.

PubMed

Lazarovici, Philip; Lahiani, Adi; Gincberg, Galit; Haham, Dikla; Fluksman, Arnon; Benny, Ofra; Marcinkiewicz, Cezary; Lelkes, Peter I

2018-01-01

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

Low oxygen tension enhances endothelial fate of human pluripotent stem cells.

PubMed

Kusuma, Sravanti; Peijnenburg, Elizabeth; Patel, Parth; Gerecht, Sharon

2014-04-01

A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor β+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.

INDUCTION OF IMMUNOLOGIC TOLERANCE IN OLDER NEW ZEALAND MICE REPOPULATED WITH YOUNG SPLEEN, BONE MARROW, OR THYMUS

PubMed Central

Staples, Parker J.; Steinberg, Alfred D.; Talal, Norman

1970-01-01

Newborn, 7–9 day, and 16–18 day old NZB and B/W mice were, unlike older New Zealand mice, rendered tolerant to single doses of 8–10 mg of soluble BGG. After challenge, this tolerance was of short duration and escape occurred rapidly. Age-matched and similarly treated C3H, Balb/c and C57Bl mice did not escape from tolerance. Partial tolerance could be maintained by repeated injections of BGG. Biofiltration ruled out hyperphagocytosis as an explanation for this resistance to tolerance. Tolerance could be induced in older B/W mice if they were thymectomized, irradiated, and repopulated with young (12–15 day), but not old (2–3 month), spleen or bone marrow cells. Old bone marrow cells gave a non-tolerant response even when combined with young thymic grafts. Young bone marrow gave a tolerant response which was followed by the expected rapid escape only if a young thymus graft was also present. Escape was retarded if old thymus, or old irradiated thymus, was combined with young bone marrow. These results are best explained by abnormalities of both lymphoid precursors and thymic regulation. PMID:4192570

Differential procoagulant activity of microparticles derived from monocytes, granulocytes, platelets and endothelial cells: impact of active tissue factor.

PubMed

Shustova, Olga N; Antonova, Olga A; Golubeva, Nina V; Khaspekova, Svetlana G; Yakushkin, Vladimir V; Aksuk, Svetlana A; Alchinova, Irina B; Karganov, Mikhail Y; Mazurov, Alexey V

2017-07-01

: Microparticles released by activated/apoptotic cells exhibit coagulation activity as they express phosphatidylserine and some of them - tissue factor. We compared procoagulant properties of microparticles from monocytes, granulocytes, platelets and endothelial cells and assessed the impact of tissue factor in observed differences. Microparticles were sedimented (20 000g, 30 min) from the supernatants of activated monocytes, monocytic THP-1 cells, granulocytes, platelets and endothelial cells. Coagulation activity of microparticles was examined using plasma recalcification assay. The size of microparticles was evaluated by dynamic light scattering. Tissue factor activity was measured by its ability to activate factor X. All microparticles significantly accelerated plasma coagulation with the shortest lag times for microparticles derived from monocytes, intermediate - for microparticles from THP-1 cells and endothelial cells, and the longest - for microparticles from granulocytes and platelets. Average diameters of microparticles ranged within 400-600 nm. The largest microparticles were produced by endothelial cells and granulocytes, smaller - by monocytes, and the smallest - by THP-1 cells and platelets. The highest tissue factor activity was detected in microparticles from monocytes, lower activity - in microparticles from endothelial cells and THP-1 cells, and no activity - in microparticles from platelets and granulocytes. Anti-tissue factor antibodies extended coagulation lag times for microparticles from monocytes, endothelial cells and THP-1 cells and equalized them with those for microparticles from platelets and granulocytes. Higher coagulation activity of microparticles from monocytes, THP-1 cells and endothelial cells in comparison with microparticles from platelets and granulocytes is determined mainly by the presence of active tissue factor.

Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.

PubMed

Hamed, Saher; Ben-Nun, Ohad; Egozi, Dana; Keren, Aviad; Malyarova, Nastya; Kruchevsky, Danny; Gilhar, Amos; Ullmann, Yehuda

2012-10-01

Bone marrow-derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that was isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell-treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell-treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.

A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.

PubMed

Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun

2018-07-01

Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland-Altman plot shows that 95% of the data are between the 2SD agreement lines. We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.

Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

PubMed Central

2014-01-01

Background Bone marrow mesenchymal stem cells (BM-MSCs) are capable of differentiating into endothelial cells in vitro and acquire major characteristics of mature endothelial-like expression of vWF and CD31. SFAs and lipid oxidation products have been linked with postprandial endothelial dysfunction. Consumption of SFAs impairs arterial endothelial function, while a Mediterranean-type MUFA-diet has a beneficial effect on endothelial function by producing a decrease in levels of vWF, TFPI and PAI-1. Stearoyl-CoA desaturase 1 (SCD1), which converts SFA to MUFA, is involved in the cellular biosynthesis of MUFAs from SFA substrates. High expression of SCD1 is corresponded with low rates of fatty acid oxidation, therefore it might reduce inflammatory responses and be beneficial for the growth of induced endothelial cells. Overexpression of SCD1 in BM-MSCs might increase the growth of induced endothelial cells. The goal of this research is to study the relationship between overexpression of SCD1 and the expression of induced endothelial cells in BM-MSCs in vitro. Methods The gene SCD1 was integrated into a lentiviral vector, and then 293 T cells were transfected by the connected product to produce a packaged virus. BM-MSCs were infected by the packaged virus. Cell culture and endothelial induction were performed. Fluorescent quantitative PCR of CD31, vWF and VE-cad was performed after 1 week and 2 weeks to test the growth of induced endothelial cells. Results The mRNA amount of CD31, vWF and VE-cad of the SCD1 overexpressed group was statistically higher than that of the empty vector (EV) group and that of the normal group after 1 week and 2 weeks, respectively (p < 0.05). Immunocytochemical staining of CD31 or vWF was detected by visualizing red color. Conclusions This study suggested that overexpression of SCD1 in BM-MSCs could increase the expression of induced endothelial cells in vitro. PMID:24650127

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394

Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development.

PubMed

Georgescu, Adriana; Alexandru, Nicoleta; Andrei, Eugen; Dragan, Emanuel; Cochior, Daniel; Dias, Sérgio

2016-08-01

Atherosclerosis is an inflammatory disease, in which risk factors such as hyperlipidemia and hypertension affect the arterial endothelium, resulting in dysfunction, cell damage or both. The number of circulating endothelial progenitor cells and microparticles provides invaluable outcome prediction for atherosclerosis disease. However, evidence for the therapeutic potential of endothelial progenitor cells and microparticles in atherosclerosis development is limited. Our study was designed to investigate the possible protective role of a cell therapy-based approach, using endothelial progenitor cells and the dual behaviour of circulating platelet microparticles, on atherosclerosis development in hypertensive-hypercholesterolemic hamster model. Consequently, control hamsters received four intravenous inoculations of: (1) 1×10(5) endothelial progenitor cells of healthy origins in one dose per month, during four months of diet-induced atherosclerosis, and after hypertensive-hypercholesterolemic diet for further four months; (2) in a second set of experiments, 1×10(5) endothelial progenitor cells of healthy origins or/and 1×10(5) platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive-hypercholesterolemic diet. Endothelial progenitor cell treatment had the following effects: (1) re-established plasmatic parameters: cholesterol and triglyceride concentrations, blood pressure, heart rate, cytokine and chemokine profiles, platelet microparticle pro-thrombotic activity and endothelial progenitor cell paracrine activity reflected by cytokine/chemokine detection; (2) reduced lipid, macrophage and microparticle accumulation in liver; (3) reduced atherosclerosis development, revealed by decreased lipid, macrophage and microparticle content of arterial wall; (4) induced the recruitment and incorporation of endothelial progenitor cells into liver and arterial wall; (5) improved arterial dysfunction by increasing contraction and relaxation; (6) reduced the protein expression of specific pro-inflammatory molecules in liver and arterial wall. Platelet microparticle transplantation aggravated the above-mentioned biomarkers and atherosclerosis process, which were partially reverted with co-inoculation of platelet microparticles and endothelial progenitor cells. With this study, we demonstrate in a hypertensive-hypercholesterolemic hamster model, that the endothelial progenitor cell-based therapy suppresses the development of atherosclerosis and reduces hepatic lipid and macrophage accumulation with the consequent alleviation of dyslipidaemia and hypertension. Our results support the notion that increasing the number of circulating endothelial progenitor cells by different ways could be a promising therapeutic tool for atherosclerosis. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

PubMed

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

PubMed Central

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer. PMID:21915264

Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells

PubMed Central

Garner, Omai B.; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C.; Park, Arnold; Bowden, Thomas A.; Freiberg, Alexander N.

2014-01-01

ABSTRACT Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. IMPORTANCE Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. PMID:25505064

Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish

PubMed Central

Kanada, Masamitsu; Zhang, Jinyan; Yan, Libo; Sakurai, Takashi

2014-01-01

The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies. PMID:25551022

Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish.

PubMed

Kanada, Masamitsu; Zhang, Jinyan; Yan, Libo; Sakurai, Takashi; Terakawa, Susumu

2014-01-01

The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies.

A versatile microfluidic platform for the study of cellular interactions between endothelial cells and neutrophils.

PubMed

Wu, Xiaojie; Newbold, Molly A; Gao, Zhe; Haynes, Christy L

2017-05-01

Endothelial migration is a critical physiological process during vascular angiogenesis, growth and development, as well as in various disease conditions, such as cancer and cardiovascular diseases. Neutrophil migration, known as the important characteristic of immune responses, is also recognized as a contributor to the diseases involving endothelial migration. Herein, the mutually dependent relationship between neutrophil recruitment and endothelial migration was studied on a microfluidic platform for the first time. An in vivo-like microenvironment is created inside microfluidic devices by embedding a gel scaffold into the micro-chambers. This approach, with controllable stable chemical gradients and the ability to quantitate interaction characteristics, overcomes the limitations of the current in vivo and in vitro assays for cell migration studies. The number of neutrophils migrating through the endothelial cell layer is heavily influenced by the concentration of vascular endothelial growth factor (VEGF) that induces endothelial cell migration in the gel scaffold, and is not as correlated to the concentration of chemokine solution used for initiating neutrophil migration. More importantly, neutrophil migration diminishes the effects of the drug that inhibits endothelial migration and this process is regulated by the concentration of chemokine molecules instead of VEGF concentration. The results presented herein demonstrate the complicated cellular interactions between endothelial cells and neutrophils: endothelial migration delicately regulates neutrophil migration while the presence of neutrophils stabilizes the structures of endothelial migration. This study provides deeper understanding of the dynamic cellular interactions between neutrophils and endothelial cells as well as the pathogenesis of relevant diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3.

PubMed

Koenig, M N; Naik, E; Rohrbeck, L; Herold, M J; Trounson, E; Bouillet, P; Thomas, T; Voss, A K; Strasser, A; Coultas, L

2014-11-01

The growth of new blood vessels by angiogenesis is essential for normal development, but can also cause or contribute to the pathology of numerous diseases. Recent studies have shown that BIM, a pro-apoptotic BCL2-family protein, is required for endothelial cell apoptosis in vivo, and can contribute to the anti-angiogenic effect of VEGF-A inhibitors in certain tumor models. Despite its importance, the extent to which BIM is autonomously required for physiological endothelial apoptosis remains unknown and its regulation under such conditions is poorly defined. While the transcription factor FOXO3 has been proposed to induce Bim in response to growth factor withdrawal, evidence for this function is circumstantial. We report that apoptosis was reduced in Bim(-/-) primary endothelial cells, demonstrating a cell-autonomous role for BIM in endothelial death following serum and growth factor withdrawal. In conflict with in vitro studies, BIM-dependent endothelial death in vivo did not require FOXO3. Moreover, endothelial apoptosis proceeded normally in mice lacking FOXO-binding sites in the Bim promoter. Bim mRNA was upregulated in endothelial cells starved of serum and growth factors and this was accompanied by the downregulation of miRNAs of the miR-17∼92 cluster. Bim mRNA levels were also elevated in miR-17∼92(+/-) endothelial cells cultured under steady-state conditions, suggesting that miR-17∼92 cluster miRNAs may contribute to regulating overall Bim mRNA levels in endothelial cells.

Diverse Functional Outcomes of Plasmodium falciparum Ligation of EPCR: Potential Implications for Malarial Pathogenesis

PubMed Central

Gillrie, Mark R.; Avril, Marion; Brazier, Andrew J.; Davis, Shevaun P.; Stins, Monique F.; Smith, Joseph D.; Ho, May

2015-01-01

Summary P. falciparum-infected erythrocytes (IRBC) expressing the domain cassettes (DC) 8 and 13 of the cytoadherent ligand PfEMP1 adhere to the endothelial protein C receptor (EPCR). By interfering with EPCR anti-coagulant and pro-endothelial barrier functions, IRBC adhesion could promote coagulation and vascular permeability that contribute to the pathogenesis of cerebral malaria. In this study, we examined adhesion of DC8- and DC13-expressing parasite lines to endothelial cells from different microvasculature, and the consequences of EPCR engagement on endothelial cell function. We found that IRBC from IT4var19 (DC8) and IT4var07 (DC13) parasite lines adhered to human brain, lung, and dermal endothelial cells under shear stress. However, the relative contribution of EPCR to parasite cytoadherence on the different types of endothelial cell varied. We also observed divergent functional outcomes for DC8 CIDRα1.1 and DC13 CIDRα1.4 domains. IT4var07 CIDRα1.4 inhibited generation of activated protein C (APC) on lung and dermal endothelial cells and blocked the APC-EPCR binding interaction on brain endothelial cells. IT4var19 CIDRα1.1 inhibited thrombin-induced endothelial barrier dysfunction in lung endothelial cells, while IT4var07 CIDRα1.4- inhibited the protective effect of APC on thrombin-induced permeability. Overall, these findings reveal a much greater complexity of how CIDRα1-expressing parasites may modulate malaria pathogenesis through EPCR adhesion. PMID:26119044

Intestinal and peri-tumoral lymphatic endothelial cells are resistant to radiation-induced apoptosis

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

Sung, Hoon Ki; Department of Anatomy, Yeung Nam University Medical School, Daegu 705-717; Morisada, Tohru

2006-06-30

Radiation therapy is a widely used cancer treatment, but it is unable to completely block cancer metastasis. The lymphatic vasculature serves as the primary route for metastatic spread, but little is known about how lymphatic endothelial cells respond to radiation. Here, we show that lymphatic endothelial cells in the small intestine and peri-tumor areas are highly resistant to radiation injury, while blood vessel endothelial cells in the small intestine are relatively sensitive. Our results suggest the need for alternative therapeutic modalities that can block lymphatic endothelial cell survival, and thus disrupt the integrity of lymphatic vessels in peri-tumor areas.

Patients with Dry Eye Disease and Low Subbasal Nerve Density are at High Risk for an Accelerated Corneal Endothelial Cell Loss

PubMed Central

Kheirkhah, Ahmad; Satitpitakul, Vannarut; Hamrah, Pedram; Dana, Reza

2016-01-01

Purpose To evaluate the changes in corneal endothelial cell density (CECD) over time in patients with dry eye disease (DED) and to correlate the endothelial cell loss with corneal subbasal nerve density. Methods This retrospective study included 40 eyes of 20 patients with DED. Laser in vivo confocal microscopy had been performed in the central cornea of both eyes at an initial visit and repeated after a mean follow-up of 33.2 ± 10.2 months. The densities of corneal endothelial cells and subbasal nerves were measured in both visits and compared with 13 eyes of 13 normal age-matched controls. Results At the initial visit, the DED group had lower densities of corneal endothelial cells (2620 ± 386 cells/mm2) and subbasal nerves (17.8 ± 7.5 mm/mm2) compared with the control group (2861 ± 292 cells/mm2 and 22.8 ± 3.0 mm/mm2, with P=0.08 and P=0.01, respectively). At the end of follow-up, although there was no significant change in subbasal nerve density (16.7 ± 7.2 mm/mm2, P=0.43), the mean CECD significantly decreased to 2465 ± 391 cells/mm2 (P=0.01), with a mean corneal endothelial cell loss of 2.1 ± 3.6% per year. The endothelial cell loss showed a statistically significant negative correlation with the initial subbasal nerve density (Rs= −0.55, P=0.02). Conclusion Patients with DED have an accelerated corneal endothelial cell loss which is more than what has been reported in the literature for normal aging. Those with lower subbasal nerve density, in particular, are at a higher risk for endothelial cell loss over time. PMID:28060067

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression.

PubMed

Xia, Zhengyuan; Liu, Min; Wu, Yong; Sharma, Vijay; Luo, Tao; Ouyang, Jingping; McNeill, John H

2006-11-21

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.

[Isolation and culture of bovine choriocapillary endothelial cells using paramagnetic beads coated with Lycopersicon esculentum].

PubMed

Swiech-Zubilewicz, A; Soubrane, G; Mascarelli, F

2000-01-01

To establish a pure culture of choriocapillary endothelial cells as a model of angiogenesis in vitro. Bovine choriocapillary endothelial cells (BCEC) were obtained by the method described by Hoffmann et al. (6) using the polystyrene paramagnetic beads coated with Lycopersicon esculentum, which attach specifically to the rest of fucose on the surface of microvascular endothelial cells. The endothelial characteristic of the cultured cells was evaluated by immunocytochemistry using anti von Willebrand factor and anti-CD 31 antibodies. Proliferation and survival of BCEC were tested using haemacytometer of Mallasez. The purity of obtained BCEC culture was confirmed by positive immunocytochemical staining with anti von Willebrand and anti factor CD 31 antibodies in more than 95% of cells. The proliferation of cells in Endothelial Cell Medium resulted in twofold increase of number of cells during 4-day observation period. After reaching the confluence, the cells continued to proliferate with increase of the cell number by 60% during 4-day observation. The use of paramagnetic beads coated with specific lectine provide a pure isolation of BCEC, which can be maintained in culture with preservation of their characteristic.

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

2013-10-29

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2

PubMed Central

Eichmann, Anne; Corbel, Catherine; Nataf, Valérie; Vaigot, Pierre; Bréant, Christiane; Le Douarin, Nicole M.

1997-01-01

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation. PMID:9144204

Establishment and characterization of an angiosarcoma-derived cell line, AS-M.

PubMed

Krump-Konvalinkova, Vera; Bittinger, Fernando; Olert, Jürgen; Bräuninger, Wolfgang; Brunner, Joachim; Kirkpatrick, C James

2003-01-01

A novel human endothelial cell line, AS-M, has been established from a cutaneous angiosarcoma on the scalp. The cells expressing platelet endothelial cell adhesion molecule-1 (CD31) were isolated using magnetic beads and subsequently cultured for a year. To date, the cells have undergone more than 100 population doublings (PDs). The AS-M cells manifested endothelial characteristics, such as active uptake of acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL), capacity to bind the Ulex europeaus agglutin-I (UEA-I), and expression of von Willebrand factor (vWF) and CD31. The single cell-derived clone, AS-M.5, showed a constitutive expression of CD31, vWF, angiotensin-converting enzyme (ACE), endoglin (CD105), and the endothelial cell receptor tyrosine kinases KDR and Tie-1. Similarly to freshly isolated endothelial cells, the AS-M.5 responded to induction by bacterial lipopolysaccharide (LPS) by increased transcription of cell adhesion molecules and cytokines. The AS-M.5 cultures required endothelial growth supplements for optimal growth and long-term propagation in vitro. However, in contrast to normal endothelial cells, p53 gene products were detected in nuclei of AS-M.5 cells. Cytogenetic analyses consistently revealed a hypodiploid karyotype with complete loss of one homologue of several chromosomes and a homogeneous pattern of distinct karyotypic changes. Although the AS-M.5 presented characteristics suggestive of tumor cells, they did not develop into tumors when inoculated subcutaneously into nude mice. The cell line AS-M.5 could be a useful model system to study endothelial pathobiology in vitro.

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

Infection of endothelial cells by common human viruses.

PubMed

Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

Pulmonary endothelial pavement patterns.

PubMed Central

Kibria, G; Heath, D; Smith, P; Biggar, R

1980-01-01

The appearance of the endothelial pavement pattern was studied in the pulmonary trunk, pulmonary veins, aorta, and inferior vena cava of the rat by means of silver staining of the cell borders. The endothelial cell in each of the four blood vessels was found to have its own distinctive shape, fusiform and pointed in the direction of blood flow in the case of the aorta and larger and more rectangular in the pulmonary trunk and pulmonary veins. Detailed quantitation of the dimensions and surface area of the endothelial cells in each blood vessel was carried out by a photographic technique. Pulmonary hypertension was induced in one group of rats by feeding them on Crotalaria spectabilis seeds. The endothelial pavement pattern in their pulmonary trunks became disrupted with many of the cells assuming a fusiform shape reminiscent of aortic endothelium. Many small, new endothelial cells formed in the pulmonary trunk suggesting division of cells to line the enlarging blood vessels. In contrast the endothelial cells of the inferior vena cava merely increased in size to cope with the dilatation of this vein. Images PMID:7385090

Femtosecond laser cutting of endothelial grafts: comparison of endothelial and epithelial applanation.

