Hemostatic Factors and Inflammatory Disease

1999 ◽  
Vol 82 (08) ◽  
pp. 858-864 ◽  
Author(s):  
Jay Degen
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Biological Activities ◽  
Bacterial Species ◽  
Physiological Role ◽  
Muscle Cells ◽  
Cell Types ◽  
Hemostatic Factors

IntroductionVascular integrity is preserved by a sophisticated system of circulating and cell-associated hemostatic factors that control local thrombin generation, platelet deposition, and the conversion of soluble fibrinogen to an insoluble fibrin matrix.1,2 However, there is considerable evidence that hemostatic factors play both a wider physiological role than simply controlling blood loss, and a wider pathological role than simply triggering inopportune thrombotic events, such as myocardial infarction and stroke. In tissue repair, a crucial physiological process, fibrin(ogen) is thought to provide a critical provisional matrix on which cells can proliferate, organize, and carry out specialized functions. A variety of cell types specifically bind to and migrate on fibrin(ogen) matrices. These include endothelial cells, macrophages, neutrophils, smooth muscle cells, fibroblasts, and keratinocytes.3-8 Direct binding to fibrin(ogen) through both integrin [e.g., αvβ3, α1β5, αMβ2 (CD11b/CD18, Mac-1)] and non-integrin receptors (e.g., intercellular adhesion molecule (ICAM-1)) appears to contribute to these cell-fibrin interactions.8-11 Fibrin(ogen) degradation products have also been reported to have an impressive array of biological activities, including mitogenic, angiogenic, chemotactic, and immunosuppressive activities.12-14 There are now substantial data indicating that fibrin(ogen) may plays an important role in the inflammatory response15,16 and that it may, in fact, direct leukocyte transendothelial cell migration.11 Similarly, through several G-protein coupled protease-activated receptors on fibroblasts, endothelial cells, leukocytes, smooth muscle cells, and other cell types, thrombin is thought to play an important role in inflammatory and fibroproliferative responses.17 Fibrinolytic factors, such as plasmin(ogen), also appear to be important modulators of inflammation.18 Finally, host fibrinogen, prothrombin, plasminogen, plasminogen activator, and other hemostatic factors appear to be crucial to the pathogenesis and virulence of many bacterial species.19-21 Unfortunately, despite a myriad of provocative observations made using in vitro systems, there is little direct in vivo evidence supporting an important role of fibrin(ogen) or other hemostatic factors in either the inflammatory response or disease progression. Direct and definitive analyses have been hampered by the lack of an experimental means to specifically manipulate the level or structure of selected hemostatic factors in vivo. Fortunately, this experimental roadblock has been effectively removed by the development of gene-targeting and gene transfer technologies in mice (see below).

scholarly journals An integrin receptor on normal and thrombasthenic platelets that binds thrombospondin [see comments]

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2022-2027 ◽  
Author(s):  
J Lawler ◽  
RO Hynes
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Alpha Subunit ◽  
Adhesive Proteins ◽  
Alpha V Beta 3

Abstract The members of the integrin family of membrane glycoprotein heterodimer complexes function as cell surface receptors for adhesive proteins. We report here on the identification of two integrins on the surface of human platelets that bind to thrombospondin. When platelet membrane proteins are radiolabeled with 125I-lactoperoxidase, solubilized in n- octylglucoside, (Boehringer Mannheim Biochemicals, Indianapolis, IN), and applied to a column of thrombospondin-Sepharose, both complexes are bound to the column and specifically eluted with the peptide GRGDSP. One of these integrins, glycoprotein (GP) IIb-IIIa, appears to bind relatively weakly. The second integrin shares the same beta subunit (beta 3 or GPIIIa), but has a distinct alpha subunit that comigrates with the alpha subunit (alpha v) of the vitronectin receptor (VnR) on endothelial cells and reacts with a monoclonal antibody, LM142, which was raised against an integrin from M21 melanoma cells. The alpha v beta 3 integrin is present on a variety of cell types and appears to act as a receptor for thrombospondin on endothelial and smooth muscle cells. On endothelial and M21 melanoma cells this receptor is also involved in adhesion to fibrinogen, vitronectin, and von Willebrand factor (vWF). The alpha v beta 3 integrin is present at approximately equal levels on normal and thrombasthenic platelets, whereas levels of GPIIb-IIIa are greatly reduced on thrombasthenic platelets. The alpha v beta 3 integrin on thrombasthenic platelets also binds to thrombospondin-Sepharose and can be eluted with the peptide GRGDSP. These data indicate that the alpha v beta 3 integrin on platelets, endothelial cells, and smooth muscle cells functions as an Arg-Gly-Asp (RGD)-dependent receptor for thrombospondin.

