Ascorbic Acid Increases the Thrombogenicity of Cellular Matrices

1991 ◽  
Vol 66 (04) ◽  
pp. 505-509 ◽  
Author(s):  
Georg A Hindriks ◽  
Jan J Sixma ◽  
Philip G de Groot
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Strong Increase ◽  
Aggregate Formation ◽  
Lower Shear ◽  
The Matrix ◽  
Proline Incorporation

SummaryWe have studied the influence of ascorbate on extracellular matrix formation in cultured human endothelial cells, smooth muscle cells and fibroblasts and measured the influence of the changed composition of their isolated extracellular matrices on their affinity for platelets. When endothelial cells were grown for a week in the presence of ascorbate, no influence on proline incorporation in their extracellular matrix was found. In accordance, no influence on platelet adhesion or aggregate formation on these matrices was detected. When smooth muscle cells were cultured in the presence of ascorbate, a strong increase in the amount of collagen types I and III in the extracellular matrix was found. When these matrices were perfused with whole blood, a significant enhanced increase in aggregate formation was observed. No influence was seen on the total coverage of the matrix with platelets. When fibroblasts were grown in the presence of ascorbate, no significant increase in proline incorporation in their matrix was measured. However, an increased adhesion of platelets was seen to the matrices at lower shear rates. We conclude that ascorbate feeding has a significant effect on endogenous deposited matrices of smooth muscle cells and fibroblasts, and that the changed composition had profound effects on platelet interaction with these matrices.

Comparison of Platelet Interaction with Subendothelium of Human Renal and Umbilical Arteries and the Extracellular Matrix Produced by Human Venous Endothelial Cells

1984 ◽  
Vol 52 (01) ◽  
pp. 060-065 ◽  
Author(s):  
Kjell S Sakariassen ◽  
Jan Dirk Banga ◽  
Philip G de Groot ◽  
Jan J Sixma
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Aggregate Formation ◽  
Disease Subtype ◽  
Elastic Membranes ◽  
Platelet Aggregate ◽  
Umbilical Arteries

SummaryPlatelet interaction with subendothelium of human renal and umbilical arteries and with the extracellular matrix produced by cultured human venous endothelial cells was compared in flowing citrated blood by using an annular and a rectangular perfusion chamber.The renal arteries were post mortem specimens from adults showing, often pronounced, intimal fibrosis, whereas the umbilical arteries had well organized parallel arranged smooth muscle cells, without elastic membranes. The extracellular matrix obtained after removal of endothelial cells with Triton X-100 was homogenously attached to its substratum.Significantly more platelets adhered to the extracellular matrix than to the subendothelia. This discrepancy was most pronounced in reconstituted blood with plasma from a patient with homozygous severe von Willebrand’s disease (subtype III). No differences in platelet adherence and platelet aggregate formation were noted between the subendothelia. Platelet aggregate formation was poor on all surfaces.These data indicate that the extracellular matrix produced by endothelial cells is at least as reactive for the interaction with platelets as subendothelium, probably partly synthesized by smooth muscle cells.

scholarly journals Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

1982 ◽  
Vol 92 (2) ◽  
pp. 462-470 ◽  
Author(s):  
E Schwartz ◽  
R S Bienkowski ◽  
B Coltoff-Schiller ◽  
S Goldfischer ◽  
O O Blumenfeld
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Time Course ◽  
Cultured Cells ◽  
Growth Parameters ◽  
Muscle Cells ◽  
Cellular Protein ◽  
Collagen Accumulation ◽  
The Matrix

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

Analysis of the glycopeptides derived from the extracellular matrix secreted by cultured bovine aortic smooth muscle cells

1987 ◽  
Vol 65 (7) ◽  
pp. 595-601
Author(s):  
James R. A. Leushner
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Minor Components ◽  
Proliferating Cells ◽  
Cell Behaviour ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
The Matrix

Modulation of smooth muscle cell behaviour in culture has been associated with changes in the extracellular matrix. In the present study cultures of bovine aortic smooth muscle cells were compared in the rapidly proliferating and confluent phases of growth. The extracellular matrix was similar in both phases of growth and consisted of glycoproteins ranging from molecular weight 20 000 to over 200 000. The glycopeptides derived from these components displayed several differences. N-linked heteropolysaccharides of the biantennary and complex (more than two branches) types were predominant in the matrix of the confluent phase. Larger amounts of high mannose glycopeptides were present in the preparations from proliferating cells. O-Glycosidic glycopeptides were minor components in both preparations, but a slight increase was noted in the confluent phase of growth. Some of the changes in glycopeptides were interpreted in terms of the levels of the major components of the matrix such as the interstitial procollagens and fibronectin. The results indicate that processing of oligosaccharides associated with secreted glycoproteins of the extracellular matrix correlates with the state of growth of smooth muscle cells in culture.

scholarly journals Inhibition of eNOS by L-NAME resulting in rat hind limb developmental defects through PFKFB3 mediated angiogenetic pathway

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ziqiang Wu ◽  
Huan Yao ◽  
Huan Xu ◽  
Yang Wang ◽  
Wangming Hu ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Limb Development ◽  
Hind Limb ◽  
Critical Role ◽  
Muscle Cells ◽  
Developmental Defects ◽  
Pregnant Rat

Abstract l-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether l-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that l-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.