PubMed

Bernard, Aurélien; He, Zhiguo; Gauthier, Anne Sophie; Trone, Marie Caroline; Baubeau, Emmanuel; Forest, Fabien; Dumollard, Jean Marc; Peocʼh, Michel; Thuret, Gilles; Gain, Philippe

2015-02-01

Stromal surface quality of endothelial lamellae cut for endothelial keratoplasty with a femtosecond laser (FSL) with epithelial applanation remains disappointing. Applanation of the endothelial side of the cornea, mounted inverted on an artificial chamber, has therefore been proposed to improve cut quality. We compared lamellar quality after FSL cutting using epithelial versus endothelial applanation. Lamellae were cut with an FSL from organ-cultured corneas. After randomization, 7 were cut with epithelial applanation and 7 with endothelial applanation. Lamellae of 50-, 75-, and 100-μm thickness were targeted. Thickness was measured by optical coherence tomography before and immediately after cutting. Viable endothelial cell density was quantified immediately after cutting using triple labeling with Hoechst/ethidium/calcein-AM coupled with image analysis with ImageJ. The stromal surface was evaluated by 9 masked observers using semiquantitative scoring of scanning electronic microscopy images. Histology of 2 samples was also analyzed before lamellar detachment. Precision (difference in target/actual thickness) and thickness regularity [coefficient of variation (CV) of 10 measurements] were significantly better with endothelial applanation (precision: 18 μm; range, 10-30; CV: 11%; range, 8-12) than with epithelial applanation (precision: 84 μm; range, 54-107; P = 0.002; CV: 24%; range, 13-47; P = 0.001). Endothelial applanation provided thinner lamellae. However, viable endothelial cell density was significantly lower after endothelial applanation (1183 cells/mm2; range, 787-1725 versus 1688 cells/mm2; range, 1288-2025; P = 0.018). FSL cutting of endothelial lamellae using endothelial applanation provides thinner more regular grafts with more predictable thickness than with conventional epithelial applanation but strongly reduces the pool of viable endothelial cells.

Endothelial cell density after photorefractive keratectomy for moderate myopia using a 213 nm solid-state laser system.

PubMed

Tsiklis, Nikolaos S; Kymionis, George D; Pallikaris, Aristofanis I; Diakonis, Vasilios F; Ginis, Harilaos S; Kounis, George A; Panagopoulou, Sophia I; Pallikaris, Ioannis G

2007-11-01

To evaluate whether photorefractive keratectomy (PRK) for moderate myopia using a solid-state laser with a wavelength of 213 nm alters the corneal endothelial cell density. University refractive surgery center. The corneal endothelium was analyzed preoperatively and 1, 6, and 12 months postoperatively using corneal confocal microscopy (modified HRT II with a Rostock Cornea Module, Heidelberg Engineering) in 60 eyes (30 patients). Patients were randomized to have myopic PRK using a 213 nm wavelength solid-state laser (study group) or a conventional 193 nm wavelength excimer laser (control group). Three endothelial images were acquired in each of 30 preoperative normal eyes to evaluate the repeatability of endothelial cell density measurements. Repeated-measures analysis of variance was used to compare the variations in endothelial cell density between the 2 lasers and the changes in endothelial cell density over time. There were no statistically significant differences in sex, age, corneal pachymetry, attempted correction, preoperative endothelial cell density, or postoperative refractive outcomes (uncorrected visual acuity, best spectacle-corrected visual acuity, and spherical equivalent refraction) between the 2 groups (P>.05). The coefficient of repeatability of endothelial cell density was 131 cells/mm(2). The measured endothelial cell count per 1.0 mm(2) did not significantly change up to 1 year postoperatively in either group (both P>.05). No statistically significant difference was found between the 2 groups in any postoperative interval (P>.05). Photorefractive keratectomy for moderate myopia using a 213 nm wavelength solid-state laser or a conventional 193 nm wavelength excimer laser did not significantly affect corneal endothelial density during the 1-year postoperative period.

A Pilot Study Linking Endothelial Injury in Lungs and Kidneys in Chronic Obstructive Pulmonary Disease

PubMed Central

Laucho-Contreras, Maria E.; Petersen, Hans; Bijol, Vanesa; Sholl, Lynette M.; Choi, Mary E.; Divo, Miguel; Pinto-Plata, Victor; Chetta, Alfredo; Tesfaigzi, Yohannes; Celli, Bartolomé R.

2017-01-01

Rationale: Patients with chronic obstructive pulmonary disease (COPD) frequently have albuminuria (indicative of renal endothelial cell injury) associated with hypoxemia. Objectives: To determine whether (1) cigarette smoke (CS)-induced pulmonary and renal endothelial cell injury explains the association between albuminuria and COPD, (2) CS-induced albuminuria is linked to increases in the oxidative stress–advanced glycation end products (AGEs) receptor for AGEs (RAGE) pathway, and (3) enalapril (which has antioxidant properties) limits the progression of pulmonary and renal injury by reducing activation of the AGEs–RAGE pathway in endothelial cells in both organs. Methods: In 26 patients with COPD, 24 ever-smokers without COPD, 32 nonsmokers who underwent a renal biopsy or nephrectomy, and in CS-exposed mice, we assessed pathologic and ultrastructural renal lesions, and measured urinary albumin/creatinine ratios, tissue oxidative stress levels, and AGEs and RAGE levels in pulmonary and renal endothelial cells. The efficacy of enalapril on pulmonary and renal lesions was assessed in CS-exposed mice. Measurements and Main Results: Patients with COPD and/or CS-exposed mice had chronic renal injury, increased urinary albumin/creatinine ratios, and increased tissue oxidative stress and AGEs-RAGE levels in pulmonary and renal endothelial cells. Treating mice with enalapril attenuated CS-induced increases in urinary albumin/creatinine ratios, tissue oxidative stress levels, endothelial cell AGEs and RAGE levels, pulmonary and renal cell apoptosis, and the progression of chronic renal and pulmonary lesions. Conclusions: Patients with COPD and/or CS-exposed mice have pulmonary and renal endothelial cell injury linked to increased endothelial cell AGEs and RAGE levels. Albuminuria could identify patients with COPD in whom angiotensin-converting enzyme inhibitor therapy improves renal and lung function by reducing endothelial injury. PMID:28085500

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells.

PubMed

Becher, Tobias; Schulze, Torsten J; Schmitt, Melanie; Trinkmann, Frederik; El-Battrawy, Ibrahim; Akin, Ibrahim; Kälsch, Thorsten; Borggrefe, Martin; Stach, Ksenija

2017-01-15

Lipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. After a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. In this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Copyright © 2016. Published by Elsevier Ireland Ltd.

Angiogenesis in the reparatory mucosa of the mandibular edentulous ridge is driven by endothelial tip cells.

PubMed

Stănescu, Ruxandra; Didilescu, Andreea Cristiana; Jianu, Adelina Maria; Rusu, M C

2012-01-01

Sprouting angiogenesis is led by specialized cell--the endothelial tip cells (ETCs) which can be targeted by pro- or anti-angiogenic therapies. We aimed to perform a qualitative study in order to assess the guidance by tip cells of the endothelial sprouts in the repairing mucosa of the edentulous mandibular crest. Mucosa of the mandibular edentulous ridge was collected from six adult patients, prior to healing abutment placement (second surgery). Slides were prepared and immunostained with antibodies for CD34 and Ki67. The abundant vasculature of the lamina propria was observed on slides and the CD34 antibodies labeled endothelial tip cells in various stages of the endothelial sprouts. Ki67 identified positive endothelial cells, confirming the proliferative status of the microvascular bed. According to the results, the in situ sprouting angiogenesis is driven by tip cells in the oral mucosa of the edentulous ridge and these cells can be targeted by various therapies, as required by the local pathologic or therapeutic conditions.

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

PubMed Central

Larochelle, Andre; Choi, Uimook; Shou, Yan; Naumann, Nora; Loktionova, Natalia A.; Clevenger, Joshua R.; Krouse, Allen; Metzger, Mark; Donahue, Robert E.; Kang, Elizabeth; Stewart, Clinton; Persons, Derek; Malech, Harry L.; Dunbar, Cynthia E.; Sorrentino, Brian P.

2009-01-01

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen. PMID:19509470

Establishment of pancreatic microenvironment model of ER stress: Quercetin attenuates β-cell apoptosis by invoking nitric oxide-cGMP signaling in endothelial cells.

PubMed

Suganya, Natarajan; Mani, Krishna Priya; Sireesh, Dornadula; Rajaguru, Palanisamy; Vairamani, Mariappanadar; Suresh, Thiruppathi; Suzuki, Takayoshi; Chatterjee, Suvro; Ramkumar, Kunka Mohanram

2018-05-01

The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic β-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 μM) confers protection of pancreatic β-cells against ER stress through nitric oxide (NO ∙ ) signaling. In addition, increased intracellular insulin and NO ∙ -mediated cyclic 3',5'-guanosine monophosphate (cGMP) levels in pancreatic β-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic β-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators. Copyright © 2018 Elsevier Inc. All rights reserved.

Kisspeptin-10 induces endothelial cellular senescence and impaired endothelial cell growth.

PubMed

Usui, Sayaka; Iso, Yoshitaka; Sasai, Masahiro; Mizukami, Takuya; Mori, Hiroyoshi; Watanabe, Takuya; Shioda, Seiji; Suzuki, Hiroshi

2014-07-01

The KPs (kisspeptins) are a family of multifunctional peptides with established roles in cancer metastasis, puberty and vasoconstriction. The effects of KPs on endothelial cells have yet to be determined. The aim of the present study was to investigate the effects of KP-10 on endothelial cell growth and the mechanisms underlying those effects. The administration of recombinant KP-10 into the hindlimbs of rats with ischaemia significantly impaired blood flow recovery, as shown by laser Doppler, and capillary growth, as shown using histology, compared with the controls. HUVECs (human umbilical vein endothelial cells) express the KP receptor and were treated with KP-10 in culture studies. KP-10 inhibited endothelial cell tube formation and proliferation in a significant and dose-dependent manner. The HUVECs treated with KP exhibited the senescent phenotype, as determined using a senescence-associated β-galactosidase assay, cell morphology analysis, and decreased Sirt1 (sirtuin 1) expression and increased p53 expression shown by Western blot analysis. Intriguingly, a pharmacological Rho kinase inhibitor, Y-27632, was found to increase the proliferation of HUVECs and to reduce the number of senescent phenotype cells affected by KP-10. In conclusion, KP-10 suppressed endothelial cells growth both in vivo and in vitro in the present study. The adverse effect of KP on endothelial cells was attributable, at least in part, to the induction of cellular senescence.

Systemic administration of thrombin peptide TP508 enhances VEGF-stimulated angiogenesis and attenuates effects of chronic hypoxia

PubMed Central

Olszewska-Pazdrak, Barbara; Carney, Darrell H.

2015-01-01

Revascularization of chronic wounds and ischemic tissue is attenuated by endothelial dysfunction and the inability of angiogenic factors to stimulate angiogenesis. We recently showed that TP508, a nonproteolytic thrombin peptide, increases perfusion and NO-dependent vasodilation in hearts with chronic ischemia and stimulates NO production by endothelial cells. In this study, we investigated systemic in vivo effects of TP508 on VEGF-stimulated angiogenesis in vitro using aortic explants in normoxic and hypoxic conditions. Mice were injected with saline or TP508 and 24h later aortas were removed and cultured to quantify endothelial sprouting. TP508 injection increased endothelial sprouting and potentiated the in vitro response to VEGF. Exposure of control explants to hypoxia inhibited basal and VEGF-stimulated endothelial cell sprouting. This effect of hypoxia was significantly prevented by TP508 injection. Thus, TP508 systemic administration increases responsiveness of aortic endothelial cells to VEGF and diminishes the effect of chronic hypoxia on endothelial cell sprouting. Studies using human endothelial cells in culture suggest that protective effects of TP508 during hypoxia may involve stimulation of endothelial cell NO production. These data suggest potential clinical benefit of using a combination of systemic TP508 and local VEGF as a therapy for revascularization of ischemic tissue. PMID:23594718

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

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

Vallon, Mario, E-mail: m.vallon@arcor.de; Rohde, Franziska; Janssen, Klaus-Peter

2010-02-01

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile,more » an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.« less

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

PubMed

Williamson, Matthew R; Shuttleworth, Adrian; Canfield, Ann E; Black, Richard A; Kielty, Cay M

2007-12-01

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Downregulation of endothelial adhesion molecules by dimethylfumarate, but not monomethylfumarate, and impairment of dynamic lymphocyte-endothelial cell interactions.

PubMed

Wallbrecht, Katrin; Drick, Nora; Hund, Anna-Carina; Schön, Michael P

2011-12-01

Although fumaric acid esters (FAE) have a decade-long firm place in the therapeutic armamentarium for psoriasis, their pleiotropic mode of action is not yet fully understood. While most previous studies have focused on the effects of FAE on leucocytes, we have addressed their activity on macro- and microvascular endothelial cells. As detected both on mRNA and protein levels, dimethylfumarate effected a profound reduction of TNFα-induced expression of E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106) on two different endothelial cell populations in a concentration-dependent manner. This reduction of several endothelial adhesion molecules was accompanied by a dramatic diminution of both rolling and firm adhesive interactions between endothelial cells and lymphocytes in a dynamic flow chamber system. Dimethylfumarate, at a concentration of 50 μm, reduced lymphocyte rolling on endothelial cells by 85.9% (P<0.001 compared to untreated controls), and it diminished the number of adherent cells by 88% (P<0.001). In contrast, monomethylfumarate (MMF) influenced neither surface expression of adhesion molecules nor interactions between endothelial cells and lymphocytes. These observations demonstrate that endothelial cells, in addition to the known effects on leucocytes, undergo profound functional changes in response to dimethylfumarate. These changes are accompanied by severely impaired dynamic interactions with lymphocytes, which constitute the critical initial step of leucocyte recruitment to inflamed tissues in psoriasis and other TNF-related inflammatory disorders. © 2011 John Wiley & Sons A/S.

G-protein-coupled receptor 30 mediates the effects of estrogen on endothelial cell tube formation in vitro.

PubMed

Zhou, Liyuan; Chen, Hong; Mao, Xun; Qi, Hongbo; Baker, Philip N; Zhang, Hua

2017-06-01

The placenta is the exchange organ between the mother and the fetus. The inadequate function of this organ is associated with a number of pregnancy disorders. Hypoxia and oxidative stress during placental development may induce endothelial dysfunction, resulting in the reduction in the perfusion of the placenta. During pregnancy, the levels of estrogen are increased. Decreased estrogen levels have been reported in women with preeclampsia. However, whether estrogen is involved in placental angiogenesis remains unclear. In this study, we aimed to investigate the effects of estrogen on endothelial cell tube formation and to elucidate the underlying mechanisms. For this purpose, human umbilical vein endothelial cells (HUVECs) were cultured with 17‑β‑estradiol under conditions of hypoxia/reoxygenation (H/R). The total pipe length of the tube‑like structure on endothelial cells was measured. The expression levels of G‑protein‑coupled receptor 30 (GPR30) and endothelial nitric oxide synthase (eNOS) and Akt were also measured in the endothelial cells following treatment with 17‑β‑estradiol under H/R conditions by western blot analysis and immunostaining. We found that the total pipe length of the tube‑like structure on endothelial cells was significantly reduced. This reduction was reversed by treatment with 17‑β‑estradiol. The expression of GPR30 in endothelial cells was significantly increased following treatment with 17‑β‑estradiol under H/R conditions. Furthermore, the levels of eNOS and Akt in endothelial cells were also significantly increased following treatment with 17-β-estradiol under H/R conditions. The activation of eNOS was inhibited by wortmannin, an inhibitor of PI3K/Akt. Our data thus demonstrate that estrogen prevents the failure of endothelial cell tube formation induced by H/R. GPR30 plays an important role in these protective effects through the activation of eNOS and Akt in endothelial cells. Our data suggest that increased levels of estrogen are important for placental angiogenesis.

Endothelial glycocalyx: permeability barrier and mechanosensor.

PubMed

Curry, F E; Adamson, R H

2012-04-01

Endothelial cells are covered with a polysaccharide rich layer more than 400 nm thick, mechanical properties of which limit access of circulating plasma components to endothelial cell membranes. The barrier properties of this endothelial surface layer are deduced from the rate of tracer penetration into the layer and the mechanics of red and white cell movement through capillary microvessels. This review compares the mechanosensor and permeability properties of an inner layer (100-150 nm, close to the endothelial membrane) characterized as a quasi-periodic structure which accounts for key aspects of transvascular exchange and vascular permeability with those of the whole endothelial surface layers. We conclude that many of the barrier properties of the whole surface layer are not representative of the primary fiber matrix forming the molecular filter determining transvascular exchange. The differences between the properties of the whole layer and the inner glycocalyx structures likely reflect dynamic aspects of the endothelial surface layer including tracer binding to specific components, synthesis and degradation of key components, activation of signaling pathways in the endothelial cells when components of the surface layer are lost or degraded, and the spatial distribution of adhesion proteins in microdomains of the endothelial cell membrane.

Inhibition of tumor necrosis factor-{alpha}-induced expression of adhesion molecules in human endothelial cells by the saponins derived from roots of Platycodon grandiflorum

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

Kim, Ji Young; Kim, Dong Hee; Kim, Hyung Gyun

2006-01-15

Adhesion molecules play an important role in the development of atherogenesis and are produced by endothelial cells after being stimulated with various inflammatory cytokines. This study examined the effect of saponins that were isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. CKS significantly inhibited the TNF{alpha}-induced increase in monocyte adhesion to endothelial cells as well as decreased the protein and mRNA expression levels of vascular adhesion molecule-1 and intercellular cell adhesion molecule-1 on endothelial cells. Furthermore, CKS significantly inhibited themore » TNF{alpha}-induced production of intracellular reactive oxygen species (ROS) and activation of NF-{kappa}B by preventing I{kappa}B degradation and inhibiting I{kappa}B kinase activity. Overall, CKS has anti-atherosclerotic and anti-inflammatory activity, which is least in part the result of it reducing the cytokine-induced endothelial adhesion to monocytes by inhibiting intracellular ROS production, NF-{kappa}B activation, and cell adhesion molecule expression in endothelial cells.« less

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

PubMed

Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

2013-05-03

Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice1

PubMed Central

Sasaki, Takamitsu; Kitadai, Yasuhiko; Nakamura, Toru; Kim, Jang-Seong; Tsan, Rachel Z; Kuwai, Toshio; Langley, Robert R; Fan, Dominic; Kim, Sun-Jin; Fidler, Isaiah J

2007-01-01

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), and VEGFR. Double immunofluorescence staining revealed that tumor-associated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), and phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, and increased the level of apoptosis in both tumor-associated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer. PMID:18084614

Endothelial keratoplasty with infant donor tissue

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Yamazaki, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2014-01-01

Here we report a case of endothelial keratoplasty with infant donor tissue obtained after brain death. A 52-year-old man with endothelial dysfunction of unknown cause in the right eye underwent non-Descemet stripping automated endothelial keratoplasty (nDSAEK) with tissue from an infant donor (2 years). Intraoperative and postoperative complications were recorded. Best corrected visual acuity and donor central endothelial cell density were recorded preoperatively and postoperatively. Infant donor tissue preparation with a microkeratome set at 300 μm was successful; the donor tissue was extremely elastic and soft compared with adult tissue. The central endothelial cell density of the infant donor tissue was as high as 4,291 cells/mm2. No complications were observed during donor tissue (8.0 mm in diameter) insertion with the double-glide technique (Busin glide with intraocular lens sheet glide) or any of the other procedures. Best corrected visual acuity improved from 1.7 logMAR (logarithm of the minimum angle of resolution; 0.02 decimal visual acuity) preoperatively to 0.2 logMAR (0.6) after 6 months and 0.1 logMAR (0.8) after 1 year. The central endothelial cell density after 6 months was 4,098 cells/mm2 (representing a 4.5% cell loss from preoperative donor cell measurements), and the central endothelial cell density after 1 year was 4,032 cells/mm2 (6.0% decrease). Infant donor tissue may be preferably used for DSAEK/nDASEK, since it may not be suitable for penetrating keratoplasty or Descemet membrane endothelial keratoplasty. PMID:25246761

Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells.

PubMed

Krump-Konvalinkova, Vera; Yasuda, Satoshi; Rubic, Tina; Makarova, Natalia; Mages, Jörg; Erl, Wolfgang; Vosseler, Claudia; Kirkpatrick, C James; Tigyi, Gabor; Siess, Wolfgang

2005-03-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.

Nesting of colon and ovarian cancer cells in the endothelial niche is associated with alterations in glycan and lipid metabolism

PubMed Central

Halama, Anna; Guerrouahen, Bella S.; Pasquier, Jennifer; Satheesh, Noothan J.; Suhre, Karsten; Rafii, Arash

2017-01-01

The metabolic phenotype of a cancer cell is determined by its genetic makeup and microenvironment, which dynamically modulates the tumor landscape. The endothelial cells provide both a promoting and protective microenvironment – a niche for cancer cells. Although metabolic alterations associated with cancer and its progression have been fairly defined, there is a significant gap in our understanding of cancer metabolism in context of its microenvironment. We deployed an in vitro co-culture system based on direct contact of cancer cells with endothelial cells (E4+EC), mimicking the tumor microenvironment. Metabolism of colon (HTC15 and HTC116) and ovarian (OVCAR3 and SKOV3) cancer cell lines was profiled with non-targeted metabolic approaches at different time points in the first 48 hours after co-culture was established. We found significant, coherent and non-cell line specific changes in fatty acids, glycerophospholipids and carbohydrates over time, induced by endothelial cell contact. The metabolic patterns pinpoint alterations in hexosamine biosynthetic pathway, glycosylation and lipid metabolism as crucial for cancer – endothelial cells interaction. We demonstrated that “Warburg effect” is not modulated in the initial stage of nesting of cancer cell in the endothelial niche. Our study provides novel insight into cancer cell metabolism in the context of the endothelial microenvironment. PMID:28051182

Endothelial cell responses in terms of adhesion, proliferation, and morphology to stiffness of polydimethylsiloxane elastomer substrates.