The Proadhesive Properties of Multimerin in Supporting Cellular Adhesion: Evidence for RGD and Non-RGD Dependent Adhesive Mechanisms.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3917-3917
Author(s):  
Frederic Adam ◽  
Shilun Zheng ◽  
Nilesh Joshi ◽  
Youko Suehiro ◽  
David S. Kelton ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Structural Features ◽  
Cellular Adhesion ◽  
Cellular Activation ◽  
Rgd Sequence ◽  
Rgd Site

Abstract Multimerin is a large soluble protein, with an uncertain function, found in platelets, megakaryocytes, endothelium and extracellular matrix fibers but not in plasma. The observation that multimerin contains structural features of an adhesive protein, including an Arg-Gly-Asp (RGD) sequence, led us to investigate its ability to support adhesion of platelets, megakaryocytes, endothelial cells and other cell types. Multimerin had the ability to support the adhesion of both platelets and megakaryocytes and this required cellular activation and the multimerin RGD site. Studies of normal and Glanzmann platelets indicated that multimerin interacted with the major platelet integrin receptor, αIIbβ3 and radioimmunoprecipitation analyses confirmed that multimerin bound to αIIbβ3. Multimerin also supported adhesion of endothelial cells, neutrophils and other cells including smooth muscle cells, fibroblast cells, human embryonic kidney (HEK293) and epithelial cells. Unlike platelets, these cells do not express αIIbβ3; this indicated that other integrin or non-integrin receptors could be involved in cellular adhesion to multimerin. Comparisons of cell adhesion to wild-type and RGE-multimerin indicated that unlike platelets and megakaryocytes, some other cell types (e.g. endothelial cells, smooth muscle cells and neutrophils) were capable of adhering to RGE-multimerin. This suggested that cellular adhesion to multimerin occurs by both RGD and non-RGD dependent mechanisms. Finally, unlike platelets, megakaryocytes and neutrophils, adhesion of other cell types to multimerin did not require cellular activation. In conclusion, our data indicate multimerin has fairly broad proadhesive properties, involving RGD and non-RGD dependent mechanisms, and that cellular receptors including αIIbβ3 interact with multimerin to mediate its binding to activated platelets, endothelial cells and potentially other cell types.

scholarly journals Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors.

1988 ◽  
Vol 107 (6) ◽  
pp. 2351-2361 ◽  
Author(s):  
J Lawler ◽  
R Weinstein ◽  
R O Hynes
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Attachment ◽  
Solid Phase ◽  
Rat Kidney ◽  
Muscle Cells ◽  
Cell Types ◽  
Adhesive Properties ◽  
Adhesive Proteins