THROMBOSPONDIN: CELL BIOLOGY OF AN ADHESIVE GLYCOPROTEIN

1987 ◽  
Author(s):  
R L Nachman ◽  
R L Silverstein ◽  
A S Asch
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tumor Cells ◽  
Glial Cell ◽  
Muscle Cells ◽  
Major Constituent ◽  
U937 Cells ◽  
Activated Platelets

Thrombospondin (TSP), a multifunctional 450 KD glycoprotein is a secretory product of thrombin stimulated platelets. It is a major component of the platelets alpha granule constituting approximately 3% of total platelet protein. Thrombospondin does not circulate in appreciable concentrations ∽0 100 ng/ml); however, the tissue distribution is broad. In addition to its expression on the membrane of activated platelets, the protein is synthesized by fibroblasts endothelial cells, glial cell smooth muscle cells alveolar pneumocytes mononuclear phagocytes and various tumor cells. TSP is a major constituent of the extracellular matrix and has been demonstrated in the vessel wall, basement membrane and glandular connective tissue. Fibroblasts, smooth muscle cells and endothelial cells in tissue culture incorporate TSP into the extracellular matrix. Matrix TSP is under cell-cycle regulatory control. Mesenchymal cells in the proliferative phase synthesize greater amounts of TSP than non growing cells. Platelet derived growth factor induces smooth muscle cell and glial cell synthesis of TSP. Atheromatous lesions contain increased amounts of TSP compared to normal vessels emphasizing the potential role of TSP in the interaction of proliferating cells with the matrix. TSP binds specifically, saturably, and reversibly to mouse peritoneal macrophages and to cells of the monocyte-like human cell line U937. Binding was time dependent and was optimal in the presence of both Ca++ and Mg++. PMA stimulated U937 cells and activated macrophages bound TSP to an equivalent extent as resting cells. The TSP binding site on the surface of U937 cells and peripheral blood monocytes mediates the adhesive interaction between these cells and thrombin-stimulated platelets. Using a sensitive rosetting assay we found that monocytes were not rosetted by resting platelets while >90% were rosetted by thrombin-stimulated platelets. Monoclonal and polyclonal anti-TSP antibodies markedly inhibited rosetting as did TSP itself. Antifibronectin or non-immune control antibodies did not inhibit rosetting, nor did fibronectin, fibrinogen, the fibronectinadhesion tetrapeptide arg-gly-asp-ser (RGDS), or heparin. The TSP membrane receptor, an 88 KD glycoprotein, formely known as GPIV has been identified in platelets, endothelial cells, monocytes and a variety of tumor cells. TSP may thus serve as a molecular bridge linking activated platelets with monocytes at sites of early vascular injury. Such interactions involving the TSP receptor complex may be of critical importance in the regulation of thrombosis and the initiation of atherosclerosis.

scholarly journals Inhibition of leptin-induced vascular extracellular matrix remodelling by adiponectin

2014 ◽  
Vol 53 (2) ◽  
pp. 145-154 ◽  
Author(s):  
Zhi Zhang ◽  
Fang Wang ◽  
Bing-jian Wang ◽  
Guang Chu ◽  
Qunan Cao ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Culture Vessel ◽  
Collagen Types ◽  
Tissue Fibrosis

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

scholarly journals Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins, and collagen by proteinases isolated from macrophages.