PubMed

Ataollahi, Forough; Pramanik, Sumit; Moradi, Ali; Dalilottojari, Adel; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar; Abu Osman, Noor Azuan

2015-07-01

Extracellular environments can regulate cell behavior because cells can actively sense their mechanical environments. This study evaluated the adhesion, proliferation and morphology of endothelial cells on polydimethylsiloxane (PDMS)/alumina (Al2 O3 ) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 2.5, 5, 7.5, and 10 wt % Al2 O3 at a curing temperature of 50°C for 4 h. The substrates were then characterized by mechanical, structural, and morphological analyses. The cell adhesion, proliferation, and morphology of cultured bovine aortic endothelial (BAEC) cells on substrate materials were evaluated by using resazurin assay and 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The composites (PDMS/2.5, 5, 7.5, and 10 wt % Al2 O3 ) exhibited higher stiffness than the pure PDMS substrate. The results also revealed that stiffer substrates promoted endothelial cell adhesion and proliferation and also induced spread morphology in the endothelial cells compared with lesser stiff substrates. Statistical analysis showed that the effect of time on cell proliferation depended on stiffness. Therefore, this study concludes that the addition of different Al2 O3 percentages to PDMS elevated substrate stiffness which in turn increased endothelial cell adhesion and proliferation significantly and induced spindle shape morphology in endothelial cells. © 2014 Wiley Periodicals, Inc.

Improvement of the in vivo cellular repopulation of decellularized cardiovascular tissues by a detergent-free, non-proteolytic, actin-disassembling regimen.

PubMed

Assmann, Alexander; Struß, Marc; Schiffer, Franziska; Heidelberg, Friederike; Munakata, Hiroshi; Timchenko, Elena V; Timchenko, Pavel E; Kaufmann, Tim; Huynh, Khon; Sugimura, Yukiharu; Leidl, Quentin; Pinto, Antonio; Stoldt, Volker R; Lichtenberg, Artur; Akhyari, Payam

2017-12-01

Low immunogenicity and high repopulation capacity are crucial determinants for the functional and structural performance of acellular cardiovascular implants. The present study evaluates a detergent-free, non-proteolytic, actin-disassembling regimen (BIO) for decellularization of heart valve and vessel grafts, particularly focusing on their bio-functionality. Rat aortic conduits (rAoC; n = 89) and porcine aortic valve samples (n = 106) are decellularized using detergents (group DET) or the BIO regimen. BIO decellularization results in effective elimination of cellular proteins and significantly improves removal of DNA as compared with group DET, while the extracellular matrix (ECM) structure as well as mechanical properties are preserved. The architecture of rAoC in group BIO allows for improved bio-functionalization with fibronectin (FN) in a standardized rat implantation model: BIO treatment significantly increases speed and amount of autologous medial cellular repopulation in vivo (p < 0.001) and decreases the formation of hyperplastic intima (p < 0.001) as compared with FN-coated DET-decellularized grafts. Moreover, there are no signs of infiltration with inflammatory cells. The present biological, detergent-free, non-proteolytic regimen balances effective decellularization and ECM preservation in cardiovascular grafts, and provides optimized bio-functionality. Additionally, this study implies that the actin-disassembling regimen may be a promising approach for bioengineering of acellular scaffolds from other muscular tissues, as for example myocardium or intestine. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Inducible Knockdown of Endothelial Protein Tyrosine Phosphatase-1B Promotes Neointima Formation in Obese Mice by Enhancing Endothelial Senescence.

PubMed

Jäger, Marianne; Hubert, Astrid; Gogiraju, Rajinikanth; Bochenek, Magdalena L; Münzel, Thomas; Schäfer, Katrin

2018-02-01

Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of receptor tyrosine kinase signaling. In this study, we determined the importance of PTP1B expressed in endothelial cells for the vascular response to arterial injury in obesity. Morphometric analysis of vascular lesions generated by 10% ferric chloride (FeCl 3 ) revealed that tamoxifen-inducible endothelial PTP1B deletion (Tie2.ER T2 -Cre × PTP1B fl/fl ; End.PTP1B knockout, KO) significantly increased neointima formation, and reduced numbers of (endothelial lectin-positive) luminal cells in End.PTP1B-KO mice suggested impaired lesion re-endothelialization. Significantly higher numbers of proliferating cell nuclear antigen (PCNA)-positive proliferating cells as well as smooth muscle actin (SMA)-positive or vascular cell adhesion molecule-1 (VCAM1)-positive activated smooth muscle cells or vimentin-positive myofibroblasts were detected in neointimal lesions of End.PTP1B-KO mice, whereas F4/80-positive macrophage numbers did not differ. Activated receptor tyrosine kinase and transforming growth factor-beta (TGFβ) signaling and oxidative stress markers were also significantly more abundant in End.PTP1B-KO mouse lesions. Genetic knockdown or pharmacological inhibition of PTP1B in endothelial cells resulted in increased expression of caveolin-1 and oxidative stress, and distinct morphological changes, elevated numbers of senescence-associated β-galactosidase-positive cells, and increased expression of tumor suppressor protein 53 (p53) or the cell cycle inhibitor cyclin-dependent kinase inhibitor-2A (p16INK4A) suggested senescence, all of which could be attenuated by small interfering RNA (siRNA)-mediated downregulation of caveolin-1. In vitro, senescence could be prevented and impaired re-endothelialization restored by preincubation with the antioxidant Trolox. Our results reveal a previously unknown role of PTP1B in endothelial cells and provide mechanistic insights how PTP1B deletion or inhibition may promote endothelial senescence. Absence of PTP1B in endothelial cells impairs re-endothelialization, and the failure to induce smooth muscle cell quiescence or to protect from circulating growth factors may result in neointimal hyperplasia. Antioxid. Redox Signal. 00, 000-000.

Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries.

PubMed

Budel, S; Schuster, A; Stergiopoulos, N; Meister, J J; Bény, J L

2001-09-01

We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

Sildenafil Inhibits the Proliferation of Cultured Human Endothelial Cells

PubMed Central

Erdogan, Ali; Luedders, Doerte Wiebke; Muenz, Benedikt Manuel; Schaefer, Christian Alexander; Tillmanns, Harald; Wiecha, Johannes; Kuhlmann, Christoph Ruediger Wolfram

2007-01-01

The proliferation of endothelial cells plays a crucial role in the development of intraplaque angiogenesis (IPA). IPA is a major source of intraplaque hemorrhage and therefore contributes to the destabilization of atherosclerotic plaques. Therefore, the aim of the present study was to examine, whether sildenafil inhibits endothelial cell growth. The proliferation of human endothelial cells derived from umbilical cord veins (HUVEC) was examined on DNA level by measurements of (3H)-thymidine incorporation. Cell viability was analyzed using trypan blue staining. The proliferation of cultured human endothelial cells was significantly decreased by 1 μmol/l (-48.4%) and 10 μmol/l (-89.6%) sildenafil (n=10, p<0.05). This was not a cytotoxic effect, because cell viability was only reduced at sildenafil concentrations of 50 μmol/l or greater. In addition sildenafil significantly reduced endothelial proliferation induced by bFGF (n=10, p<0.05). The presented results demonstrate an antiangiogenic effect of sildenafil that might be useful in the prevention of atherosclerotic plaque vascularization. PMID:23675029

Association of Plasmodium falciparum with Human Endothelial Cells in vitro

PubMed Central

Utter, Christopher; Serrano, Adelfa E.; Glod, John W.; Leibowitz, Michael J.

2017-01-01

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria. PMID:28656007

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

PubMed Central

Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

2013-01-01

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value. PMID:23613998

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3

PubMed Central

Koenig, M N; Naik, E; Rohrbeck, L; Herold, M J; Trounson, E; Bouillet, P; Thomas, T; Voss, A K; Strasser, A; Coultas, L

2014-01-01

The growth of new blood vessels by angiogenesis is essential for normal development, but can also cause or contribute to the pathology of numerous diseases. Recent studies have shown that BIM, a pro-apoptotic BCL2-family protein, is required for endothelial cell apoptosis in vivo, and can contribute to the anti-angiogenic effect of VEGF-A inhibitors in certain tumor models. Despite its importance, the extent to which BIM is autonomously required for physiological endothelial apoptosis remains unknown and its regulation under such conditions is poorly defined. While the transcription factor FOXO3 has been proposed to induce Bim in response to growth factor withdrawal, evidence for this function is circumstantial. We report that apoptosis was reduced in Bim−/− primary endothelial cells, demonstrating a cell-autonomous role for BIM in endothelial death following serum and growth factor withdrawal. In conflict with in vitro studies, BIM-dependent endothelial death in vivo did not require FOXO3. Moreover, endothelial apoptosis proceeded normally in mice lacking FOXO-binding sites in the Bim promoter. Bim mRNA was upregulated in endothelial cells starved of serum and growth factors and this was accompanied by the downregulation of miRNAs of the miR-17∼92 cluster. Bim mRNA levels were also elevated in miR-17∼92+/− endothelial cells cultured under steady-state conditions, suggesting that miR-17∼92 cluster miRNAs may contribute to regulating overall Bim mRNA levels in endothelial cells. PMID:24971484

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

PubMed

Schmidt, Dörthe; Asmis, Lars M; Odermatt, Bernhard; Kelm, Jens; Breymann, Christian; Gössi, Matthias; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2006-10-01

Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

PubMed

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte–endothelial cell coculture model

PubMed Central

Liu, Xin; Sun, Jiao

2014-01-01

Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte–endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease. PMID:24648726

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

PubMed

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

PubMed Central

Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

2015-01-01

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified. PMID:25769545

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

PubMed Central

Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

2013-01-01

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy. PMID:23626771

P-selectin mediates neutrophil adhesion to endothelial cell borders.

PubMed

Burns, A R; Bowden, R A; Abe, Y; Walker, D C; Simon, S I; Entman, M L; Smith, C W

1999-03-01

During an acute inflammatory response, endothelial P-selectin (CD62P) can mediate the initial capture of neutrophils from the free flowing bloodstream. P-selectin is stored in secretory granules (Weibel-Palade bodies) and is rapidly expressed on the endothelial surface after stimulation with histamine or thrombin. Because neutrophil transmigration occurs preferentially at endothelial borders, we wished to determine whether P-selectin-dependent neutrophil capture (adhesion) occurs at endothelial cell borders. Under static or hydrodynamic flow (2 dyn/cm2) conditions, histamine (10(-4) M) or thrombin (0.2 U/mL) treatment induced preferential (> or = 75%) neutrophil adhesion to the cell borders of endothelial monolayers. Blocking antibody studies established that neutrophil adhesion was completely P-selectin dependent. P-selectin surface expression increased significantly after histamine treatment and P-selectin immunostaining was concentrated along endothelial borders. We conclude that preferential P-selectin expression along endothelial borders may be an important mechanism for targeting neutrophil migration at endothelial borders.

Inhibition of TGF-β Signaling in SHED Enhances Endothelial Differentiation.

PubMed

Xu, J G; Gong, T; Wang, Y Y; Zou, T; Heng, B C; Yang, Y Q; Zhang, C F

2018-02-01

Low efficiency of deriving endothelial cells (ECs) from adult stem cells hampers their utilization in tissue engineering studies. The purpose of this study was to investigate whether suppression of transforming growth factor beta (TGF-β) signaling could enhance the differentiation efficiency of dental pulp-derived stem cells into ECs. We initially used vascular endothelial growth factor A (VEGF-A) to stimulate 2 dental pulp-derived stem cells (dental pulp stem cells and stem cells from human exfoliated deciduous teeth [SHED]) and compared their differentiation capacity into ECs. We further evaluated whether the vascular endothelial growth factor receptor I (VEGF-RI)-specific ligand placental growth factor-1 (PlGF-1) could mediate endothelial differentiation. Finally, we investigated whether the TGF-β signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than dental pulp stem cells. VEGFR1-specific ligand-PLGF exerted a negligible effect on SHED-ECs differentiation. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-β-SMAD2/3 signaling.

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

PubMed Central

van den Biggelaar, Maartje; Bouwens, Eveline A.M.; Kootstra, Neeltje A.; Hebbel, Robert P.; Voorberg, Jan; Mertens, Koen

2009-01-01

Background Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion. Design and Methods Human blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human B-domain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy. Results Blood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×106 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6- to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15–22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies. Conclusions We conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A. PMID:19336741

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

PubMed

Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

2013-07-21

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.

PubMed

Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C

1997-01-01

Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P < .001) before but not after delivery. Expression of the integrin counter receptors on leukocytes was similarly increased in preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum factor which increases endothelial cell [Ca2+]i and enhances adhesion molecule expression.

Multiple protocadherins are expressed in brain microvascular endothelial cells and might play a role in tight junction protein regulation.

PubMed

Dilling, Christina; Roewer, Norbert; Förster, Carola Y; Burek, Malgorzata

2017-10-01

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood-brain barrier.

Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury.

PubMed

Lu, Naihao; Sui, Yinhua; Tian, Rong; Peng, Yi-Yuan

2018-05-16

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

Catheter-directed Intraportal Delivery of Endothelial Cell Therapy for Liver Regeneration: A Feasibility Study in a Large-Animal Model of Cirrhosis.

PubMed

Lee, Kyungmouk Steve; Santagostino, Sara F; Li, David; Ramjit, Amit; Serrano, Kenneth; Ginsberg, Michael D; Ding, Bi-Sen; Rafii, Shahin; Madoff, David C

2017-10-01

Purpose To demonstrate the feasibility of imaging-guided catheter-directed delivery of endothelial cell therapy in a porcine model of cirrhosis for liver regeneration. Materials and Methods After approval from the institutional animal care and use committee, autologous liver endothelial cells were grown from core hepatic specimens from swine. Cirrhosis was induced in swine by means of transcatheter infusion of ethanol and iodized oil into the hepatic artery. Three weeks after induction of cirrhosis, the swine were randomly assigned to receive autologous cell therapy (endothelial cells, n = 4) or control treatment (phosphate-buffered saline, n = 4) by means of imaging-guided transhepatic intraportal catheterization. Fluorescence-activated cell sorting analysis was performed on biopsy samples 1 hour after therapy. Three weeks after intraportal delivery of endothelial cells, the swine were euthanized and the explanted liver underwent quantitative pathologic examination. Statistical analysis was performed with an unpaired t test by using unequal variance. Results Liver endothelial cells were successfully isolated, cultured, and expanded from eight 20-mm, 18-gauge hepatic core samples to 50 × 10 6 autologous cells per pig. Intraportal delivery of endothelial cell therapy or saline was technically successful in all eight swine, with no complications. Endothelial cells were present in the liver for a minimum of 1 hour after intraportal infusion. Swine treated with endothelial cell therapy showed mean levels of surrogate markers of hepatobiliary injury that were consistent with decreases in hepatic fibrosis and biliary ductal damage relative to the control animals, although statistical significance was not met in this pilot study: The mean percentage of positive pixels at Masson trichrome staining was 7.28% vs 5.57%, respectively (P = .20), the mean proliferation index with cytokeratin wide-spectrum was 2.55 vs 1.13 (P = .06), and the mean proliferation index with Ki67 was 7.08 vs 4.96 (P = .14). Conclusion The results confirm the feasibility of imaging-guided catheter-directed endothelial cell therapy with an intraportal technique for the treatment of cirrhosis in a porcine model. A trend toward decreased liver fibrosis with endothelial cell therapy was observed. Larger animal studies and human studies are necessary to confirm significance. © RSNA, 2017.

Signaling hierarchy regulating human endothelial cell development

USDA-ARS?s Scientific Manuscript database

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

The role of corneal endothelial morphology in graft assessment and prediction of endothelial cell loss during organ culture of human donor corneas.

PubMed

Hermel, Martin; Salla, Sabine; Fuest, Matthias; Walter, Peter

2017-03-01

Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. I-ECD was 2812 ± 360/mm 2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm 2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm 2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve graft quality. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

PubMed

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

Presentation, diagnosis and management of limbal stem cell deficiency.

PubMed

Sejpal, Kunjal; Bakhtiari, Pejman; Deng, Sophie X

2013-01-01

The human corneal surface epithelium is continuously repopulated by the limbal stem cells (LSCs). Limbal Stem Cell Deficiency (LSCD) can lead to corneal opacity and vascularization, with consequent visual impairment or blindness. Many acquired and congenital diseases can lead to LCSD by direct injury to the LSCs, destruction of LSC niche, or both. Based on the severity of the disease, LSCD can present with various symptoms and signs. Although LSCD can be detected clinically, laboratory tests are necessary to confirm the diagnosis and monitor the disease progression. This article concisely reviews the clinical presentation, techniques for diagnosis and management of LSCD.

Identification and Characterization of a Dendritic Cell Precursor in Parenchymal Lung Tissue.

PubMed

von Garnier, Christophe; Blank, Fabian; Rothen-Rutishauser, Barbara; Goethert, Joachim R; Holt, Patrick G; Stumbles, Philip A; Strickland, Deborah H

2017-03-01

The pulmonary parenchymal and mucosal microenvironments are constantly exposed to the external environment and thus require continuous surveillance to maintain steady-state immunological homeostasis. This is achieved by a mobile network of pulmonary dendritic cells (DC) and macrophages (mø) that constantly sample and process microenvironmental antigens into signals that can initiate or dampen inflammation, either locally or after onward migration to draining lymph nodes. The constant steady-state turnover of pulmonary DC and mø requires replenishment from bone marrow precursors; however, the nature of the pulmonary precursor cell (PC) remains unclear, although recent studies suggest that subsets of pulmonary DC may derive from circulating monocytic precursors. In the current study, we describe a population of cells in steady-state mouse lung tissue that has the surface phenotypic and ultrastructural characteristics of a common DC progenitor. Irradiation and reconstitution studies confirmed the bone marrow origins of this PC and showed that it had rapid depletion and reconstitution kinetics that were similar to those of DC, with a 50% repopulation by donor-derived cells by Days 7-9 after reconstitution. This was significantly faster than the rates observed for mø, which showed 50% repopulation by donor-derived cells beyond Days 16-21 after reconstitution. Purified PC gained antigen-presenting function and a cell surface phenotype similar to that of pulmonary DC after maturation in vitro, with light and electron microscopy confirming a myeloid DC morphology. To the best of our knowledge, this is the first study to describe a PC for DC in lung tissue; the findings have implications for the restoration of pulmonary immunological homeostasis after bone marrow transplant.

Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2.

PubMed

Bugarcic, Andrea; Vetter, Irina; Chalmers, Silke; Kinna, Genevieve; Collins, Brett M; Teasdale, Rohan D

2015-11-01

Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex. © 2015 International Federation for Cell Biology.

NaK-ATPase pump sites in cultured bovine corneal endothelium of varying cell density at confluence.

PubMed

Crawford, K M; Ernst, S A; Meyer, R F; MacCallum, D K

1995-06-01

The driving force for ion and water flow necessary for efficient deturgesence of the corneal stroma resides in the ouabain-sensitive sodium (Na) pump of corneal endothelial cells. Using a cell culture model of corneal endothelial cell hypertrophy, the authors examined the expression of Na pumps at the cell surface to see how this central element of the endothelial pump changed as corneal endothelial cell density decreased to a level associated with corneal decompensation in vivo. 3H-ouabain binding to NaK-ATPase at saturating conditions was used to quantitate the number of Na pump sites on cultured bovine corneal endothelial cells as the confluent density decreased from approximately 2750 cells/mm2 to approximately 275 cells/mm2. The mean number of Na pump sites per cell at confluence (1.92 +/- 0.07 x 10(6)) did not change as the cell density decreased 2.7-fold from 2763 cells/mm2 to 1000 cells/mm2. However, pump site expression doubled to approximately 4 x 10(6) sites/cell as the cell density decreased from 1000 cells/mm2 to 275 cells/mm2. Despite the incremental increase in Na pump site expression that occurred as the cells hypertrophied below a density of 1000/mm2 to achieve confluence, this increase was insufficient to prevent a decrease in Na pump site density of the intact monolayer, expressed as pump sites/mm2. The confluent cell density of cultured bovine corneal endothelial cells can be varied from that found in the normal native cornea to that associated with corneal decompensation. In confluent cultures with cell densities ranging from 2750 cells/mm2 to 1000 cells/mm2, the number of pump sites per cell remains relatively unchanged. Below cell densities of 1000 cells/mm2, the number of pump sites per cell progressively increases. The increased Na pump site abundance in markedly hypertrophied endothelial cells cannot adequately compensate for the progressive reduction in the number of transporting cells per unit area within the intact monolayer. Even when considered with the decrease in the size of the paracellular ion conductive pathway that is a consequence of progressive endothelial hypertrophy, the overall pumping capacity of the intact endothelial monolayer declines.

Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

NASA Astrophysics Data System (ADS)

Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

1989-06-01

The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells.

PubMed

Garner, Omai B; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C; Park, Arnold; Bowden, Thomas A; Freiberg, Alexander N; Lee, Benhur; Baum, Linda G

2015-03-01

Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Circulating endothelial progenitor cells and cardiovascular outcomes.

PubMed

Werner, Nikos; Kosiol, Sonja; Schiegl, Tobias; Ahlers, Patrick; Walenta, Katrin; Link, Andreas; Böhm, Michael; Nickenig, Georg

2005-09-08

Endothelial progenitor cells derived from bone marrow are believed to support the integrity of the vascular endothelium. The number and function of endothelial progenitor cells correlate inversely with cardiovascular risk factors, but the prognostic value associated with circulating endothelial progenitor cells has not been defined. The number of endothelial progenitor cells positive for CD34 and kinase insert domain receptor (KDR) was determined with the use of flow cytometry in 519 patients with coronary artery disease as confirmed on angiography. After 12 months, we evaluated the association between baseline levels of endothelial progenitor cells and death from cardiovascular causes, the occurrence of a first major cardiovascular event (myocardial infarction, hospitalization, revascularization, or death from cardiovascular causes), revascularization, hospitalization, and death from all causes. A total of 43 participants died, 23 from cardiovascular causes. A first major cardiovascular event occurred in 214 patients. The cumulative event-free survival rate increased stepwise across three increasing baseline levels of endothelial progenitor cells in an analysis of death from cardiovascular causes, a first major cardiovascular event, revascularization, and hospitalization. After adjustment for age, sex, vascular risk factors, and other relevant variables, increased levels of endothelial progenitor cells were associated with a reduced risk of death from cardiovascular causes (hazard ratio, 0.31; 95 percent confidence interval, 0.16 to 0.63; P=0.001), a first major cardiovascular event (hazard ratio, 0.74; 95 percent confidence interval, 0.62 to 0.89; P=0.002), revascularization (hazard ratio, 0.77; 95 percent confidence interval, 0.62 to 0.95; P=0.02), and hospitalization (hazard ratio, 0.76; 95 percent confidence interval, 0.63 to 0.94; P=0.01). Endothelial progenitor-cell levels were not predictive of myocardial infarction or of death from all causes. The level of circulating CD34+KDR+ endothelial progenitor cells predicts the occurrence of cardiovascular events and death from cardiovascular causes and may help to identify patients at increased cardiovascular risk. Copyright 2005 Massachusetts Medical Society.