Thrombospondin is a 420,000-D glycoprotein that has recently been shown to have several properties in common with the members of a class of adhesive proteins. To characterize further the adhesive properties of thrombospondin, we have studied its ability to support cell attachment. Thrombospondin adsorbed to plastic dishes supports the attachment of human endothelial and smooth muscle cells and the monocyte-like cell line (U937) as well as normal rat kidney cells. The majority of attached cells do not spread on the solid-phase thrombospondin. The attachment of all four cell types to thrombospondin is abolished if the assay is performed in the presence of EGTA, although the cells still attach to fibronectin. If thrombospondin is adsorbed to the dishes in the presence of EGTA and then washed with buffer containing calcium before addition of the cells, attachment is still markedly inhibited, indicating that calcium affects the conformation and function of thrombospondin. Attachment of all four cell types is also markedly inhibited by the synthetic peptides gly-arg-gly-asp-ser-pro (GRG-DSP) and gly-arg-gly-asp-ala-cys (GRGDAC) but not by the control peptide gly-arg-gly-glu-ser-pro (GRG-ESP). Affinity chromatography of n-octylglucoside extracts of surface-labeled endothelial cells or smooth muscle cells on thrombospondin-Sepharose and GRG-DSP-Affigel columns was used to identify an integrin complex related to glycoprotein IIb-IIIa as an RGD-dependent receptor for thrombospondin. In addition, a monoclonal antibody (LM609) that blocks attachment of endothelial cells to vitronectin, fibrinogen, and von Willebrand factor also inhibits attachment of endothelial cells to thrombospondin. These data indicate that the attachment of cells to thrombospondin is mediated by RGD and calcium-dependent mechanisms and is consistent with the hypothesis that the GRGDAC sequence in thrombospondin is a site for interaction with an integrin receptor of the beta 3 subclass.

Abstract 296: Bone Marrow Derived Cells Contribute to Cell Turnover in Ageing Murine Hearts

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Helge Möllmann ◽  
Holger M Nef ◽  
Christian Troidl ◽  
Sandra Voss ◽  
Mathias Heil ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Sorting ◽  
Physiological Role ◽  
Muscle Cells ◽  
Left Ventricular ◽  
Cell Turnover ◽  
Bone Marrow Derived Cells

Background: The paradigm that the heart is a terminally differentiated organ has recently been challenged since some studies reported the ability of bone marrow (BM) derived cells to transdifferentiate into cardiomyocytes after myocardial damage. However, the physiological role of bone marrow derived cells during the lifespan of an undamaged heart is widely unknown. We therefore examined the quantity and phenotype of bone marrow derived cells in aged murine hearts. Methods: 12-week-old mice (n=20) were sublethally irradiated and BM from enhanced green fluorescent-transgenic (eGFP) littermates was transplanted. After 1 month 5 mice were sacrificed and served as control. The remaining mice were sacrificed after 18.2±1.1 months. Immunohistochemistry was performed in 10 hearts using titin antibodies to identify cardiomyocytes, vimentin for fibroblasts, sMemb for myofibroblasts, α-smooth muscle actin for smooth muscle cells, F4/80 for macrophages, BS-1 and CD31 for endothelial cells. Additionally, anti-eGFP immunostaining was used to exclude autofluorescence. Sections were analyzed using fluorescence and confocal laser microscopy. The remaining 5 hearts were digested with collagenase and cell sorting was performed for a quantification of BM-derived cells in relation to eGFP negative cells. Results: BM transplantation was successful as FACS analysis showed 92±5% eGFP expressing leukocytes after 1 month and 78±6% after 18 months. In the juvenile hearts only few eGFP-positive cells were detected (<1 cell/mm2). Numerous eGFP-positive cells were found in left ventricular sections in the old hearts. Histological quantification revealed 9.3±3.3 cells/mm2 to be derived from BM cells. Most of these cells were fibroblasts and myofibroblasts. In addition, numerous endothelial cells and smooth muscle cells contributing to neoangiogenesis were detected. Few eGFP-positive cardiomyocytes could be identified. The cell sorting of eGFP-positive cells documented 4.8±1.9% of all cardiac cells to be derived of BM cells. Conclusion: The present study demonstrates for the first time a substantial recruitment and accumulation of BM derived cells in the ageing myocardium suggesting their contribution in cell turnover of the heart during the lifespan of mice.

Abstract 180: The Myristolated Alanine-Rich C Kinase Substrate (MARCKS) Differentially Regulates Proliferation of Vascular Smooth Muscle Cells and Endothelial Cells through Affecting Protein Stability and Proteolytic Processing of the Kinase Interacting with Stathmin (KIS)

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Dan Yu ◽  
Charles Drucker ◽  
Rajabrata Sarkar ◽  
Dudley K Strickland ◽  
Thomas S Monahan
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Protein Stability ◽  
Muscle Cells ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Functional Domain ◽  
Human Coronary Artery