1980 ◽  
Vol 152 (5) ◽  
pp. 1340-1357 ◽  
Author(s):  
Z Werb ◽  
M J Banda ◽  
P A Jones
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Connective Tissue ◽  
Smooth Muscle Cells ◽  
Conditioned Medium ◽  
Proteolytic Enzymes ◽  
Muscle Cells ◽  
Granulocyte Elastase ◽  
Elastin Degradation ◽  
The Matrix

We have investigated the ability of neutral and lysosomal enzymes of mouse macrophages to degrade the insoluble extracellular matrices secreted by smooth muscle cells, endothelial cells, and fibroblasts. Matrices produced by smooth muscle cells contained glycoproteins, elastin, and collagens, but matrices of endothelial cells and fibroblasts contained no elastin. Sequential enzyme digestion of residual matrix revealed that plasmin, a product of macrophage plasminogen activation, degraded 50-70% of the glycoprotein in the matrices but did not degrade the elastin or the collagens. Purified macrophage elastase degraded glycoprotein and elastin components but had no effect on the collagens. The rate of elastin degradation by macrophage elastase was decreased in the presence of the glycoproteins. In contrast, human granulocyte elastase effectively degraded the matrix glycoproteins, elastin, and, to a lesser extent, collagens, Mammalian collagenase degraded only collagens. Conditioned medium from resident and inflammatory macrophages, containing mixtures of the secreted proteinases, degraded the glycoprotein and elastin components of the matrices. However, conditioned medium was less effective in degrading matrix than comparable amounts of purified macrophage elastase because > 90% of the elastase in the medium was in a latent form. Inclusion of plasminogen in the assays accelerated degradation. In the presence of plasminogen, glycoproteins were degraded readily by medium from P388D1, pyran copolymer-, thioglycollate-, and periodate-elicited macrophages and, to a lesser extent, by medium from endotoxin-elicited and resident macrophages; medium from P388D1, thioglycollate-, and periodate-elicited macrophages was most effective in elastin degradation, and resident, endotoxin-elicited and pyran copolymer-elicited macrophages degraded almost no elastin. The macrophage cathepsins D and B degraded all the matrix components at an optimum pH of 5.5 and acted with the secreted neutral proteinases to degrade the connective tissue macromolecules to amino acids and oligopeptides. These data indicate that macrophages at inflammatory sites contain and secrete proteolytic enzymes that could degrade the extracellular matrix.

Plasminogen Activator Inhibitor-1 Expression in Human Liver and Healthy or Atherosclerotic Vessel Walls

1994 ◽  
Vol 72 (01) ◽  
pp. 044-053 ◽  
Author(s):  
N Chomiki ◽  
M Henry ◽  
M C Alessi ◽  
F Anfosso ◽  
I Juhan-Vague
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Plasminogen Activator ◽  
Human Liver ◽  
Plasminogen Activator Inhibitor ◽  
Muscle Cells ◽  
Vessel Walls ◽  
Activator Inhibitor ◽  
Pai 1

SummaryIndividuals with elevated levels of plasminogen activator inhibitor type 1 are at risk of developing atherosclerosis. The mechanisms leading to increased plasma PAI-1 concentrations are not well understood. The link observed between increased PAI-1 levels and insulin resistance has lead workers to investigate the effects of insulin or triglyceride rich lipoproteins on PAI-1 production by cultured hepatocytes or endothelial cells. However, little is known about the contribution of these cells to PAI-1 production in vivo. We have studied the expression of PAI-1 in human liver sections as well as in vessel walls from different territories, by immunocytochemistry and in situ hybridization.We have observed that normal liver endothelial cells expressed PAI-1 while parenchymal cells did not. However, this fact does not refute the role of parenchymal liver cells in pathological states.In healthy vessels, PAI-1 mRNA and protein were detected primarily at the endothelium from the lumen as well as from the vasa vasorum. In normal arteries, smooth muscle cells were able to produce PAI-1 depending on the territory tested. In deeply altered vessels, PAI-1 expression was observed in neovessels scattering the lesions, in some intimal cells and in smooth muscle cells. Local increase PAI-1 mRNA described in atherosclerotic lesions could be due to the abundant neovascularization present in the lesion as well as a raised expression in smooth muscle cells. The increased PAI-1 in atherosclerosis could lead to fibrin deposit during plaque rupture contributing further to the development and progression of the lesion.

Thrombin Inhibition and Thrombin Generation by Cultured Aortic Endothelial and Smooth Muscle Cells from Minipig

1982 ◽  
Vol 48 (01) ◽  
pp. 101-103 ◽  
Author(s):  
B Kirchhof ◽  
J Grünwald
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vessel Wall ◽  
Thrombin Generation ◽  
Muscle Cells ◽  
Thrombin Inhibition ◽  
Generating Capacity ◽  
Wall Interaction ◽  
Cells Cultured

SummaryEndothelial and smooth muscle cells cultured from minipig aorta were examined for their inhibitory activity on thrombin and for their thrombin generating capacity.Endothelial cells showed both a thrombin inhibition and an activation of prothrombin in the presence of Ca++, which was enhanced in the presence of phospholipids. Smooth muscle cells showed an activation of prothrombin but at a lower rate. Both coagulation and amidolytic micro-assays were suitable for studying the thrombin-vessel wall interaction.

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