Factor X/Xa elicits protective signaling responses in endothelial cells directly via PAR-2 and indirectly via endothelial protein C receptor-dependent recruitment of PAR-1.

PubMed

Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R

2010-11-05

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Phloretin attenuates hyperuricemia-induced endothelial dysfunction through co-inhibiting inflammation and GLUT9-mediated uric acid uptake.

PubMed

Liu, Shuyun; Yuan, Yujia; Zhou, Yijie; Zhao, Meng; Chen, Younan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

2017-10-01

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti-inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA-induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP-1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor-kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose-dependent manner. In contrast, phloretin significantly attenuated pro-inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor-kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA-induced endothelial injury via a synergic mechanism including direct anti-inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia-related cardiovascular diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Expansion and cryopreservation of porcine and human corneal endothelial cells.

PubMed

Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

2017-08-01

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function.

PubMed

Li, Xiaocong; Jiang, Chunyu; Zhao, Jungong

2016-08-01

Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) through paracrine secretion has been reported in various tissue repair models. Exosomes, key components of cell paracrine mechanism, have been rarely reported in wound healing. Exosomes were isolated from the media of EPCs obtained from human umbilical cord blood. Diabetic rats wound model was established and treated with exosomes. The in vitro effects of exosomes on the proliferation, migration and angiogenic tubule formation of endothelial cells were investigated. We revealed that human umbilical cord blood EPCs derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that exosomes enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. Taken together, our findings indicated that EPCs-derived exosomes facilitate wound healing by positively modulating vascular endothelial cells function. Copyright © 2016 Elsevier Inc. All rights reserved.

Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma.

PubMed

Way, D; Hendrix, M; Witte, M; Witte, C; Nagle, R; Davis, J

1987-09-01

An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium.

Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.

PubMed

Zeiger, A S; Liu, F D; Durham, J T; Jagielska, A; Mahmoodian, R; Van Vliet, K J; Herman, I M

2016-08-16

Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.

Magnolol inhibits angiogenesis by regulating ROS-mediated apoptosis and the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells.

PubMed

Kim, Gi Dae; Oh, Jedo; Park, Hyen-Joo; Bae, Kihwan; Lee, Sang Kook

2013-08-01

Magnolol, a neolignan from the traditional medicinal plant Magnolia obovata, has been shown to possess neuroprotective, anti-inflammatory, anticancer and anti-angiogenic activities. However, the precise mechanism of the anti-angiogenic activity of magnolol remains to be elucidated. In the present study, the anti-angiogenic effect of magnolol was evaluated in mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial-like cells. The endothelial-like cells were obtained by differentiation from mES/EB cells. Magnolol (20 µM) significantly suppressed the transcriptional and translational expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES/EB-derived endothelial-like cells. To further understand the molecular mechanism of the suppression of PECAM expression, signaling pathways were analyzed in the mES/EB-derived endothelial-like cells. Magnolol induced the generation of reactive oxygen species (ROS) by mitochondria, a process that was associated with the induction of apoptosis as determined by positive Annexin V staining and the activation of cleaved caspase-3. The involvement of ROS generation by magnolol was confirmed by treatment with an antioxidant, N-acetyl-cysteine (NAC). NAC inhibited the magnolol-mediated induction of ROS generation and suppression of PECAM expression. In addition, magnolol suppressed the activation of MAPKs (ERK, JNK and p38) and the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells. Taken together, these findings demonstrate for the first time that the anti-angiogenic activity of magnolol may be associated with ROS-mediated apoptosis and the suppression of the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells.

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

NASA Technical Reports Server (NTRS)

McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

2003-01-01

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

Restoration of Autophagy in Endothelial Cells from Patients with Diabetes Mellitus Improves Nitric Oxide Signaling

PubMed Central

Fetterman, Jessica L.; Holbrook, Monica; Flint, Nir; Feng, Bihua; Bretón-Romero, Rosa; Linder, Erika A.; Berk, Brittany D.; Duess, Mai-Ann; Farb, Melissa G.; Gokce, Noyan; Shirihai, Orian S.; Hamburg, Naomi M.; Vita, Joseph A.

2016-01-01

Background Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. Methods and Results We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n=45) and non-diabetic controls (n=41). p62 levels were higher in cells from diabetics (34.2±3.6 vs. 20.0±1.6, P=0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (−21±5% vs. 64±22%, P=0.003) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P=0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P=0.01) in cells from diabetics to a lesser extent than in cells from controls (P=0.04), suggesting ongoing, but inadequate autophagic clearance. Conclusion Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease. PMID:26926601

Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.

PubMed

Fetterman, Jessica L; Holbrook, Monica; Flint, Nir; Feng, Bihua; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Duess, Mai-Ann; Farb, Melissa G; Gokce, Noyan; Shirihai, Orian S; Hamburg, Naomi M; Vita, Joseph A

2016-04-01

Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Vascular Procr+ stem cells: Finding new branches while looking for the roots.

PubMed

Gur-Cohen, Shiri; Lapidot, Tsvee

2016-10-01

Generation and growth of the blood vasculature network is a highly synchronized process, requiring coordinated efforts of endothelial cells and pericytes to maintain blood vessel integrity and regeneration. In a recent paper published in Cell Research, Yu et al. identified and characterized bipotent Procr-expressing vascular endothelial stem cells, which give rise to both endothelial cells and pericytes.

Diesel Exhaust Particle Exposure Causes Redistribution of Endothelial Tube VE-Cadherin

PubMed Central

Chao, Ming-Wei; Kozlosky, John; Po, Iris P.; Strickland, Pamela Ohman; Svoboda, Kathy K. H.; Cooper, Keith; Laumbach, Robert; Gordon, Marion K.

2010-01-01

Whether diesel exhaust particles (DEPs) potentially have a direct effect on capillary endothelia was examined by following the adherens junction component, vascular endothelial cell cadherin (VE-cadherin). This molecule is incorporated into endothelial adherens junctions at the cell surface, where it forms homodimeric associations with adjacent cells and contributes to the barrier function of the vasculature (Dejana et al., 2008; Venkiteswaran et al., 2002; Villasante et al., 2007). Human umbilical vein endothelial cells (HUVECs) that were pre-formed into capillary-like tube networks in vitro were exposed to DEPs for 24 hr. After exposure, the integrity of VE-cadherin in adherens junctions was assessed by immunofluorescence analysis, and demonstrated that increasing concentrations of DEPs caused increasing redistribution of VE-cadherin away from the cell-cell junctions toward intracellular locations. Since HUVEC tube networks are three-dimensional structures, whether particles entered the endothelial cells or tubular lumens was also examined. The data indicate that translocation of the particles does occur. The results, obtained in a setting that removes the confounding effects of inflammatory cells or blood components, suggest that if DEPs encounter alveolar capillaries in vivo, they may be able to directly affect the endothelial cell-cell junctions. PMID:20887764

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

PubMed

Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

2013-01-01

The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Oral Mucosa Harbors a High Frequency of Endothelial Cells: A Novel Postnatal Cell Source for Angiogenic Regeneration.

PubMed

Zhou, Jian; Rogers, Jason H; Lee, Scott H; Sun, DongMing; Yao, Hai; Mao, Jeremy J; Kong, Kimi Y

2017-01-15

Endothelial progenitor cells/endothelial cells (EPCs/ECs) have great potential to treat pathological conditions such as cardiac infarction, muscle ischemia, and bone fractures, but isolation of EPC/ECs from existing cell sources is challenging due to their low EC frequency. We have isolated endothelial progenitor (EP)-like cells from rat oral mucosa and characterized their yield, immunophenotype, growth, and in vivo angiogenic potential. The frequency of EP-like cells derived from oral mucosa is thousands of folds higher than EPCs derived from donor-match bone marrow samples. EP-like cells from oral mucosa were positive for EC markers CD31, VE-Cadherin, and VEGFR2. Oral mucosa-derived EP-like cells displayed robust uptake of acetylated low-density lipoprotein and formed stable capillary networks in Matrigel. Subcutaneously implanted oral mucosa-derived EP-like cells anastomosed with host blood vessels, implicating their ability to elicit angiogenesis. Similar to endothelial colony-forming cells, EP-like cells from oral mucosa have a significantly higher proliferative rate than human umbilical vein endothelial cells. These findings identify a putative EPC source that is easily accessible in the oral cavity, potentially from discarded tissue specimens, and yet with robust yield and potency for angiogenesis in tissue and organ regeneration.

Oral Mucosa Harbors a High Frequency of Endothelial Cells: A Novel Postnatal Cell Source for Angiogenic Regeneration

PubMed Central

Zhou, Jian; Rogers, Jason H.; Lee, Scott H.; Sun, DongMing; Yao, Hai; Mao, Jeremy J.

2017-01-01

Endothelial progenitor cells/endothelial cells (EPCs/ECs) have great potential to treat pathological conditions such as cardiac infarction, muscle ischemia, and bone fractures, but isolation of EPC/ECs from existing cell sources is challenging due to their low EC frequency. We have isolated endothelial progenitor (EP)-like cells from rat oral mucosa and characterized their yield, immunophenotype, growth, and in vivo angiogenic potential. The frequency of EP-like cells derived from oral mucosa is thousands of folds higher than EPCs derived from donor-match bone marrow samples. EP-like cells from oral mucosa were positive for EC markers CD31, VE-Cadherin, and VEGFR2. Oral mucosa-derived EP-like cells displayed robust uptake of acetylated low-density lipoprotein and formed stable capillary networks in Matrigel. Subcutaneously implanted oral mucosa-derived EP-like cells anastomosed with host blood vessels, implicating their ability to elicit angiogenesis. Similar to endothelial colony-forming cells, EP-like cells from oral mucosa have a significantly higher proliferative rate than human umbilical vein endothelial cells. These findings identify a putative EPC source that is easily accessible in the oral cavity, potentially from discarded tissue specimens, and yet with robust yield and potency for angiogenesis in tissue and organ regeneration. PMID:27832737

Involvement of adhesion molecules (CD11a-ICAM-1) in vascular endothelial cell injury elicited by PMA-stimulated neutrophils.

PubMed

Fujita, H; Morita, I; Murota, S

1991-06-14

Protective effect of anti-CD11a and anti-ICAM-1 antibodies on the cytotoxicity induced by PMA-stimulated neutrophils was studied using cultured endothelial cells isolated from bovine carotid artery. Anti-CD11a antibody and anti-ICAM-1 antibody inhibited the endothelial cell injury induced by the activated neutrophils in a dose dependent manner. On the other hand, both antibodies themselves had no effect on either the luminol chemiluminescence released out of the activated neutrophils or the adhesion of the neutrophils to the endothelial cell monolayer. These data suggest that these adhesion molecules play some important roles in the vascular endothelial cell injury elicited by activated neutrophils.

Iron ion irradiation increases promotes adhesion of monocytic cells to arterial vascular endothelium

NASA Astrophysics Data System (ADS)

Kucik, Dennis; Khaled, Saman; Gupta, Kiran; Wu, Xing; Yu, Tao; Chang, Polly; Kabarowski, Janusz

Radiation causes inflammation, and chronic, low-level vascular inflammation is a risk factor for atherosclerosis. Consistent with this, exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Part of the inflammatory response to radiation is a change in the adhesiveness of the endothelial cells that line the blood vessels, triggering inappropriate accumulation of leukocytes, leading to later, damaging effects of inflammation. Although some studies have been done on the effects of gamma irradiation on vascular endothelium, the response of endothelium to heavy ion radiation likely to be encountered in prolonged space flight has not been determined. We investigated how irradiation of aortic endothelial cells with iron ions affects adhesiveness of cultured aortic endothelial cells for monocytic cells and the consequences of this for development of atherosclerosis. Aortic endothelial cells were irradiated with 600 MeV iron ions at Brookhaven National Laboratory and adhesion-related changes were measured. Cells remained viable for at least 72 hours, and were even able to repair acute damage to cell junctions. We found that iron ion irradiation altered expression levels of specific endothelial cell adhesion molecules. Further, these changes had functional consequences. Using a flow chamber adhesion assay to measure adhesion of monocytic cells to endothelial cells under physiological shear stress, we found that adhesivity of vascular endothelium was enhanced in as little as 24 hours after irradiation. Further, the radiation dose dependence was not monotonic, suggesting that it was not simply the result of endothelial cell damage. We also irradiated aortic arches and carotid arteries of Apolipoprotein-E-deficient mice. Histologic analysis of these mice will be conducted to determine whether effects of radiation on endothelial adhesiveness result in consequences for development of atherosclerosis. (Supported by NSBRI: NCC-9-58-162)

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo.

PubMed

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg H W

2009-10-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo

PubMed Central

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg HW

2009-01-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc. PMID:18774958

Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

PubMed

Rahman, Nurul Adhwa; Rasil, Alifah Nur'ain Haji Mat; Meyding-Lamade, Uta; Craemer, Eva Maria; Diah, Suwarni; Tuah, Ani Afiqah; Muharram, Siti Hanna

2016-07-01

Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

The adaptor CRADD/RAIDD controls activation of endothelial cells by proinflammatory stimuli.

PubMed

Qiao, Huan; Liu, Yan; Veach, Ruth A; Wylezinski, Lukasz; Hawiger, Jacek

2014-08-08

A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Upregulation of microRNA-876 Induces Endothelial Cell Apoptosis by Suppressing Bcl-Xl in Development of Atherosclerosis.

PubMed

Xu, Kaicheng; Liu, Peng; Zhao, Yue

2017-01-01

The injury and apoptotic cell death of endothelial cells hallmark the development of atherosclerosis (AS), characterized by dysregulation of lipid homeostasis, immune responses, and formation of coronary plaques. However, the mechanisms underlying the initiation of endothelial cell apoptosis remain ill-defined. Recent evidence suggests a role of microRNAs in the processes of AS-associated endothelial cell apoptosis. Thus, we studied this question in the current study. AS was developed in ApoE (-/-) mice suppled with high-fat diet (HFD), compared to ApoE (-/-) mice suppled with normal diet (ND). Mouse endothelial cells were isolated from the aortic arch using flow cytometry based on their expression of Pecam-1. Oxidized low-density lipoprotein (ox-LDL) were used to treat human aortic endothelial cells (HAECs) as an in vitro model for AS. Gene expression was quantified by RT-qPCR and protein levels were analyzed by Western blotting. Apoptosis was evaluated by FITC Annexin V Apoptosis essay and by TUNEL staining. Prediction of the binding between miRNAs and 3'-UTR of mRNA from the target gene was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. HFD mice, but not ND mice, developed AS in 12 weeks. Significantly reduced endothelial cell marks and significantly increased mesenchymal cell marks were detected in the aortic arch of the HFD mice, compared to the ND mice. The endothelial cell apoptosis was significantly higher in HFD mice, seemingly due to functional suppression of protein translation of anti-apoptotic Bcl-Xl protein through upregulation of miR-876. Similar results were obtained from in vitro study. Inhibition of miR-876 abolished the effects of ox-LDL-induced apoptotic cell death of HAECs. AS-associated endothelial cell apoptosis may partially result from downregulation of Bcl-Xl, through upregulation of miR-876 that binds and suppresses translation of Bcl-Xl mRNA. © 2017 The Author(s). Published by S. Karger AG, Basel.

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice

PubMed Central

Yao, Longbiao; Yago, Tadayuki; Shao, Bojing; Liu, Zhenghui; Silasi-Mansat, Robert; Setiadi, Hendra; Lupu, Florea

2013-01-01

Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin–dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti–P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation. PMID:24081661

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation.

PubMed

Watanabe, Ryou; Hayashi, Ryuhei; Kimura, Yu; Tanaka, Yuji; Kageyama, Tomofumi; Hara, Susumu; Tabata, Yasuhiko; Nishida, Kohji

2011-09-01

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

Atherosclerosis-Associated Endothelial Cell Apoptosis by MiR-429-Mediated Down Regulation of Bcl-2.

PubMed

Zhang, Tao; Tian, Feng; Wang, Jing; Jing, Jing; Zhou, Shan-Shan; Chen, Yun-Dai

2015-01-01

Endothelial cell injury and subsequent apoptosis play a key role in the development and pathogenesis of atherosclerosis, which is hallmarked by dysregulated lipid homeostasis, aberrant immunity and inflammation, and plaque-instability-associated coronary occlusion. Nevertheless, our understanding of the mechanisms underlying endothelial cell apoptosis is still limited. MicroRNA-429 (miR-29) is a known cancer suppressor that promotes cancer cell apoptosis. However, it is unknown whether miR-429 may be involved in the development of atherosclerosis through similar mechanisms. We addressed these questions in the current study. We examined the levels of endothelial cell apoptosis in ApoE (-/-) mice suppled with high-fat diet (HFD), a mouse model for atherosclerosis (simplified as HFD mice). We analyzed the levels of anti-apoptotic protein Bcl-2 and the levels of miR-429 in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-429 and 3'-UTR of Bcl-2 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-429 were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs). HFD mice developed atherosclerosis in 12 weeks, while the control ApoE (-/-) mice that had received normal diet (simplified as NOR mice) did not. HFD mice had significantly lower percentage of endothelial cells and significantly higher percentage of mesenchymal cells in the aorta than NOR mice. Significantly higher levels of endothelial cell apoptosis were detected in HFD mice, resulting from decreases in Bcl-2 protein, but not mRNA. The decreases in Bcl-2 in endothelial cells were due to increased levels of miR-429, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Atherosclerosis-associated endothelial cell apoptosis may result from down regulation of Bcl-2, through increased miR-429 that binds and suppresses translation of Bcl-2 mRNA. © 2015 The Author(s) Published by S. Karger AG, Basel.

Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells.

PubMed

Nylund, Reetta; Kuster, Niels; Leszczynski, Dariusz

2010-10-18

Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE). There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed). These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the proteomes were examined immediately after the end of the exposure and when the false discovery rate correction was applied to analysis. This observation agrees with our earlier study showing that the 1800 MHz GSM radiation exposure had only very limited effect on the proteome of human endothelial cell line EA.hy926, as compared with the effect of 900 MHz GSM radiation.

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

PubMed Central

Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

2016-01-01

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters. PMID:26936382

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

PubMed

Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

2016-03-03

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

PubMed

Schweitzer, Kelly S; Chen, Steven X; Law, Sarah; Van Demark, Mary; Poirier, Christophe; Justice, Matthew J; Hubbard, Walter C; Kim, Elena S; Lai, Xianyin; Wang, Mu; Kranz, William D; Carroll, Clinton J; Ray, Bruce D; Bittman, Robert; Goodpaster, John; Petrache, Irina

2015-07-15

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

An EMMPRIN-γ-catenin-Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions.

PubMed

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S; Enríquez, José A; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G

2014-09-01

Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. © 2014. Published by The Company of Biologists Ltd.

Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.

PubMed Central

Usuki, K; Heldin, N E; Miyazono, K; Ishikawa, F; Takaku, F; Westermark, B; Heldin, C H

1989-01-01

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis. Images PMID:2678104

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow

PubMed Central

Vion, Anne-Clemence; Hammoutene, Adel; Poisson, Johanne; Lasselin, Juliette; Devue, Cecile; Pic, Isabelle; Dupont, Nicolas; Busse, Johanna; Stark, Konstantin; Lafaurie-Janvore, Julie; Barakat, Abdul I.; Loyer, Xavier; Souyri, Michele; Viollet, Benoit; Julia, Pierre; Tedgui, Alain; Codogno, Patrice; Rautou, Pierre-Emmanuel

2017-01-01

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α–induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation. PMID:28973855

Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle.

PubMed

Tamaki, Tetsuro; Akatsuka, Akira; Ando, Kiyoshi; Nakamura, Yoshihiko; Matsuzawa, Hideyuki; Hotta, Tomomitsu; Roy, Roland R; Edgerton, V Reggie

2002-05-13

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

PubMed

Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

2013-09-01

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

Endothelium as a transducing surface.

PubMed

Ryan, U S

1989-02-01

Endothelial cells responses to a variety of agonists include release of endothelium dependent vasodilators, such as endothelium dependent relaxing factor (EDRF) and prostacyclin (PGI2). These substances act on vascular smooth muscle to cause relaxation and also have potent anti-aggregatory effects on platelets. A study of the mechanisms of signal transduction involved in these processes was undertaken. An investigation of intracellular calcium using FURA-2 and INDO-1 loaded endothelial cells shows transient elevation in response to vasodilator agonists. The calcium content of endothelial cells calculated using 45Ca flux techniques is increased in response to bradykinin and thrombin. Receptor activation leads to increased phosphoinositide turnover in endothelial cells and activates protein kinase C, the latter may be involved in feedback regulation. Patch clamp studies have demonstrated receptor-operated ionic channels in the endothelial cell membrane. Thus, intracellular calcium concentration is elevated in response to receptor activation, both as a result of liberation of calcium from intracellular stores and calcium entry from extracellular sources. Endothelial cells also respond to particulate stimuli. They can selectively bind and phagocytize bacteria. Phagocytosis leads to generation of superoxide aionin, a process which also seems to be controlled by elevation of intracellular calcium and activation of protein kinase C. In addition phagocytosis activates endothelial cells resulting in increased migration, division and further phagocytosis. All in all, the plethora of different endothelial responses to a variety of stimuli suggests a complex and multipotent cell type.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth factor-induced morphological, physiological and molecular characteristics in cerebral endothelial cells.