Objective: Presently, the antiproliferative agents used in drug eluting stents and drug coated balloons inhibit both VSMC and endothelial cell (EC) proliferation, and thus these patients require dual antiplatelet therapy indefinitely. Identification of a VSMC-specific target to prevent proliferation represents a significant unmet clinical need. Previously we found that knockdown of MARCKS arrests VSMC proliferation through a p27 kip1 -dependent mechanism. Interestingly MARCKS knockdown increases EC proliferation. p27 kip1 is phosphorylated by KIS allowing it to exit the nucleus and be degraded. Here we seek to understand how MARCKS influences KIS protein expression in these two cell types. Approach and Results: We performed siRNA-mediated knock down of MARCKS in human coronary artery endothelial cells (CAECs) and human coronary artery smooth muscle cells (CASMCs). MARCKS knockdown did not affect KIS mRNA expression as determined with RT-PCR in either cell type. KIS protein stability was evaluated in the presence of cyclohexamide with Western blot. In CAECs, MARCKS knockdown increased KIS stability, however, in CASMCs, MARCKS knockdown significantly decreased KIS protein stability. In CASMCs, MARCKS knockdown significantly increased KIS ubiquitinization where as in CAECs, MARCKS knockdown decreased KIS ubiquitinization. Interestingly, the well-studied functional domain of MARCKS(ED domain) is not directly involved in KIS regulation. MARCKS mutants (S4G and S4D) rescued proliferation in VSMCs. MARCKS knockdown in vivo in the murine femoral wire injury model resulted in decreased medial bromodeoxyuridine (BrdU) integration and neointima formation. MARCKS knockdown enhanced endothelial barrier function recovery four days after injury as assessed by Evans Blue integration. Conclusions: MARCKS differentially regulates the protein stability and proteolytic processing of KIS in VSMCs and ECs. The differential interaction of MARCKS and KIS likely explains the observed difference in proliferation observed with MARCKS knockdown in these two cell types.

scholarly journals Human Cytomegalovirus Reduces Endothelin-1 Expression in Both Endothelial and Vascular Smooth Muscle Cells

2021 ◽  
Vol 9 (6) ◽  
pp. 1137
Author(s):  
Koon-Chu Yaiw ◽  
Abdul-Aleem Mohammad ◽  
Chato Taher ◽  
Huanhuan Leah Cui ◽  
Helena Costa ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Human Cytomegalovirus ◽  
Muscle Cells ◽  
Opportunistic Pathogen ◽  
Cell Types ◽  
Precursor Protein ◽  
Endothelin 1

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

scholarly journals Characterization of ectonucleotidases on vascular smooth-muscle cells

1985 ◽  
Vol 230 (2) ◽  
pp. 503-507 ◽  
Author(s):  
J D Pearson ◽  
S B Coade ◽  
N J Cusack
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Competitive Inhibitor ◽  
Aortic Endothelial Cells ◽  
Aortic Smooth Muscle ◽  
Cell Enzyme

We compared the properties of the ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5′-nucleotidase, EC 3.1.3.5) in intact pig aortic smooth-muscle cells in culture with the properties that we previously investigated for ectonucleotidases of aortic endothelial cells [Cusack, Pearson & Gordon (1983) Biochem. J. 214, 975-981]. In experiments with nucleotide phosphorothioate diastereoisomers, stereoselective catabolism of adenosine 5′-[β-thio]triphosphate, but not of adenosine 5′-[α-thio]triphosphate, by the triphosphatase and stereoselective catabolism of adenosine 5′-[α-thio]diphosphate by the diphosphatase were found, as occurs in endothelial cells. In contrast with endothelial ecto-5′-nucleotidase, the smooth-muscle-cell enzyme catabolized adenosine 5′-monophosphorothioate (AMPS) to adenosine: the affinity of the enzyme for AMPS was greater than for AMP, and Vmax for AMPS was about one-sixth that for AMP. In both cell types AMPS was an apparently competitive inhibitor of AMP catabolism by 5′-nucleotidase. The relative rates of catabolism of nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety were similar to those in endothelial cells. No ectopyrophosphatase activity was detected in smooth-muscle cells, in contrast with endothelial cells, where modest activity is present.

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