PubMed

Krizbai, I A; Bauer, H; Amberger, A; Hennig, B; Szabó, H; Fuchs, R; Bauer, H C

2000-09-01

The capacity of vascular endothelial cells to modulate their phenotype in response to changes in environmental conditions is one of the most important characteristics of this cell type. Since different growth factors may play an important signalling role in this adaptive process we have investigated the effect of endothelial cell growth factor (ECGF) on morphological, physiological and molecular characteristics of cerebral endothelial cells (CECs). CECs grown in the presence of ECGF and its cofactor heparin exhibit an epithelial-like morphology (type I CECs). Upon removal of growth factors, CECs develop an elongated spindle-like shape (type II CECs) which is accompanied by the reorganization of actin filaments and the induction of alpha-actin expression. Since one of the most important functions of CECs is the creation of a selective diffusion barrier between the blood and the central nervous system (CNS), we have studied the expression of junction-related proteins in both cell types. We have found that removal of growth factors from endothelial cultures leads to the downregulation of cadherin and occludin protein levels. The loss of junctional proteins was accompanied by a significant increase in the migratory activity and an altered protease activity profile of the cells. TGF-beta1 suppressed endothelial migration in all experiments. Our data provide evidence to suggest that particular endothelial functions are largely controlled by the presence of growth factors. The differences in adhesiveness and migration may play a role in important physiological and pathological processes of endothelial cells such as vasculogenesis or tumor progression.

Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro

PubMed Central

Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

2014-01-01

Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-l-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. PMID:25339691

Depletion of CD52-positive cells inhibits the development of central nervous system autoimmune disease, but deletes an immune-tolerance promoting CD8 T-cell population. Implications for secondary autoimmunity of alemtuzumab in multiple sclerosis.

PubMed

von Kutzleben, Stephanie; Pryce, Gareth; Giovannoni, Gavin; Baker, David

2017-04-01

The objective was to determine whether CD52 lymphocyte depletion can act to promote immunological tolerance induction by way of intravenous antigen administration such that it could be used to either improve efficiency of multiple sclerosis (MS) inhibition or inhibit secondary autoimmunities that may occur following alemtuzumab use in MS. Relapsing experimental autoimmune encephalomyelitis was induced in ABH mice and immune cell depletion was therapeutically applied using mouse CD52 or CD4 (in conjunction with CD8 or CD20) depleting monoclonal antibodies. Immunological unresponsiveness was then subsequently induced using intravenous central nervous system antigens and responses were assessed clinically. A dose-response of CD4 monoclonal antibody depletion indicated that the 60-70% functional CD4 T-cell depletion achieved in perceived failed trials in MS was perhaps too low to even stop disease in animals. However, more marked (~75-90%) physical depletion of CD4 T cells by CD4 and CD52 depleting antibodies inhibited relapsing disease. Surprisingly, in contrast to CD4 depletion, CD52 depletion blocked robust immunological unresponsiveness through a mechanism involving CD8 T cells. Although efficacy was related to the level of CD4 T-cell depletion, the observations that CD52 depletion of CD19 B cells was less marked in lymphoid organs than in the blood provides a rationale for the rapid B-cell hyper-repopulation that occurs following alemtuzumab administration in MS. That B cells repopulate in the relative absence of T-cell regulatory mechanisms that promote immune tolerance may account for the secondary B-cell autoimmunities, which occur following alemtuzumab treatment of MS. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

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

Latham, Antony M.; Odell, Adam F.; Mughal, Nadeem A.

2012-11-01

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a highmore » VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states. -- Highlights: Black-Right-Pointing-Pointer Endothelial cells mount a stress response under conditions of low serum. Black-Right-Pointing-Pointer Endothelial VEGFR levels are modulated during this response. Black-Right-Pointing-Pointer The cell regulates VEGF-A bioavailability and cell survival. Black-Right-Pointing-Pointer This may partly underlie endothelial dysfunction seen in many pathologies.« less

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

PubMed

van Lessen, Max; Shibata-Germanos, Shannon; van Impel, Andreas; Hawkins, Thomas A; Rihel, Jason; Schulte-Merker, Stefan

2017-05-12

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Automated Deep Learning-Based System to Identify Endothelial Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Kusumoto, Dai; Lachmann, Mark; Kunihiro, Takeshi; Yuasa, Shinsuke; Kishino, Yoshikazu; Kimura, Mai; Katsuki, Toshiomi; Itoh, Shogo; Seki, Tomohisa; Fukuda, Keiichi

2018-06-05

Deep learning technology is rapidly advancing and is now used to solve complex problems. Here, we used deep learning in convolutional neural networks to establish an automated method to identify endothelial cells derived from induced pluripotent stem cells (iPSCs), without the need for immunostaining or lineage tracing. Networks were trained to predict whether phase-contrast images contain endothelial cells based on morphology only. Predictions were validated by comparison to immunofluorescence staining for CD31, a marker of endothelial cells. Method parameters were then automatically and iteratively optimized to increase prediction accuracy. We found that prediction accuracy was correlated with network depth and pixel size of images to be analyzed. Finally, K-fold cross-validation confirmed that optimized convolutional neural networks can identify endothelial cells with high performance, based only on morphology. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Ammonia promotes endothelial cell survival via the heme oxygenase-1-mediated release of carbon monoxide.

PubMed

Liu, Xiao-Ming; Peyton, Kelly J; Durante, William

2017-01-01

Although endothelial cells produce substantial quantities of ammonia during cell metabolism, the physiologic role of this gas in these cells is not known. In this study, we investigated if ammonia regulates the expression of heme oxygenase-1 (HO-1), and if this enzyme influences the biological actions of ammonia on endothelial cells. Exogenously administered ammonia, given as ammonium chloride or ammonium hydroxide, or endogenously generated ammonia stimulated HO-1 protein expression in cultured human and murine endothelial cells. Dietary supplementation of ammonia also induced HO-1 protein expression in murine arteries. The increase in HO-1 protein by ammonia in endothelial cells was first detected 4h after ammonia exposure and was associated with the induction of HO-1 mRNA, enhanced production of reactive oxygen species (ROS), and increased expression and activity of NF-E2-related factor-2 (Nrf2). Ammonia also activated the HO-1 promoter and this was blocked by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. The induction of HO-1 expression by ammonia was dependent on ROS formation and prevented by N-acetylcysteine or rotenone. Finally, prior treatment of endothelial cells with ammonia inhibited tumor necrosis factor-α-stimulated cell death. However, silencing HO-1 expression abrogated the protective action of ammonia and this was reversed by the administration of carbon monoxide but not bilirubin or iron. In conclusion, this study demonstrates that ammonia stimulates the expression of HO-1 in endothelial cells via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cytoprotective action of ammonia by generating carbon monoxide. Moreover, it identifies ammonia as a potentially important signaling gas in the vasculature that promotes endothelial cell survival. Copyright © 2016 Elsevier Inc. All rights reserved.

Angiogenesis in mucous retention cyst: a human in vivo-like model of endothelial cell differentiation in mucous substrate.

PubMed

Swelam, Wael; Ida-Yonemochi, Hiroko; Saku, Takashi

2005-01-01

Mucous retention cysts contain a mucous pool in the lumina, in which pure angiogenic processes are occasionally observed. By using this unique human material, our aim was to understand the in vivo angiogenic process. Fifteen surgical tissue samples of mucous retention cysts of the lip were examined for expression of vascular endothelial markers and extracellular matrix molecules by immunohistochemistry and in situ hybridization (ISH). Endothelial cells forming new vascular channels showed immunopositivities for CD31, CD34, vascular endothelial growth factor (VEGF), and von Willebrand factor (vWF). These newly formed capillaries were surrounded by tenascin-positive matrices and further by a dense infiltration of CD68-positive cells with foamy to epitheloid appearances. Some of these cells were simultaneously positive for CD34, VEGF, and one of its receptors, Flk-1, and they showed definite mRNA as well as protein signals for tenascin. In addition, these cells often tended to be aligned, which suggested tubule formation. The results suggest that monocyte/macrophage lineage cells are a major source for endothelial cells at least in mucous retention cysts and that tenascin produced by those cells plays an important role in differentiation of endothelial cells.

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases1

PubMed Central

Zozulya, Alla L.; Reinke, Emily; Baiu, Dana C.; Karman, Jozsef; Sandor, Matyas; Fabry, Zsuzsanna

2007-01-01

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α -induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS. PMID:17182592

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

PubMed

Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

2002-09-04

The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

PubMed

Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

2002-11-01

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

NASA Astrophysics Data System (ADS)

Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

2016-03-01

The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.

PubMed

Cui, Jiajia; Qin, Lingfeng; Zhang, Junwei; Abrahimi, Parwiz; Li, Hong; Li, Guangxin; Tietjen, Gregory T; Tellides, George; Pober, Jordan S; Mark Saltzman, W

2017-08-04

Human endothelial cells are initiators and targets of the rejection response. Pre-operative modification of endothelial cells by small interfering RNA transfection could shape the nature of the host response post-transplantation. Ablation of endothelial cell class II major histocompatibility complex molecules by small interfering RNA targeting of class II transactivator can reduce the capacity of human endothelial cells to recruit and activate alloreactive T cells. Here, we report the development of small interfering RNA-releasing poly(amine-co-ester) nanoparticles, distinguished by their high content of a hydrophobic lactone. We show that a single transfection of small interfering RNA targeting class II transactivator attenuates major histocompatibility complex class II expression on endothelial cells for at least 4 to 6 weeks after transplantation into immunodeficient mouse hosts. Furthermore, silencing of major histocompatibility complex class II reduces allogeneic T-cell responses in vitro and in vivo. These data suggest that poly(amine-co-ester) nanoparticles, potentially administered during ex vivo normothermic machine perfusion of human organs, could be used to modify endothelial cells with a sustained effect after transplantation.The use of gene silencing techniques in the treatment of post-transplantation host rejection is not long lasting and can have systemic effects. Here, the authors utilize a nanocarrier for siRNA for treatment of arteries ex vivo prior to implantation subsequently attenuating immune reaction in vivo.

Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles

NASA Astrophysics Data System (ADS)

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Andrée, Birgit; Cebotari, Serghei; Boyle, Erin C.; Haverich, Axel; Hilfiker, Andres

2017-08-01

In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

PubMed

Chrifi, Ihsan; Louzao-Martinez, Laura; Brandt, Maarten; van Dijk, Christian G M; Burgisser, Petra; Zhu, Changbin; Kros, Johan M; Duncker, Dirk J; Cheng, Caroline

2017-06-01

Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1 + endothelial progenitor cells during embryonic development. Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin + vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin + , EEA1 + , Rab11 + , Rab5 + , and Rab7 + vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell-cell adhesion at adherens junctions and contributes to the control of vascular sprouting. © 2017 American Heart Association, Inc.

Generation and characterization of a spontaneously immortalized endothelial cell line from mice microcirculation.

PubMed

Loiola, Rodrigo A; Torres, Tathiany C; Aburaya, Carla M; Landgraf, Maristella A; Landgraf, Richardt G; Bosco Pesquero, João; Fernandes, Liliam

2013-05-01

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation. Copyright © 2013 Elsevier Inc. All rights reserved.

Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

PubMed

Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

2017-11-01

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation

PubMed Central

Garner, Omai B.; Aguilar, Hector C.; Fulcher, Jennifer A.; Levroney, Ernest L.; Harrison, Rebecca; Wright, Lacey; Robinson, Lindsey R.; Aspericueta, Vanessa; Panico, Maria; Haslam, Stuart M.; Morris, Howard R.; Dell, Anne

2010-01-01

Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell. PMID:20657665

Traction force dynamics predict gap formation in activated endothelium

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

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

Neuropilin2 expressed in gastric cancer endothelial cells increases the proliferation and migration of endothelial cells in response to VEGF

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

Kim, Woo Ho; Lee, Sun Hee; Jung, Myung Hwan

2009-08-01

The structure and characteristics of the tumor vasculature are known to be different from those of normal vessels. Neuropilin2 (Nrp2), which is expressed in non-endothelial cell types, such as neuronal or cancer cells, functions as a receptor for both semaphorin and vascular endothelial growth factor (VEGF). After isolating tumor and normal endothelial cells from advanced gastric cancer tissue and normal gastric mucosa tissues, respectively, we identified genes that were differentially expressed in gastric tumor endothelial (TEC) and normal endothelial cells (NEC) using DNA oligomer chips. Using reverse transcriptase-PCR, we confirmed the chip results by showing that Nrp2 gene expression ismore » significantly up-regulated in TEC. Genes that were found to be up-regulated in TEC were also observed to be up-regulated in human umbilical vein endothelial cells (HUVECs) that were co-cultured with gastric cancer cells. In addition, HUVECs co-cultured with gastric cancer cells showed an increased reactivity to VEGF-induced proliferation and migration. Moreover, overexpression of Nrp2 in HUVECs significantly enhanced the proliferation and migration induced by VEGF. Observation of an immunohistochemical analysis of various human tumor tissue arrays revealed that Nrp2 is highly expressed in the tumor vessel lining and to a lesser extent in normal tissue microvessels. From these results, we suggest that Nrp2 may function to increase the response to VEGF, which is more significant in TEC than in NEC given the differential expression, leading to gastric TEC with aggressive angiogenesis phenotypes.« less

Surface modification and endothelialization of biomaterials as potential scaffolds for vascular tissue engineering applications.

PubMed

Ren, Xiangkui; Feng, Yakai; Guo, Jintang; Wang, Haixia; Li, Qian; Yang, Jing; Hao, Xuefang; Lv, Juan; Ma, Nan; Li, Wenzhong

2015-08-07

Surface modification and endothelialization of vascular biomaterials are common approaches that are used to both resist the nonspecific adhesion of proteins and improve the hemocompatibility and long-term patency of artificial vascular grafts. Surface modification of vascular grafts using hydrophilic poly(ethylene glycol), zwitterionic polymers, heparin or other bioactive molecules can efficiently enhance hemocompatibility, and consequently prevent thrombosis on artificial vascular grafts. However, these modified surfaces may be excessively hydrophilic, which limits initial vascular endothelial cell adhesion and formation of a confluent endothelial lining. Therefore, the improvement of endothelialization on these grafts by chemical modification with specific peptides and genes is now arousing more and more interest. Several active peptides, such as RGD, CAG, REDV and YIGSR, can be specifically recognized by endothelial cells. Consequently, graft surfaces that are modified by these peptides can exhibit targeting selectivity for the adhesion of endothelial cells, and genes can be delivered by targeting carriers to specific tissues to enhance the promotion and regeneration of blood vessels. These methods could effectively accelerate selective endothelial cell recruitment and functional endothelialization. In this review, recent developments in the surface modification and endothelialization of biomaterials in vascular tissue engineering are summarized. Both gene engineering and targeting ligand immobilization are promising methods to improve the clinical outcome of artificial vascular grafts.

Increased affinity of endothelial cells to NiTi using ultraviolet irradiation: An in vitro study.

PubMed

Tateshima, Satoshi; Kaneko, Naoki; Yamada, Masahiro; Duckwiler, Gary; Vinuela, Fernando; Ogawa, Takahiro

2018-04-01

Nickel-titanium alloy (NiTi) is one of the most popular materials used endovascularly because of its shape memory and superelasticity. The NiTi device needs to be covered by endothelial cells after being placed in the blood vessel to reduce ischemic complications. The objective of this study was to examine the impact of ultraviolet (UV) irradiation on the biocompatibility of NiTi surfaces with endothelial cells. NiTi sheets were treated with UV irradiation for 48 h and human aorta derived endothelial cells were used in this study. UV irradiation converted the NiTi surface to hydrophilic state and increased albumin adsorption. The number of endothelial cell migration, attachment, proliferation as well as their metabolic activity were significantly increased on UV treated NiTi. This study provides the first evidence of the photoactivation of NiTi surfaces by UV irradiation and demonstrates improved biocompatibility of UV-treated NiTi surfaces with vascular endothelial cells. These results suggest that UV irradiation may promote endothelialization of NiTi devices in blood vessels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1034-1038, 2018. © 2017 Wiley Periodicals, Inc.

Suppression of endothelial cell adhesion by XJP-1, a new phenolic compound derived from banana peel.

PubMed

Fu, Rong; Yan, Tianhua; Wang, Qiujuan; Guo, Qinglong; Yao, Hequan; Wu, Xiaoming; Li, Yang

2012-01-01

The adhesion of monocytes to activated vascular endothelial cells is a critical event in the initiation of atherosclerosis. Adhesion is mediated by oxidized low-density lipoprotein (ox-LDL) which up-regulates inflammatory markers on endothelial cells. Here we report that (±) 7, 8-dihydroxy-3-methyl-isochromanone-4 (XJP-1), an inhibitor of ox-LDL-induced adhesion of monocytes to endothelial cells blocks cellular functions which are associated with adhesion. We show that XJP-1 down-regulates ox-LDL-induced over-expression of adhesion molecules (ICAM-1 and VCAM-1) in a dose-dependent manner in human umbilical vein endothelial cells (HUVECs), attenuates ox-LDL-induced up-regulation of low-density lipoprotein receptor (LOX)-1, decreases generation of reactive oxygen species (ROS), blocks translocation of nuclear factor-kappa B (NF-κB) activity, and prevents activation of c-Jun N-terminal kinase (JNK)/p38 pathways in endothelial cells. These findings suggest that XJP-1 may attenuate ox-LDL-induced endothelial adhesion of monocytes by blocking expression of adhesion molecules through suppressing ROS/NF-κB, JNK and p38 pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

The coffee diterpene kahweol inhibits tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in human endothelial cells

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

Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil

2006-12-15

Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B activation pathway in these cells. Overall, kahweol hasmore » anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.« less

Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells.

PubMed

Gautier, Violette; Cayrol, Corinne; Farache, Dorian; Roga, Stéphane; Monsarrat, Bernard; Burlet-Schiltz, Odile; Gonzalez de Peredo, Anne; Girard, Jean-Philippe

2016-10-03

IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells.

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation.

PubMed

Cai, Shijie; Alp, Nicholas J; McDonald, Denise; Smith, Ian; Kay, Jonathan; Canevari, Laura; Heales, Simon; Channon, Keith M

2002-09-01

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

Effect of incision width on graft survival and endothelial cell loss after DSAEK

PubMed Central

Price, Marianne O.; Bidros, Maria; Gorovoy, Mark; Price, Francis W.; Benetz, Beth A.; Menegay, Harry J.; Debanne, Sara M.; Lass, Jonathan H.

2009-01-01

Purpose To assess the effect of incision width (5.0 and 3.2 mm) on graft survival and endothelial cell loss six months and one year after Descemet stripping automated endothelial keratoplasty (DSAEK). Methods One hundred sixty-seven subjects with endothelial decompensation from a moderate-risk condition (principally Fuchs’ dystrophy or pseudophakic corneal edema) underwent DSAEK by two experienced surgeons. The donor was folded over and inserted with single point fixation forceps. This retrospective analysis assessed graft survival, complications, and endothelial cell loss, which was calculated from baseline donor and 6-month and 1-year postoperative central endothelial images evaluated by an independent specular microscopy reading center. Results No primary graft failures occurred in either group. One-year graft survival rates were comparable (98% vs. 97%) in the 5.0- and 3.2-mm groups, respectively (P=1.0). Complications included graft dislocation, graft rejection episodes, and elevated intraocular pressure and occurred at similar rates in both groups (P ≥ 0.28). Pupillary block glaucoma did not occur in either group. Mean baseline donor endothelial cell density did not differ: 2782 cells/mm2 in the 5.0-mm (n=64) and 2784 cells/mm2 in the 3.2-mm (n=103) groups. Percent endothelial cell loss was 27±20% (n=55) vs. 40±22% (n=71; 6 months) and 31±19% (n=45) vs. 44±22% (n=62; 12 months) in the 5.0-mm and 3.2-mm incision groups, respectively (both P<0.001). Conclusions One year after DSAEK, overall graft success was comparable for the two groups; however, the 5.0-mm incision width resulted in substantially lower endothelial cell loss at 6 and 12 months. PMID:20299973

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

PubMed

Yan, Meiping; Zhang, Xinhua; Chen, Ao; Gu, Wei; Liu, Jie; Ren, Xiaojiao; Zhang, Jianping; Wu, Xiaoxiong; Place, Aaron T; Minshall, Richard D; Liu, Guoquan

2017-11-01

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2- via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction. © FASEB.

Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells: new insight into the pathogenesis of systemic sclerosis.

PubMed

Cipriani, P; Guiducci, S; Miniati, I; Cinelli, M; Urbani, S; Marrelli, A; Dolo, V; Pavan, A; Saccardi, R; Tyndall, A; Giacomelli, R; Cerinic, M Matucci

2007-06-01

Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair. MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony-forming unit-fibroblastoid colonies was determined. After culture in endothelial-specific medium, the endothelial-like MSC (EL-MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed. MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR-2+, CXCR4+, VEGFR-2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL-MSCs showed increased expression of VEGFR-1, VEGFR-2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell-derived factor 1 to cultured SSc EL-MSCs increased their angiogenic potential less than that in controls. Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes

PubMed Central

Joshi, Meghnad; Patil, Pradeep B.; He, Zhong; Holgersson, Jan; Olausson, Michael; Sumitran-Holgersson, Suchitra

2012-01-01

Background aims One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Methods Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. Results After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Conclusions Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy. PMID:22424216

Endothelial necrosis at 1h post-burn predicts progression of tissue injury

PubMed Central

Hirth, Douglas; McClain, Steve A.; Singer, Adam J.; Clark, Richard A.F.

2013-01-01

Burn injury progression has not been well characterized at the cellular level. To define burn injury progression in terms of cell death, histopathologic spatiotemporal relationships of cellular necrosis and apoptosis were investigated in a validated porcine model of vertical burn injury progression. Cell necrosis was identified by High Mobility Group Box 1 protein and apoptosis by Caspase 3a staining of tissue samples taken 1h, 24h and 7 days post-burn. Level of endothelial cell necrosis at 1h was predictive of level of apoptosis at 24h (Pearson's r=0.87) and of level of tissue necrosis at 7 days (Pearson's r=0.87). Furthermore, endothelial cell necrosis was deeper than interstitial cell necrosis at 1h (p<0.001). Endothelial cell necrosis at 1h divided the zone of injury progression (Jackson's zone of stasis) into an upper subzone with necrotic endothelial cells and initially viable adnexal and interstitial cells at 1h that progressed to necrosis by 24h, and a lower zone with initially viable endothelial cells at 1h, but necrosis and apoptosis of all cell types by 24h. Importantly, this spatiotemporal series of events and rapid progression resembles myocardial infarction and stroke, and implicates mechanisms of these injuries, ischemia, ischemia reperfusion, and programmed cell death, in burn progression. PMID:23627744

Subregional localization and characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head.

PubMed

Li, Zhuan; Vink, Chris S; Mariani, Samanta A; Dzierzak, Elaine

2016-08-01

Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Genetic and live imaging data have supported this. Recently, the embryonic head has been shown to contain fully functional hematopoietic stem cells (HSC). By lineage tracing, cerebrovascular specific endothelial cells were shown to contribute to the postnatal mouse hematopoietic system. Since Ly6aGFP is a marker of all HSCs, some hematopoietic cluster cells and hemogenic endothelial cells in the midgestation mouse aorta, we examine here whether embryonic head HSCs and vascular endothelial cells are positive for this marker. Whereas some head vasculature, single hematopoietic cells and all HSCs are Ly6aGFP expressing, we do not find clusters of hematopoietic cells emerging from the cerebrovasculature that are characteristic of endothelial-to-hematopoietic transition. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration.

PubMed

Grote, Karsten; Salguero, Gustavo; Ballmaier, Matthias; Dangers, Marc; Drexler, Helmut; Schieffer, Bernhard

2007-08-01

Tissue regeneration involves the formation of new blood vessels regulated by angiogenic factors. We reported recently that the expression of the angiogenic factor CCN1 is up-regulated under various pathophysiologic conditions within the cardiovascular system. Because CD34+ progenitor cells participate in cardiovascular tissue regeneration, we investigated whether CCN1-detected for the first time in human plasma-promotes the recruitment of CD34+ progenitor cells to endothelial cells, thereby enhancing endothelial proliferation and neovascularization. In this study, we demonstrated that CCN1 and supernatants from CCN1-stimulated human CD34+ progenitor cells promoted proliferation of endothelial cells and angiogenesis in vitro and in vivo. In addition, CCN1 induced migration and transendothelial migration of CD34+ cells and the release of multiple growth factors, chemokines, and matrix metalloproteinase-9 (MMP-9) from these cells. Moreover, the CCN1-specific integrins alpha(M)beta(2) and alpha(V)beta(3) are expressed on CD34+ cells and CCN1 stimulated integrin-dependent signaling. Furthermore, integrin antagonists (RGD-peptides) suppressed both binding of CCN1 to CD34+ cells and CCN1-induced adhesion of CD34+ cells to endothelial cells. These data suggest that CCN1 promotes integrin-dependent recruitment of CD34+ progenitor cells to endothelial cells, which may contribute to paracrine effects on angiogenesis and tissue regeneration.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

PubMed

Radwanska, Agata; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Ciais, Delphine; Rekima, Samah; Rupp, Tristan; Sudaka, Anne; Orend, Gertraud; Van Obberghen-Schilling, Ellen

2017-10-06

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro.

PubMed

Schlegel, Nicolas; Baumer, Yvonne; Drenckhahn, Detlev; Waschke, Jens

2009-05-01

To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. Experimental laboratory research. Research laboratory. Wistar rats and cultured human microvascular endothelial cells. Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

PubMed Central

2013-01-01

Background Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Conclusions Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery. PMID:23773766

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

PubMed

Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

2013-06-18

Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Endothelial Dysfunction in Human Diabetes Is Mediated by Wnt5a-JNK Signaling.

PubMed

Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G; Fetterman, Jessica L; Linder, Erika A; Berk, Brittany D; Masaki, Nobuyuki; Weisbrod, Robert M; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J; Walsh, Kenneth; Hamburg, Naomi M

2016-03-01

Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus. © 2016 American Heart Association, Inc.

Haploinsufficiency of the insulin-like growth factor-1 receptor enhances endothelial repair and favorably modifies angiogenic progenitor cell phenotype.

PubMed

Yuldasheva, Nadira Y; Rashid, Sheikh Tawqeer; Haywood, Natalie J; Cordell, Paul; Mughal, Romana; Viswambharan, Hema; Imrie, Helen; Sukumar, Piruthivi; Cubbon, Richard M; Aziz, Amir; Gage, Matthew; Mbonye, Kamatamu Amanda; Smith, Jessica; Galloway, Stacey; Skromna, Anna; Scott, D Julian A; Kearney, Mark T; Wheatcroft, Stephen B

2014-09-01

Defective endothelial regeneration predisposes to adverse arterial remodeling and is thought to contribute to cardiovascular disease in type 2 diabetes mellitus. We recently demonstrated that the type 1 insulin-like growth factor receptor (IGF1R) is a negative regulator of insulin sensitivity and nitric oxide bioavailability. In this report, we examined partial deletion of the IGF1R as a potential strategy to enhance endothelial repair. We assessed endothelial regeneration after wire injury in mice and abundance and function of angiogenic progenitor cells in mice with haploinsufficiency of the IGF1R (IGF1R(+/-)). Endothelial regeneration after arterial injury was accelerated in IGF1R(+/-) mice. Although the yield of angiogenic progenitor cells was lower in IGF1R(+/-) mice, these angiogenic progenitor cells displayed enhanced adhesion, increased secretion of insulin-like growth factor-1, and enhanced angiogenic capacity. To examine the relevance of IGF1R manipulation to cell-based therapy, we transfused IGF1R(+/-) bone marrow-derived CD117(+) cells into wild-type mice. IGF1R(+/-) cells accelerated endothelial regeneration after arterial injury compared with wild-type cells and did not alter atherosclerotic lesion formation. Haploinsufficiency of the IGF1R is associated with accelerated endothelial regeneration in vivo and enhanced tube forming and adhesive potential of angiogenic progenitor cells in vitro. Partial deletion of IGF1R in transfused bone marrow-derived CD117(+) cells enhanced their capacity to promote endothelial regeneration without altering atherosclerosis. Our data suggest that manipulation of the IGF1R could be exploited as novel therapeutic approach to enhance repair of the arterial wall after injury. © 2014 American Heart Association, Inc.

Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone.

PubMed

Harazin, András; Bocsik, Alexandra; Barna, Lilla; Kincses, András; Váradi, Judit; Fenyvesi, Ferenc; Tubak, Vilmos; Deli, Maria A; Vecsernyés, Miklós

2018-01-01

The blood-brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which α-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-α and IL-1β, we hypothesized a protective effect of α-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide α-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-α and IL-1β induced cell damage, measured by impedance and MTT assay, which was attenuated by α-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and β-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-κB were also reduced by α-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of α-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB.

Protection of cultured brain endothelial cells from cytokine-induced damage by α-melanocyte stimulating hormone

PubMed Central

Barna, Lilla; Kincses, András; Váradi, Judit; Fenyvesi, Ferenc; Tubak, Vilmos

2018-01-01

The blood–brain barrier (BBB), an interface between the systemic circulation and the nervous system, can be a target of cytokines in inflammatory conditions. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce damage in brain endothelial cells and BBB dysfunction which contribute to neuronal injury. The neuroprotective effects of α-melanocyte stimulating hormone (α-MSH) were investigated in experimental models, but there are no data related to the BBB. Based on our recent study, in which α-MSH reduced barrier dysfunction in human intestinal epithelial cells induced by TNF-α and IL-1β, we hypothesized a protective effect of α-MSH on brain endothelial cells. We examined the effect of these two pro-inflammatory cytokines, and the neuropeptide α-MSH on a culture model of the BBB, primary rat brain endothelial cells co-cultured with rat brain pericytes and glial cells. We demonstrated the expression of melanocortin-1 receptor in isolated rat brain microvessels and cultured brain endothelial cells by RT-PCR and immunohistochemistry. TNF-α and IL-1β induced cell damage, measured by impedance and MTT assay, which was attenuated by α-MSH (1 and 10 pM). The peptide inhibited the cytokine-induced increase in brain endothelial permeability, and restored the morphological changes in cellular junctions visualized by immunostaining for claudin-5 and β-catenin. Elevated production of reactive oxygen species and the nuclear translocation of NF-κB were also reduced by α-MSH in brain endothelial cells stimulated by cytokines. We demonstrated for the first time the direct beneficial effect of α-MSH on cultured brain endothelial cells, indicating that this neurohormone may be protective at the BBB. PMID:29780671

Morphological Changes of Human Corneal Endothelial Cells after Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Administration: A Prospective Open-Label Clinical Study

PubMed Central

Okumura, Naoki; Suganami, Hideki; Kinoshita, Shigeru

2015-01-01

Purpose To investigate the effect and safety of a selective Rho kinase inhibitor, ripasudil 0.4% eye drops, on corneal endothelial cells of healthy subjects. Design Prospective, interventional case series. Methods In this study, 6 healthy subjects were administered ripasudil 0.4% in the right eye twice daily for 1 week. Morphological changes and corneal endothelial cell density were examined by noncontact and contact specular microscopy. Central corneal thickness and corneal volume of 5 mm-diameter area of center cornea were analyzed by Pentacam Scheimpflug topography. All the above measurements were conducted in both eyes before administration, 1.5 and 6 hours after the initial administration on day 0; and in the same manner after the final administration on day 7. Results By noncontact specular microscopy, indistinct cell borders with pseudo guttae were observed, but by contact specular microscopy, morphological changes of corneal endothelial cells were mild and pseudo guttae was not observed after single and repeated administration of ripasudil in all subjects. These changes resolved prior to the next administration, and corneal endothelial cell density, central corneal thickness and corneal volume were not changed throughout the study period. Conclusion Transient morphological changes of corneal endothelial cells such as indistinct cell borders with pseudo guttae were observed by noncontact specular microscopy in healthy subjects after ripasudil administration. Corneal edema was not observed and corneal endothelial cell density did not decrease after 1 week repetitive administration. These morphological changes were reversible and corneal endothelial cell morphology returned to normal prior to the next administration. Trial Registration JAPIC Clinical Trials Information 142705 PMID:26367375

N-Acetylcysteine Increases Corneal Endothelial Cell Survival in a Mouse Model of Fuchs Endothelial Corneal Dystrophy

PubMed Central

Kim, Eun Chul; Meng, Huan; Jun, Albert S.

2014-01-01

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo® luminescent reagent. Two-month-old homozygous knock-in Col8a2L450W/L450W mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (pitalic>0.01). Corneal endothelial cell density (CD) was higher (p=0.001) and percent polymegathism was lower (p=0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD. PMID:24952277

N-Acetylcysteine increases corneal endothelial cell survival in a mouse model of Fuchs endothelial corneal dystrophy.

PubMed

Kim, Eun Chul; Meng, Huan; Jun, Albert S

2014-10-01

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo(®) luminescent reagent. Two-month-old homozygous knock-in Col8a2(L450W/L450W) mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (p < 0.01). Corneal endothelial cell density (CD) was higher (p = 0.001) and percent polymegathism was lower (p = 0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Bony Side of Endothelial Cells in Prostate Cancer.

PubMed

Peng, Jia; Kang, Yibin

2017-06-05

Prostate cancer bone metastases are primarily osteoblastic, but the source of bone-forming cells in these lesions remains poorly defined. In this issue of Developmental Cell, Lin et al. (2017) demonstrate that tumor-associated endothelial cells can give rise to osteoblasts in prostate cancer through endothelial-to-osteoblast (EC-to-OSB) conversion. Copyright © 2017 Elsevier Inc. All rights reserved.

Defibrotide Stimulates Angiogenesis and Protects Endothelial Cells from Calcineurin Inhibitor-Induced Apoptosis via Upregulation of AKT/Bcl-xL.

PubMed

Wang, Xiangmin; Pan, Bin; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-01-01

Sinusoidal obstruction syndrome is a life-threatening complication that can occur after haematopoietic stem cell transplantation. Defibrotide (DF) has been approved for the treatment of individuals with severe sinusoidal obstruction syndrome following haematopoietic stem cell transplantation in the European Union and the United States. However, the precise mechanisms by which DF protects endothelial cells remain to be elucidated. In this study, we found that DF stimulated angiogenesis in vitro and in vivo as assessed by vascular tube formation, scratch-wound repair and Matrigel plug assays. These effects were associated with an activation of pro-survival signalling pathways, including AKT (protein kinase B), ERK (extracellular signal-regulated kinases) and p38. More importantly, DF alleviated calcineurin inhibitor-induced growth inhibition and apoptosis of human umbilical vein endothelial cells and human hepatic sinusoidal endothelial cells in parallel with upregulation of anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xL), which was mediated by AKT (protein kinase B). Notably, these effects were abrogated when Bcl-xL was depleted by small interfering RNA (ribonucleic acid). In addition, DF counteracted calcineurin inhibitor-induced activation of nuclear factor-κB and Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signalling and production of cytokines in vascular endothelial cell-derived EA.hy926 cells. Taken together, DF has pro-angiogenic, anti-apoptotic and anti-inflammatory effects on endothelial cells. DF is a potentially useful agent to prevent the development of, and treat individuals with, endothelial cell injury-related complications after haematopoietic stem cell transplantation. Schattauer GmbH Stuttgart.

Endothelial dysfunction in dengue virus pathology.

PubMed

Vervaeke, Peter; Vermeire, Kurt; Liekens, Sandra

2015-01-01

Dengue virus (DENV) is a leading cause of illness and death, mainly in the (sub)tropics, where it causes dengue fever and/or the more serious diseases dengue hemorrhagic fever and dengue shock syndrome that are associated with changes in vascular permeability. Despite extensive research, the pathogenesis of DENV is still poorly understood and, although endothelial cells represent the primary fluid barrier of the blood vessels, the extent to which these cells contribute to DENV pathology is still under debate. The primary target cells for DENV are dendritic cells and monocytes/macrophages that release various chemokines and cytokines upon infection, which can activate the endothelium and are thought to play a major role in DENV-induced vascular permeability. However, recent studies indicate that DENV also replicates in endothelial cells and that DENV-infected endothelial cells may directly contribute to viremia, immune activation, vascular permeability and immune targeting of the endothelium. Also, the viral non-structural protein-1 and antibodies directed against this secreted protein have been reported to be involved in endothelial cell dysfunction. This review provides an extensive overview of the effects of DENV infection on endothelial cell physiology and barrier function. Copyright © 2014 John Wiley & Sons, Ltd.

Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

PubMed Central

Tang, Duozhuang; Tao, Si; Chen, Zhiyang; Koliesnik, Ievgen Oleksandrovich; Calmes, Philip Gerald; Hoerr, Verena; Han, Bing; Gebert, Nadja; Zörnig, Martin; Löffler, Bettina

2016-01-01

Dietary restriction (DR) improves health, delays tissue aging, and elongates survival in flies and worms. However, studies on laboratory mice and nonhuman primates revealed ambiguous effects of DR on lifespan despite improvements in health parameters. In this study, we analyzed consequences of adult-onset DR (24 h to 1 yr) on hematopoietic stem cell (HSC) function. DR ameliorated HSC aging phenotypes, such as the increase in number of HSCs and the skewing toward myeloid-biased HSCs during aging. Furthermore, DR increased HSC quiescence and improved the maintenance of the repopulation capacity of HSCs during aging. In contrast to these beneficial effects, DR strongly impaired HSC differentiation into lymphoid lineages and particularly inhibited the proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function. The study shows that DR-dependent suppression of growth factors and interleukins mediates these divergent effects caused by DR. Supplementation of insulin-like growth factor 1 partially reverted the DR-induced quiescence of HSCs, whereas IL-6/IL-7 substitutions rescued the impairment of B lymphopoiesis exposed to DR. Together, these findings delineate positive and negative effects of long-term DR on HSC functionality involving distinct stress and growth signaling pathways. PMID:26951333

In Vivo Selection of Transplanted Hepatocytes by Pharmacological Inhibition of Fumarylacetoacetate Hydrolase in Wild-type Mice

PubMed Central

Paulk, Nicole K; Wursthorn, Karsten; Haft, Annelise; Pelz, Carl; Clarke, Gregory; Newell, Amy H; Olson, Susan B; Harding, Cary O; Finegold, Milton J; Bateman, Raymond L; Witte, John F; McClard, Ronald; Grompe, Markus

2012-01-01

Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4–6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20–100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes. PMID:22871666

Adhesion of Epstein–Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

PubMed Central

Kanno, H; Watabe, D; Shimizu, N; Sawai, T

2008-01-01

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-α, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-α or interleukin (IL)-1β, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. PMID:18190605

Neutrophil proteinase 3 (PR3) acts on protease-activated receptor-2 (PAR-2) to enhance vascular endothelial cell barrier function

PubMed Central

Kuckleburg, Christopher J.; Newman, Peter J.

2013-01-01

The principle role of the vascular endothelium is to present a semi-impermeable barrier to soluble factors and circulating cells, while still permitting the passage of leukocytes from the bloodstream into the tissue. The process of diapedesis involves the selective disruption of endothelial cell junctions, an event that could in theory compromise vascular integrity. It is therefore somewhat surprising that neutrophil transmigration does not significantly impair endothelial barrier function. We examined whether neutrophils might secrete factors that promote vascular integrity during the latter stages of neutrophil transmigration, and found that neutrophil proteinase 3 (PR3) – a serine protease harbored in azurophilic granules – markedly enhanced barrier function in endothelial cells. PR3 functioned in this capacity both in its soluble form and in a complex with cell-surface NB1. PR3-mediated enhancement of endothelial cell junctional integrity required its proteolytic activity, as well as endothelial cell expression of the protease-activated receptor, PAR-2. Importantly, PR3 suppressed the vascular permeability changes and disruption of junctional proteins induced by the action of PAR-1 agonists. These findings establish the potential for neutrophil-derived PR3 to play a role in reestablishing vascular integrity following leukocyte transmigration, and in protecting endothelial cells from PAR-1-induced permeability changes that occur during thrombotic and inflammatory events. PMID:23202369

Low-level laser irradiation effect on endothelial cells under conditions of hyperglycemia.

PubMed

Góralczyk, Krzysztof; Szymańska, Justyna; Szot, Katarzyna; Fisz, Jacek; Rość, Danuta

2016-07-01

Diabetes mellitus is considered to be a very serious lifestyle disease leading to cardiovascular complications and impaired wound healing observed in the diabetic foot syndrome. Chronic hyperglycemia is the source of the endothelial activation. The inflammatory process in diabetes is associated with the secretion of inflammatory cytokines by endothelial cells, e.g., tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). The method of phototherapy using laser beam of low power (LLLT-low-level laser therapy) effectively supports the conventional treatment of diabetic vascular complications such as diabetic foot syndrome. The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture-HUVEC line (human umbilical vein endothelial cell)-under conditions of hyperglycemia. It is considered that adverse effects of hyperglycemia on vascular endothelial cells may be corrected by the action of LLLT, especially with the wavelength of 830 nm. It leads to the reduction of TNF-α concentration in the supernatant and enhancement of cell proliferation. Endothelial cells play an important role in the pathogenesis of diabetes; however, a small number of studies evaluate an impact of LLLT on these cells under conditions of hyperglycemia. Further work on this subject is warranted.

Influence of radiographic contrast media (Iodixanol and Iomeprol) on the endothelin-1 release from human arterial and venous endothelial cells cultured on an extracellular matrix.

PubMed

Franke, R P; Fuhrmann, R; Hiebl, B; Jung, F

2012-01-01

Various radiographic contrast media (RCM) are available for visualization of blood vessels in interventional cardiology which can vary widely in their physicochemical properties thereby influencing different functions of blood cells. In the in vitro study described here the influence of two RCMs on arterial as well as on venous endothelial cells was compared to control cultures and examined under statical culture conditions, thus eliminating the influence of RCM viscosity almost completely. The supplementation of the culture medium with RCM (30% v/v) resulted in clearly different reactions of the endothelial cells exposed. Exposition to Iodixanol supplemented culture medium was followed by endothelin-1 release from venous endothelial cells which was equivalent to the endothelin-1 release from venous control cultures. Compared to control cultures, venous endothelial cells exposed to culture medium supplemented with Iomeprol displayed a completely different reaction, the increase in endothelin-1 secretion was missing completely after a 12 hours exposure. Following a 12 hours exposure to both RCMs there were no longer endothelial cells adherent, neither in venous nor in arterial endothelial cell cultures. The study showed that not the wall shear stress was responsible for the differing effects visible after 1.5 min, 5 min, and 12 hours exposure to culture media supplemented with RCM but differences in chemotoxicity of the RCM applied.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation

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

Okazaki, Hideki; Tokumaru, Sho; Hanakawa, Yasushi

2011-09-02

Highlights: {yields} VEGF-A enhanced lymphatic endothelial cell migration and increased tube formation. {yields} VEGF-A treated lymphatic endothelial cell showed activation of STAT3. {yields} Dominant-negative STAT3 inhibited VEGF-A-induced lymphatic endothelial cell migration and tube formation. -- Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube lengthmore » by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.« less

Endothelial cell density to predict endothelial graft failure after penetrating keratoplasty.

PubMed

Lass, Jonathan H; Sugar, Alan; Benetz, Beth Ann; Beck, Roy W; Dontchev, Mariya; Gal, Robin L; Kollman, Craig; Gross, Robert; Heck, Ellen; Holland, Edward J; Mannis, Mark J; Raber, Irving; Stark, Walter; Stulting, R Doyle

2010-01-01

To determine whether preoperative and/or postoperative central endothelial cell density (ECD) and its rate of decline postoperatively are predictive of graft failure caused by endothelial decompensation following penetrating keratoplasty to treat a moderate-risk condition, principally, Fuchs dystrophy or pseudophakic corneal edema. In a subset of Cornea Donor Study participants, a central reading center determined preoperative and postoperative ECD from available specular images for 17 grafts that failed because of endothelial decompensation and 483 grafts that did not fail. Preoperative ECD was not predictive of graft failure caused by endothelial decompensation (P = .91). However, the 6-month ECD was predictive of subsequent failure (P < .001). Among those that had not failed within the first 6 months, the 5-year cumulative incidence (+/-95% confidence interval) of failure was 13% (+/-12%) for the 33 participants with a 6-month ECD of less than 1700 cells/mm(2) vs 2% (+/-3%) for the 137 participants with a 6-month ECD of 2500 cells/mm(2) or higher. After 5 years' follow-up, 40 of 277 participants (14%) with a clear graft had an ECD below 500 cells/mm(2). Preoperative ECD is unrelated to graft failure from endothelial decompensation, whereas there is a strong correlation of ECD at 6 months with graft failure from endothelial decompensation. A graft can remain clear after 5 years even when the ECD is below 500 cells/mm(2).

S-nitrosylation of VASP at cysteine 64 mediates the inflammation-stimulated increase in microvascular permeability.

PubMed

Zamorano, Patricia; Marín, Natalie; Córdova, Francisco; Aguilar, Alejandra; Meininger, Cynthia; Boric, Mauricio P; Golenhofen, Nikola; Contreras, Jorge E; Sarmiento, José; Durán, Walter N; Sánchez, Fabiola A

2017-07-01

We tested the hypothesis that platelet-activating factor (PAF) induces S -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated S -nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S -nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that S -nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. S -nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist. NEW & NOTEWORTHY Here, we demonstrate that S -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that S -nitrosylation of VASP contributes to the onset of endothelial permeability. Copyright © 2017 the American Physiological Society.

Association of the Gutta-Induced Microenvironment With Corneal Endothelial Cell Behavior and Demise in Fuchs Endothelial Corneal Dystrophy.

PubMed

Kocaba, Viridiana; Katikireddy, Kishore Reddy; Gipson, Ilene; Price, Marianne O; Price, Francis W; Jurkunas, Ula V

2018-05-31

The number and size of guttae increase over time in Fuchs endothelial corneal dystrophy (FECD); however, the association between these physical parameters and disease pathogenesis is unclear. To determine the role of guttae in corneal endothelial cell function. In an in vitro model, cells from a human corneal endothelial cell line, HCENC-21T, were seeded on decellularized normal (n = 30) and FECD (n = 70) endothelial basement (Descemet) membranes (DMs). Normal human corneas were sent to our laboratory from 3 sources. The study took place at the Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, and was performed from September 2015 to July 2017. Normal DMs were obtained from 3 different tissue banks and FECD-DMs were obtained from patients undergoing endothelial keratoplasty in 2 departments. Endothelial cell shape, growth, and migration were assessed by live-cell imaging, and gene expression analysis as a function of guttae diameter was assessed by laser capture microscopy. Mean (SD) age of normal-DMs donors was 65.6 (4.4) years (16 women [53%]), and mean (SD) age of FECD-DMs donors was 68.9 (10.6) years (43 women [61%]). Cells covered a greater area (mean [SD], 97.7% [8.5%]) with a greater mean (SD) number of cells (2083[153] cells/mm2) on the normal DMs compared with the FECD DMs (72.8% [11%]; P = .02 and 1541 [221] cells/mm2 221/mm2; P = .01, respectively). Differences in endothelial cell growth over guttae were observed on FECD DMs depending on the guttae diameter. Guttae with a mean (SD) diameter of 10.5 (2.9) μm did not impede cell growth, whereas those with a diameter of 21.1 (4.9) μm were covered only by the cell cytoplasm. Guttae with the largest mean (SD) diameter, 31.8 (3.8) μm, were not covered by cells, which instead surrounded them in a rosette pattern. Moreover, cells adjacent to large guttae upregulated αSMA, N-cadherin, Snail1, and NOX4 genes compared with ones grown on normal DMs or small guttae. Furthermore, large guttae induced TUNEL-positive apoptosis in a rosette pattern, similar to ex vivo FECD specimens. These findings highlight the important role of guttae in endothelial cell growth, migration, and survival. These data suggest that cell therapy procedures in FECD might be guided by the diameter of the host guttae if subsequent clinical studies confirm these laboratory findings.

Ascorbic Acid Efflux and Re-uptake in Endothelial Cells: Maintenance of Intracellular Ascorbate

PubMed Central

May, James M.; Qu, Zhi-chao

2013-01-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70–80% of ascorbate to the medium over several hours at 37 °C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel. PMID:19148707

Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

PubMed

Sabiiti, Wilber; May, Robin C

2012-01-01

Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB.

Capsule Independent Uptake of the Fungal Pathogen Cryptococcus neoformans into Brain Microvascular Endothelial Cells

PubMed Central

Sabiiti, Wilber; May, Robin C.

2012-01-01

Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following ‘trapping’ within capillary beds of the BBB. PMID:22530025

Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate.

PubMed

May, James M; Qu, Zhi-chao

2009-05-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

An ibuprofen-antagonized plasmin inhibitor released by human endothelial cells.

PubMed

Rockwell, W B; Ehrlich, H P

1991-02-01

Serum-free culture medium harvested from endothelial cell monolayer cultures derived from human scars and dermis was examined for inhibition of fibrinolysis using a fibrin plate assay. Human cultured fibroblasts and smooth muscle cells did not produce any detectable inhibitory activity. The inhibitor is spontaneously released from the cultured endothelial cells over time. In the fibrin plate assay of plasmin-induced fibrinolysis, one nonsteroidal antiinflammatory (NSAI) drug, ibuprofen, was demonstrated to antagonize the inhibition of fibrinolysis. The antagonistic activity of ibuprofen appears unrelated to its NSAI drug activity because other NSAI drugs such as indomethacin and tolmetin have minimal antagonistic activity. Heating the cultured endothelial cells to 42 degrees C stimulates greater release of the inhibitor in a shorter period of time. This plasmin inhibitor, which is produced by endothelial cells, may contribute to postburn vascular occlusion, leading to secondary progressive necrosis in burn-traumatized patients.

Phospholipase C and perfringolysin O from Clostridium perfringens upregulate endothelial cell-leukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells.

PubMed Central

Bryant, A E; Stevens, D L

1996-01-01

Clostridium perfringens phospholipase C (PLC) and perfringolysin O (PFO) differentially induced human umbilical vein endothelial cell expression and synthesis of endothelial cell-leukocyte adherence molecule-1 (ELAM-1), intracellular leukocyte adherence molecule-1 (ICAM-1), and interleukin-8 (IL-8). PLC strongly induced expression of ELAM-1, ICAM-1, and IL-8, while PFO stimulated early ICAM-1 expression but did not promote ELAM-1 expression or IL-8 synthesis. PLC caused human umbilical vein endothelial cells to assume a fibroblastoid morphology, whereas PFO, in high concentrations or after prolonged low-dose toxin exposure, caused cell death. The toxin-induced expression of proadhesive and activational proteins and direct cytopathic effects may contribute to the leukostasis, vascular compromise, and capillary leak characteristics of C. perfringens gas gangrene. PMID:8557365

Evaluation of intracameral injection of ranibizumab and bevacizumab on the corneal endothelium by scanning electron microscopy.

PubMed

Ari, Seyhmus; Nergiz, Yusuf; Aksit, Ihsan; Sahin, Alparslan; Cingu, Kursat; Caca, Ihsan

2015-03-01

To evaluate the effects of intracameral injection of ranibizumab and bevacizumab on the corneal endothelium by scanning electron microscopy (SEM). Twenty-eight female rabbits were randomly divided into four equal groups. Rabbits in groups 1 and 2 underwent intracameral injection of 1 mg/0.1 mL and 0.5 mg/0.05 mL ranibizumab, respectively; group 3 was injected with 1.25 mg/0.05 mL bevacizumab. All three groups were injected with a balanced salt solution (BSS) into the anterior chamber of the left (fellow) eye. None of the rabbits in group 4 underwent an injection. Corneal thickness and intraocular pressure were measured before the injections, on the first day, and in the first month after injection. The rabbits were sacrificed and corneal tissues were excised in the first month after injection. Specular microscopy was used for the corneal endothelial cell count. Endothelial cell density was assessed and comparisons drawn between the groups and the control. Micrographs were recorded for SEM examination. The structure of the corneal endothelial cells, the junctional area of the cell membrane, the distribution of microvillus, and the cell morphology of the eyes that underwent intracameral injection of vascular endothelial growth factor (VEGF), BSS, and the control group were compared. Corneal thickness and intraocular pressure were not significantly different between the groups that underwent anti-VEGF or BSS injection and the control group on the first day and in the first month of injection. The corneal endothelial cell count was significantly diminished in all three groups; predominantly in group 1 and 2 (P<0.05). The SEM examination revealed normal corneal endothelial histology in group 3 and the control group. Eyes in group 1 exhibited indistinctness of corneal endothelial cell borders, microvillus loss in the luminal surface, excessive blebbing, and disintegration of intercellular junctions. In group 2, the cell structure of the corneal endothelium and intercellular junctions were normal. However, a relative reduction was observed in the microvillus density of endothelial cells. Although eyes in group 3 were morphologically similar to fellow eyes and the control group, disarrangement in endothelial cell borders was evident. The SEM examination pointed out deterioration in endothelial cell morphology after intracameral injection of 1 and 0.5 mg ranizumab. However, the effects of intracameral bevacizumab injection on corneal endothelial cells were similar to those found in fellow eyes and the control group. Further large-scale studies that examine the cellular changes by transmission electron microscopy are required to support the results of the present study that evaluates the structural changes in endothelial cells by SEM.

Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton

PubMed Central

Parker, William H.; Rhea, Elizabeth Meredith; Qu, Zhi-Chao; Hecker, Morgan R.

2016-01-01

Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species. PMID:27605450

Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation

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

Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung

2008-03-28

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expressionmore » in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.« less

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

PubMed

Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Ginkgolide B Reduces LOX-1 Expression by Inhibiting Akt Phosphorylation and Increasing Sirt1 Expression in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells

PubMed Central

Chen, Beidong; Li, Xingguang; Qi, Ruomei

2013-01-01

Oxidized low-density lipoprotein (ox-LDL) is an important risk factor in the development of atherosclerosis. LOX-1, a lectin-like receptor for ox-LDL, is present primarily on endothelial cells and upregulated by ox-LDL, tumor necrosis factor a, shear stress, and cytokines in atherosclerosis. Recent studies demonstrated that ginkgolide B, a platelet-activating factor receptor antagonist, has antiinflammatory and antioxidant effects on endothelial and nerve cells. The present study investigated the effects of ginkgolide B on LOX-1 expression and the possible mechanism of action. Our results showed that ginkgolide B inhibited LOX-1 and intercellular cell adhesion molecule-1 (ICAM-1) expression in ox-LDL-stimulated endothelial cells through a mechanism associated with the attenuation of Akt activation. Similar data were obtained by silencing Akt and LY294002. We also evaluated Sirt1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. These molecules play a protective role in endothelial cell injury. The results showed that ginkgolide B increased Sirt1 expression in ox-LDL-treated cells. The inhibitory effects of ginkgolide B on LOX-1 and ICAM-1 expression were reduced in Sirt1 siRNA-transfected cells. Nrf2 expression was increased in ox-LDL-treated cells, and ginkgolide B downregulated Nrf2 expression. These results suggest that ginkgolide B reduces Nrf2 expression by inhibiting LOX-1 expression, consequently reducing oxidative stress injury in ox-LDL-stimulated cells. Altogether, these results indicate that the protective effect of ginkgolide B on endothelial cells may be attributable to a decrease in LOX-1 expression and an increase in Sirt1 expression in ox-LDL-stimulated endothelial cells, the mechanism of which is linked to the inhibition of Akt activation. Ginkgolide B may be a multiple-target drug that exerts protective effects in ox-LDL-treated human umbilical vein endothelial cells. PMID:24069345

Carbon Ion Radiation Inhibits Glioma and Endothelial Cell Migration Induced by Secreted VEGF

PubMed Central

Liu, Yang; Liu, Yuanyuan; Sun, Chao; Gan, Lu; Zhang, Luwei; Mao, Aihong; Du, Yuting; Zhou, Rong; Zhang, Hong

2014-01-01

This study evaluated the effects of carbon ion and X-ray radiation and the tumor microenvironment on the migration of glioma and endothelial cells, a key process in tumorigenesis and angiogenesis during cancer progression. C6 glioma and human microvascular endothelial cells were treated with conditioned medium from cultures of glioma cells irradiated at a range of doses and the migration of both cell types, tube formation by endothelial cells, as well as the expression and secretion of migration-related proteins were evaluated. Exposure to X-ray radiation-conditioned medium induced dose-dependent increases in cell migration and tube formation, which were accompanied by an upregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2 and -9 expression. However, glioma cells treated with conditioned medium of cells irradiated at a carbon ion dose of 4.0 Gy showed a marked decrease in migratory potential and VEGF secretion relative to non-irradiated cells. The application of recombinant VEGF165 stimulated migration in glioma and endothelial cells, which was associated with increased FAK phosphorylation at Tyr861, suggesting that the suppression of cell migration by carbon ion radiation could be via VEGF-activated FAK signaling. Taken together, these findings indicate that carbon ion may be superior to X-ray radiation for inhibiting tumorigenesis and angiogenesis through modulation of VEGF level in the glioma microenvironment. PMID:24893038

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

PubMed

Patton, W F; Alexander, J S; Dodge, A B; Patton, R J; Hechtman, H B; Shepro, D

1991-07-01

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.

Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

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

Zhang, Wenjie; Zhang, Xiaomei, E-mail: zhangxm667@163.com; Lu, Hong

2013-05-10

Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cellmore » HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.« less

NgBR is essential for endothelial cell glycosylation and vascular development.

PubMed

Park, Eon Joo; Grabińska, Kariona A; Guan, Ziqiang; Sessa, William C

2016-02-01

NgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development. © 2016 The Authors.

Cytoprotection of Human Endothelial Cells From Menadione Cytotoxicity by Caffeic Acid Phenethyl Ester: The Role of Heme Oxygenase-1

DTIC Science & Technology

2008-06-08

reported here show that CAPE induces HO-1 in human endothelial cells. The major signaling transduction involved in HO-1 induction by those electrophilic ...phenethyl ester (CAPE) and catechol ring- fluorinated CAPE derivatives against menadione-induced oxidative stress in human endothelial cells. Bioorganic

TAK1 in brain endothelial cells mediates fever and lethargy

PubMed Central

Ridder, Dirk A.; Lang, Ming-Fei; Salinin, Sergei; Röderer, Jan-Peter; Struss, Marcel; Maser-Gluth, Christiane

2011-01-01

Systemic inflammation affects the brain, resulting in fever, anorexia, lethargy, and activation of the hypothalamus–pituitary–adrenal axis. How peripheral inflammatory signals reach the brain is still a matter of debate. One possibility is that, in response to inflammatory stimuli, brain endothelial cells in proximity to the thermoregulatory centers produce cyclooxygenase 2 (COX-2) and release prostaglandin E2, causing fever and sickness behavior. We show that expression of the MAP kinase kinase kinase TAK1 in brain endothelial cells is needed for interleukin 1β (IL-1β)–induced COX-2 production. Exploiting the selective expression of the thyroxine transporter Slco1c1 in brain endothelial cells, we generated a mouse line allowing inducible deletion of Tak1 specifically in brain endothelium. Mice lacking the Tak1 gene in brain endothelial cells showed a blunted fever response and reduced lethargy upon intravenous injection of the endogenous pyrogen IL-1β. In conclusion, we demonstrate that TAK1 in brain endothelial cells induces COX-2, most likely by activating p38 MAPK and c-Jun, and is necessary for fever and sickness behavior. PMID:22143887

Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells.

PubMed

Jellinghaus, Stefanie; Poitz, David M; Ende, Georg; Augstein, Antje; Weinert, Sönke; Stütz, Beryl; Braun-Dullaeus, Rüdiger C; Pasquale, Elena B; Strasser, Ruth H

2013-10-01

The Eph receptors represent the largest family of receptor tyrosine kinases. Both Eph receptors and their ephrin ligands are cell-surface proteins, and they typically mediate cell-to-cell communication by interacting at sites of intercellular contact. The major aim of the present study was to investigate the involvement of EphA4-ephrin-A1 interaction in monocyte adhesion to endothelial cells, as this process is a crucial step during the initiation and progression of the atherosclerotic plaque. Immunohistochemical analysis of human atherosclerotic plaques revealed expression of EphA4 receptor and ephrin-A1 ligand in major cell types within the plaque. Short-time stimulation of endothelial cells with the soluble ligand ephrin-A1 leads to a fourfold increase in adhesion of human monocytes to endothelial cells. In addition, ephrin-A1 further increases monocyte adhesion to already inflamed endothelial cells. EphrinA1 mediates its effect on monocyte adhesion via the activated receptor EphA4. This ephrinA1/EphA4 induced process involves the activation of the Rho signaling pathway and does not require active transcription. Rho activation downstream of EphA4 leads to increased polymerization of actin filaments in endothelial cells. This process was shown to be crucial for the proadhesive effect of ephrin-A1. The results of the present study show that ephrin-A1-induced EphA4 forward signaling promotes monocyte adhesion to endothelial cells via activation of RhoA and subsequent stress-fiber formation by a non-transcriptional mechanism. Copyright © 2013 Elsevier B.V. All rights reserved.

Human brain microvascular endothelial cells resist elongation due to shear stress.

PubMed

Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

2015-05-01

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

New therapeutic modality for corneal endothelial disease using Rho-associated kinase inhibitor eye drops.

PubMed

Koizumi, Noriko; Okumura, Naoki; Ueno, Morio; Kinoshita, Shigeru

2014-11-01

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal endothelial transplantation. However, despite the value and potential of endothelial graft surgery, a strictly pharmacological approach for treating corneal endothelial dysfunction remains an attractive proposition. Previously, we reported that the selective Rho-associated kinase (ROCK) inhibitor Y-27632 promotes cell adhesion and proliferation, and inhibits the apoptosis of primate corneal endothelial cells in culture. These findings have led us to develop a novel medical treatment for the early phase of corneal endothelial disease using ROCK inhibitor eye drops. In rabbit and monkey models of partial endothelial dysfunction, we showed that corneal endothelial wound healing was accelerated via the topical application of ROCK inhibitor to the ocular surface, resulting in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. Based on these animal studies, we are now attempting to advance the clinical application of ROCK inhibitor eye drops for patients with corneal endothelial dysfunction. A pilot clinical study was performed at the Kyoto Prefectural University of Medicine, and the effects of Y-27632 eye drops after transcorneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. We observed a positive effect of ROCK inhibitor eye drops in treating patients with central edema caused by Fuchs corneal endothelial dystrophy. We believe that our new findings will contribute to the establishment of a new approach for the treatment of corneal endothelial dysfunction.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

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

Alves, Tercia Rodrigues; Universidade Federal do Rio de Janeiro; Carvalho da Fonseca, Anna Carolina

2011-09-10

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached andmore » died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.« less

Regional differences in endothelial cell cytoskeleton, junctional proteins and phosphorylated tyrosine labeling in the porcine vortex vein system.

PubMed

Tan, Priscilla Ern Zhi; Yu, Paula K; Yang, Hongfang; Cringle, Stephen J; Yu, Dao-Yi

2018-07-01

We previously demonstrated endothelial phenotype heterogeneity in the vortex vein system. This study is to further determine whether regional differences are present in the cytoskeleton, junctional proteins and phosphorylated tyrosine labeling within the system. The vortex vein system of twenty porcine eyes was perfused with labels for f-actin, claudin-5, VE-Cadherin, phosphorylated tyrosine and nucleic acid. The endothelial cells of eight different regions (choroidal veins, pre-ampulla, anterior ampulla, mid-ampulla, posterior ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein) were studied using confocal microscopy. There were regional differences in the endothelial cell structures. Cytoskeleton labeling was relatively even in intensity throughout Regions 1 to 6. Overall VE-Cadherin had a non-uniform distribution and thicker width endothelial cell border staining than claudin-5. Progressing downstream there was an increased variation in thickness of VE-cadherin labeling. There was an overlap in phosphorylated tyrosine and VE-Cadherin labeling in the post-ampulla, intra-scleral canal and extra-ocular vortex vein. Intramural cells were observed that were immune-positive for VE-Cadherin and phosphorylated tyrosine. There were significant differences in the number of intramural cells in different regions. Significant regional differences with endothelial cell labeling of cytoskeleton, junction proteins, and phosphorylated tyrosine were found within the vortex vein system. These findings support existing data on endothelial cell phenotype heterogeneity, and may aid in the knowledge of venous pathologies by understanding regions of vulnerability to endothelial damage within the vortex vein system. It could be valuable to further investigate and characterize the VE-cadherin and phosphotyrosine immune-positive intramural cells. Copyright © 2018. Published by Elsevier Ltd.

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

PubMed Central

Sahara, Makoto; Hansson, Emil M; Wernet, Oliver; Lui, Kathy O; Später, Daniela; Chien, Kenneth R

2014-01-01

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. PMID:24810299

Hydroquinone stimulates inflammatory functions in microvascular endothelial cells via NF-κB nuclear activation.

PubMed

Hebeda, Cristina Bichels; Pinedo, Fernanda Júdice; Vinolo, Marco Aurélio Ramirez; Curi, Rui; Farsky, Sandra Helena Poliselli

2011-11-01

Hydroquinone impairs several leucocyte cell functions, which alter the immune response. Although endothelial cell functions are important for the development of immune responses, hydroquinone actions on endothelial cell have not been shown. Therefore, the effect of hydroquinone exposure (10 or 100 μM for 2 hr) on primary culture of microvascular endothelial cells (PMECs) obtained from the cremaster muscle of Wistar rats incubated in the presence or absence of lipopolysaccharide (LPS, 2 μg/mL) was investigated. Hydroquinone treatment induced the membrane expression of cell adhesion molecules (CAMs) from the immunoglobulin superfamilies ICAM-1 (intercellular), VCAM-1(vascular) and PECAM-1 (platelet endothelial) and induced the secretion of cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). The effects were dependent on transcriptional modifications because enhanced CAM mRNA expression as well as both cytokines and nuclear factor κB (NF-κB) nuclear activation was found. These effects may be due to the direct action of hydroquinone rather than its quinone metabolites, because endothelial cells do not present myeloperoxidase enzyme and hydroquinone incubation did not induce the expression of cytochrome P450 2E1 (CYP2E1) or prostaglandin H synthase 1. In addition, the incubation of endothelial cells with benzoquinone (10 μM, 2 hr) impaired PECAM-1 expression and did not modify NF-κB nuclear activation. Taken together, the data herein presented reveal that hydroquinone evokes pro-inflammatory properties in endothelial cells that are triggered by the enhancement of NF-κB nuclear translocation-dependent gene transcription. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

PubMed Central

Bai, Yong-Ping; Hu, Chang-Ping; Yuan, Qiong; Peng, Jun; Shi, Rui-Zheng; Yang, Tian-Lun; Cao, Ze-Hong; Li, Yuan-Jian; Cheng, Guangjie; Zhang, Guo-Gang

2013-01-01

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis. PMID:21820048

Isolated Capsulorhexis Flap Technique in Femtosecond Laser-Assisted Cataract Surgery to Protect the Corneal Endothelial Cells.

PubMed

Li, Shaowei; Chen, Xu; Zhao, Jun; Xu, Man; Yu, Zhouxing

2017-12-18

To present a novel technique that using capsulorhexis flap to protect corneal endothelial cells during femtosecond laser-assisted cataract surgery. During the procedure, an isolated capsulorhexis flap was used to lift it up carefully by using OVD until the flap attached to the corneal inner layer. It kept steady during the phaco phase, and been removed by I/A tip after intraocular lens implantation. In our pilot study, the damage to the corneal endothelial cells from the phacoemulsification procedure were no more than the traditional phaco procedure, even femtosecond laser assisted cataract surgery. Isolated capsulorhexis flap technique initiate a new approach to protect the endothelial cell. It is a simple technique with a short learning curve, but still need a further investigation to prove the protection of corneal endothelial cells.

Smad4 deletion in blood vessel endothelial cells promotes ovarian cancer metastasis.

PubMed

Yang, Jie; Wang, Ya; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

2017-05-01

SMAD4 is a critical co-smad in signal transduction pathways activated in response to transforming growth factor-β (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumors highlighted it as a tumor-suppressor gene. Herein, we report that loss of SMAD4 expression in vascular endothelial cells promotes ovarian cancer invasion. SiRNA transfer of this gene in the HUVEC reduced SMAD4 protein expression and function. Although it reduced the vessel endothelial cell tubule formation in vitro and in vivo, it did not affect the tumor growth significantly in vivo. However, it weakened the barrier integrity in endothelial cells and increased vessel permeability and the ovarian cancer liver metastasis. We documented reduced angiogenesis and increased invasion histologically and by intravital microscopy, and gained mechanistic insight at the messenger and gene level. Finally, we found a negative reciprocal regulation between SMAD4 and FYN. FYN is one of the Src family kinases (SFK), activation of which can cause dissociation of cell-cell junctions and adhesion, resulting in paracellular hypermeability. Upon SMAD4 deletion, we detected high expression levels of FYN in vessel endothelial cells, suggesting the mechanism of the ovarian tumor cells cross the endothelial barrier and transform to an invasive phenotype.

Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank.

PubMed

Krabcova, Ivana; Studeny, Pavel; Jirsova, Katerina

2013-06-01

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm(2). The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.

Facilitated Engraftment of Isolated Islets Coated With Expanded Vascular Endothelial Cells for Islet Transplantation.

PubMed

Barba-Gutierrez, D Alonso; Daneri-Navarro, A; Villagomez-Mendez, J Jesus Alejandro; Kanamune, J; Robles-Murillo, A Karina; Sanchez-Enriquez, S; Villafan-Bernal, J Rafael; Rivas-Carrillo, J D

2016-03-01

Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the β-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation. We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control. Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation. We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements. Copyright © 2016 Elsevier Inc. All rights reserved.

Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

PubMed

Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

2016-11-01

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nitric oxide releasing hydrogel promotes endothelial differentiation of mouse embryonic stem cells.

PubMed

Nie, Yan; Zhang, Kaiyue; Zhang, Shuaiqiang; Wang, Dan; Han, Zhibo; Che, Yongzhe; Kong, Deling; Zhao, Qiang; Han, Zhongchao; He, Zuo-Xiang; Liu, Na; Ma, Fengxia; Li, Zongjin

2017-11-01

Transplantation of endothelial cells (ECs) holds great promise for treating various kinds of ischemic diseases. However, the major challenge in ECs-based therapy in clinical applications is to provide high quality and enough amounts of cells. In this study, we developed a simple and efficient system to direct endothelial differentiation of mouse embryonic stem cells (ESCs) using a controllable chitosan nitric oxide (NO)-releasing hydrogel (CS-NO). ESCs were plated onto the hydrogel culture system, and the expressions of differentiation markers were measured. We found that the expression of Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) increased obviously under the controlled NO releasing environment. Moreover, the Flk-1 upregulation was accompanied by the activation of the phospho-inositide-3 kinase (PI3K)/Akt signaling. We also found that in the presence of the PI3K inhibitor (LY294002), the endothelial commitment of ESCs was abolished, indicating the importance of Akt phosphorylation in the endothelial differentiation of ESCs. Interestingly, in the absence of NO, the activation of Akt phosphorylation alone by using AKT activator (SC-79) did not profoundly promote the endothelial differentiation of ESCs, suggesting an interdependent relationship between NO and the Akt phosphorylation in driving endothelial fate specification of ESCs. Taken together, we demonstrated that NO releasing in a continuous and controlled manner is a simple and efficient method for directing the endothelial differentiation of ESCs without adding growth factors. Fascinating data continues to show that artificial stem cell niche not only serve as a physical supporting scaffold for stem cells proliferation, but also as a novel platform for directing stem cell differentiation. Because of the lack of proper microenvironment for generating therapeutic endothelial cells (ECs) in vitro, the source of ECs for transplantation is the major limitation in ECs-based therapy to clinical applications. The current study established a feeder cell-free, 2-dimensional culture system for promoting the differentiation processes of embryonic stem cells (ESCs) committed to the endothelial lineage via using a nitric oxide (NO) controlled releasing hydrogel (CS-NO). Notably, the NO releasing from the hydrogel could selectively up-regulate Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) in the absence of growth factors, which was of crucial importance in the endothelial differentiation of ESCs. In summary, the current study proposes a simple and efficient method for directing the endothelial differentiation of ESCs without extra growth factors. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

PubMed

Orynbayeva, Zulfiya; Sensenig, Richard; Polyak, Boris

2015-05-01

To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

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

Song, Xiaomin; Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084; Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084

2010-07-16

Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation andmore » migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.« less

Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components

PubMed Central

Beatty, P. Robert

2017-01-01

Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components. PMID:29121099

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors.

PubMed

Li, Yu-zhen; Liu, Xiu-hua; Rong, Fei; Hu, Sen; Sheng, Zhi-yong

2010-10-01

To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. Carbachol (2 μmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 μg/mL. These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor.

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors

PubMed Central

Li, Yu-zhen; Liu, Xiu-hua; Rong, Fei; Hu, Sen; Sheng, Zhi-yong

2010-01-01

Aim: To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. Methods: Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. Results: Carbachol (2 μmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 μg/mL. Conclusion: These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor. PMID:20871620

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

PubMed Central

Sato, Hiromi

2017-01-01

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways including endothelial barrier damage and inflammation, potentially leading to vascular hyperpermeability and severe illness in vivo. This work provides new insights into the pathophysiological mechanisms of Leptospira infection. PMID:28750011

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.

PubMed

Sato, Hiromi; Coburn, Jenifer

2017-07-01

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways including endothelial barrier damage and inflammation, potentially leading to vascular hyperpermeability and severe illness in vivo. This work provides new insights into the pathophysiological mechanisms of Leptospira infection.

A 90-Kilodalton Endothelial Cell Molecule Mediating Lymphocyte Binding in Humans

NASA Astrophysics Data System (ADS)

Salmi, Marko; Jalkanen, Sirpa

1992-09-01

Interactions between leukocyte surface receptors and their ligands on vascular endothelial cells control lymphocyte traffic between the blood and various lymphoid organs, as well as extravasation of leukocytes into sites of inflammation. A heretofore undescribed 90-kilodalton human endothelial cell adhesion molecule (VAP-1) defined by a monoclonal antibody 1B2 is described. The expression pattern, molecular mass, functional properties, and an amino-terminal amino acid sequence define VAP-1 as an endothelial ligand for lymphocytes. VAP-1 helps to elucidate the complex heterotypic cell interactions that direct tissue-selective lymphocyte migration in man.

The actin polymerization regulator WAVE2 is required for early bone marrow repopulation by hematopoietic stem cells.

PubMed

Ogaeri, Takunori; Eto, Koji; Otsu, Makoto; Ema, Hideo; Nakauchi, Hiromitsu

2009-05-01

The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34(-/low)c-Kit(+)Sca-1(+)lineage(-) HSCs (CD34(-)KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

Role of tumour necrosis factor receptor-1 and nuclear factor-κB in production of TNF-α-induced pro-inflammatory microparticles in endothelial cells.

PubMed

Lee, S K; Yang, S-H; Kwon, I; Lee, O-H; Heo, J H

2014-09-02

Tumour necrosis factor-α (TNF-α) is upregulated in many inflammatory diseases and is also a potent agent for microparticle (MP) generation. Here, we describe an essential role of TNF-α in the production of endothelial cell-derived microparticles (EMPs) in vivo and the function of TNF-α-induced EMPs in endothelial cells. We found that TNF-α rapidly increased blood levels of EMPs in mice. Treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α also induced EMP formation in a time-dependent manner. Silencing of TNF receptor (TNFR)-1 or inhibition of the nuclear factor-κB (NF-κB) in HUVECs impaired the production of TNF-α-induced EMP. Incubation of HUVECs with PKH-67-stained EMPs showed that endothelial cells readily engulfed EMPs, and the engulfed TNF-α-induced EMPs promoted the expression of pro-apoptotic molecules and upregulated intercellular adhesion molecule-1 level on the cell surface, which led to monocyte adhesion. Collectively, our findings indicate that the generation of TNF-α-induced EMPs was mediated by TNFR1 or NF-κB and that EMPs can contribute to apoptosis and inflammation of endothelial cells.

Changes in thrombospondin-1 levels in the endothelial cells of the anterior pituitary during estrogen-induced prolactin-secreting pituitary tumors

PubMed Central

Sarkar, Abby J; Chaturvedi, Kirti; Chen, Cui Ping; Sarkar, Dipak K

2010-01-01

Thrombospondin-1 (TSP-1), a multifunctional matrix glycoprotein, has been shown to control tumor growth by inhibiting angiogenesis in various tissues. However, the role of this glycoprotein in pituitary angiogenesis is not well studied. In this report, we determined the changes in the production and action of TSP-1 on endothelial cells in anterior pituitary following estradiol treatment, which is known to increase prolactin-secreting tumor growth and vascularization in this tissue. We showed that TSP-1 immunoreactive protein is distributed in the anterior pituitary, particularly in the endothelial cells. Estradiol treatment for 2 and 4 weeks decreased the total tissue immunoreactive level of TSP-1 as well as the endothelial cell-specific immunoreactive level of this protein in the anterior pituitary. The steroid treatment also decreased the protein levels of TSP-1 in anterior pituitary tissues and in purified pituitary endothelial cells in primary cultures. Determination of the effects of TSP-1 on proliferation and migration of pituitary-derived endothelial cells in primary cultures elucidated an inhibitory action of TSP-1 on these vascular cell functions. These results suggest that locally produced TSP-1 may regulate estrogen angiogenic action on the pituitary. PMID:17283240

Dehydrodiconiferyl alcohol suppresses monocyte adhesion to endothelial cells by attenuation of JNK signaling pathway.

PubMed

Tsuneyoshi, Tadamitsu; Kanamori, Yuta; Matsutomo, Toshiaki; Morihara, Naoaki

2015-09-25

Several clinical studies have shown that the intake of aged garlic extract improves endothelial dysfunction. Lignan compounds, (+)-(2S,3R)-dehydrodiconiferyl alcohol (DDC) and (-)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (DDDC), have been isolated as antioxidants in aged garlic extract. There is evidence showing the importance of oxidative stress in endothelial dysfunction. In the present study, we examined whether DDC and DDDC enhance endothelial cell function in vitro. Cell adhesion assay was performed using THP-1 monocyte and human umbilical vein endothelial cells (HUVECs) which were activated by lipopolysaccharide (LPS) or advanced glycation end products (AGEs)-BSA. Cellular ELISA method was used for the evaluation of vascular cell adhesion molecule 1 (VCAM-1) expression on HUVECs. DDC and DDDC suppressed the adhesion of THP-1 to HUVECs which was activated by LPS or AGEs-BSA. DDC and DDDC also inhibited VCAM-1 expression induced by LPS or AGEs-BSA, but DDDC was less effective than DDC. In addition, the inhibitory effect of DDC on VCAM-1 expression involved suppressing JNK/c-Jun pathway rather than NF-κB pathway. DDC has an inhibitory effect on VCAM-1 expression via JNK pathway in endothelial cells and therefore may serve as a novel pharmacological agent to improve endothelial dysfunction. Copyright © 2015 Elsevier Inc. All rights reserved.

Isolated Anxa5{sup +}/Sca-1{sup +} perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo

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

Brachvogel, Bent; Pausch, Friederike; Farlie, Peter

2007-07-15

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, {alpha}SMA, PDGFR-{beta}). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2.more » PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ{sup +} PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.« less

Transcriptional Responses of Candida albicans to Epithelial and Endothelial Cells▿ †

PubMed Central

Park, Hyunsook; Liu, Yaoping; Solis, Norma; Spotkov, Joshua; Hamaker, Jessica; Blankenship, Jill R.; Yeaman, Michael R.; Mitchell, Aaron P.; Liu, Haoping; Filler, Scott G.

2009-01-01

Candida albicans interacts with oral epithelial cells during oropharyngeal candidiasis and with vascular endothelial cells when it disseminates hematogenously. We set out to identify C. albicans genes that govern interactions with these host cells in vitro. The transcriptional response of C. albicans to the FaDu oral epithelial cell line and primary endothelial cells was determined by microarray analysis. Contact with epithelial cells caused a decrease in transcript levels of genes related to protein synthesis and adhesion, whereas contact with endothelial cells did not significantly influence any specific functional category of genes. Many genes whose transcripts were increased in response to either host cell had not been previously characterized. We constructed mutants with homozygous insertions in 22 of these uncharacterized genes to investigate their function during host-pathogen interaction. By this approach, we found that YCK2, VPS51, and UEC1 are required for C. albicans to cause normal damage to epithelial cells and resist antimicrobial peptides. YCK2 is also necessary for maintenance of cell polarity. VPS51 is necessary for normal vacuole formation, resistance to multiple stressors, and induction of maximal endothelial cell damage. UEC1 encodes a unique protein that is required for resistance to cell membrane stress. Therefore, some C. albicans genes whose transcripts are increased upon contact with epithelial or endothelial cells are required for the organism to damage these cells and withstand the stresses that it likely encounters during growth in the oropharynx and bloodstream. PMID:19700637

Inhibition of Endothelial Progenitor Cells May Explain the High Cardiovascular Event Rate in Patients with Rheumatoid Arthritis.

PubMed

Adawi, Mohamad; Pastuck, Nina; Saaida, Golan; Sirchan, Rizak; Watad, Abdalla; Blum, Arnon

2018-05-16

Rheumatoid arthritis (RA) patients may suffer cardiovascular (CV) events much more than the general population, and CV disease is the leading cause of death in patients with RA. Our hypothesis was that impaired function of endothelial progenitor cells may contribute to endothelial dysfunction and the clinical CV events of patients with RA. 27 RA patients (9 males and 18 females) with an active disease and 13 healthy subjects who served as the control group (9 males and 4 females) were enrolled to this prospective study. The ability to grow in culture colony-forming units of endothelial progenitor cells (CFU-EPCs) was measured, as well as their endothelial function using high-resolution ultrasonography of the brachial artery, and levels of C reactive protein (CRP) in the serum. For statistical analysis we used the students T-test test. As a group, patients with RA were older (p < 0.0001), had severe endothelial dysfunction (<0.0001), with impaired ability to grow CFU-EPCs (<0.0001), and a higher inflammatory state (p = 0001). No difference was observed in BMI. All RA patients had an active disease (DAS28 3.9±0.9) for 9.2±6.5 years. The same differences were observed in both genders. Patients with RA had an impaired ability to grow endothelial progenitor cells and severe endothelial dysfunction. Inability to grow colonies of endothelial progenitor cells reflects the impaired regenerative capacity of patients with RA, and may explain the endothelial dysfunction and the high CV event rate among patients with RA.

PGC-1-related coactivator (PRC) negatively regulates endothelial adhesion of monocytes via inhibition of NF κB activity

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

Chengye, Zhan; Daixing, Zhou, E-mail: dxzhou7246@hotmail.com; Qiang, Zhong

2013-09-13

Highlights: •First time to display that LPS downregulate the expression of PRC. •First time to show that PRC inhibits the induction of VCAM-1 and E-selectin. •First time to show that PRC inhibit monocytes attachment to endothelial cells. •First time to display that PRC inhibits transcriptional activity of NF-κB. •PRC protects the respiration rate and suppresses the glycolysis rate against LPS. -- Abstract: PGC-1-related coactivator (PRC) is a growth-regulated transcriptional cofactor known to activate many of the nuclear genes specifying mitochondrial respiratory function. Endothelial dysfunction is a prominent feature found in many inflammatory diseases. Adhesion molecules, such as VCAM-1, mediate themore » attachment of monocytes to endothelial cells, thereby playing an important role in endothelial inflammation. The effects of PRC in regards to endothelial inflammation remain unknown. In this study, our findings show that PRC can be inhibited by the inflammatory cytokine LPS in cultured human umbilical vein endothelial cells (HUVECs). In the presence of LPS, the expression of endothelial cell adhesion molecular, such as VCAM1 and E-selectin, is found to be increased. These effects can be negated by overexpression of PRC. Importantly, monocyte adhesion to endothelial cells caused by LPS is significantly attenuated by PRC. In addition, overexpression of PRC protects mitochondrial metabolic function and suppresses the rate of glycolysis against LPS. It is also found that overexpression of PRC decreases the transcriptional activity of NF-κB. These findings suggest that PRC is a negative regulator of endothelial inflammation.« less

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development

PubMed Central

van Lessen, Max; Shibata-Germanos, Shannon; van Impel, Andreas; Hawkins, Thomas A; Rihel, Jason; Schulte-Merker, Stefan

2017-01-01

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain. DOI: http://dx.doi.org/10.7554/eLife.25932.001 PMID:28498105

Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

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

Li Aihua; Cheng Guangli; Zhu Genghui

Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increasedmore » in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.« less

Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

PubMed Central

Lockwood, Charles J.; Krikun, Graciela; Hickey, Martha; Huang, S. Joseph; Schatz, Frederick

2011-01-01

Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interlerukin-8 synergis-tically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding. PMID:19208784

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Targeting filamin A reduces K-RAS–induced lung adenocarcinomas and endothelial response to tumor growth in mice

PubMed Central

2012-01-01

Background Many human cancer cells express filamin A (FLNA), an actin-binding structural protein that interacts with a diverse set of cell signaling proteins, but little is known about the biological importance of FLNA in tumor development. FLNA is also expressed in endothelial cells, which may be important for tumor angiogenesis. In this study, we defined the impact of targeting Flna in cancer and endothelial cells on the development of tumors in vivo and on the proliferation of fibroblasts in vitro. Methods First, we used a Cre-adenovirus to simultaneously activate the expression of oncogenic K-RAS and inactivate the expression of Flna in the lung and in fibroblasts. Second, we subcutaneously injected mouse fibrosarcoma cells into mice lacking Flna in endothelial cells. Results Knockout of Flna significantly reduced K-RAS–induced lung tumor formation and the proliferation of oncogenic K-RAS–expressing fibroblasts, and attenuated the activation of the downstream signaling molecules ERK and AKT. Genetic deletion of endothelial FLNA in mice did not impact cardiovascular development; however, knockout of Flna in endothelial cells reduced subcutaneous fibrosarcoma growth and vascularity within tumors. Conclusions We conclude that FLNA is important for lung tumor growth and that endothelial Flna impacts local tumor growth. The data shed new light on the biological importance of FLNA and suggest that targeting this protein might be useful in cancer therapeutics. PMID:22857000

Myeloperoxidase Oxidized LDL Interferes with Endothelial Cell Motility through miR-22 and Heme Oxygenase 1 Induction: Possible Involvement in Reendothelialization of Vascular Injuries

PubMed Central

Daher, Jalil; Martin, Maud; Rousseau, Alexandre; Nuyens, Vincent; Fayyad-Kazan, Hussein; Van Antwerpen, Pierre; Courbebaisse, Guy; Martiat, Philippe; Badran, Bassam; Dequiedt, Frank

2014-01-01

Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque. PMID:25530680

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

PubMed Central

Pan, Qunwen; Zhao, Yuhui; Chen, Ji; Zhao, Bin; Chen, Yanfang

2013-01-01

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs. PMID:24358213

An EMMPRIN–γ-catenin–Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

PubMed Central

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S.; Enríquez, José A.; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G.

2014-01-01

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. PMID:24994937

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

PubMed

Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

2017-03-01

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.