Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD.     

Collagen type I together with fibronectin provide a better support for endothelialization

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.     

Formation of smooth muscle alpha actin filaments in CD34+ bone marrow cells on arterial elastic laminae: potential role of SH2 domain-containing protein tyrosine phosphatase-1.

Arterial smooth muscle cells (SMCs) are present in the elastic lamina-containing media, suggesting that the elastic laminae may regulate the development of SMCs. Here, we investigated the role of elastic laminae in regulating the formation of SM alpha actin filaments in mouse CD34+ bone marrow cells and the role of a protein tyrosine phosphatase, SH2 domain-containing protein tyrosine phosphatase (SHP)-1, in the mediation of this process. Mouse CD34+ bone marrow cells were isolated by magnetic separation and used for assessing the influence of elastic laminae and collagen matrix on the formation of SM alpha actin filaments. CD34+ cells with transgenic SHP-1 knockout or siRNA-mediated SHP-1 knockdown were used to assess the role of SHP-1 in mediating the formation of SM alpha actin filaments. In cell culture tests, elastic laminae, but not collagen matrix, stimulated the formation of SM alpha actin filaments in CD34+ cells. The phosphatase SHP-1 mediated the stimulatory effect of elastic laminae. The interaction of CD34+ cells with elastic laminae, but not with collagen matrix, induced activation of SHP-1. The suppression of SHP-1 by transgenic SHP-1 knockout or siRNA-mediated SHP-1 knockdown significantly reduced the formation of SM alpha actin filaments in CD34+ cells cultured on elastic laminae. The in vitro observations were confirmed by using an in vivo model of implantation of elastic lamina and collagen matrix scaffolds into the aorta. These observations suggest that elastic laminae stimulate the formation of SM alpha actin filaments in CD34+ bone marrow cells and SHP-1 mediates the stimulatory effect of elastic laminae.     

Physiology and pathophysiology of selectins, integrins, and IgSF cell adhesion molecules focusing on inflammation. A paradigm model on infectious endocarditis

The development of adhesion bonds, either among cells or among cells and components of the extracellular matrix, is a crucial process. These interactions are mediated by some molecules collectively known as adhesion molecules (CAMs). CAMs are ubiquitously expressed proteins playing a central role in controlling cell migration, proliferation, survival, and apoptosis. Besides their key function in physiological maintenance of tissue integrity, CAMs play an eminent role in various pathological processes such as cardiovascular disorders, atherogenesis, atherosclerotic plaque progression and regulation of the inflammatory response. CAMs such as selectins, integrins, and immunoglobulin superfamily take part in interactions between leukocyte and vascular endothelium (leukocyte rolling, arrest, firm adhesion, migration). Experimental data and pathologic observations support the assumption that pathogenic microorganisms attach to vascular endothelial cells or sites of vascular injury initiating intravascular infections. In this review a paradigm focusing on cell adhesion molecules pathophysiology and infective endocarditis development is given.     

Physiology and Pathophysiology of Selectins,Integrins, and IgSf Cell Adhesion Molecules Focusing on Inflammation. A Paradigm Model on Infectious Endocarditis

Abstract

The development of adhesion bonds, either among cells or among cells and components of the extracellular matrix, is a crucial process. These interactions are mediated by some molecules collectively known as adhesion molecules (CAMs). CAMs are ubiquitously expressed proteins playing a central role in controlling cell migration, proliferation, survival, and apoptosis. Besides their key function in physiological maintenance of tissue integrity, CAMs play an eminent role in various pathological processes such as cardiovascular disorders, atherogenesis, atherosclerotic plaque progression and regulation of the inflammatory response. CAMs such as selectins, integrins, and immunoglobulin superfamily take part in interactions between leukocyte and vascular endothelium (leukocyte rolling, arrest, firm adhesion, migration). Experimental data and pathologic observations support the assumption that pathogenic microorganisms attach to vascular endothelial cells or sites of vascular injury initiating intravascular infections. In this review a paradigm focusing on cell adhesion molecules pathophysiology and infective endocarditis development is given.     

Extracellular matrix components in atherosclerotic arteries of Apo E/LDL receptor deficient mice: an immunohistochemical study

During accelerated vascular remodeling such as in atherosclerosis, the composition of the extracellular matrix becomes altered. The matrix components of the diseased artery influence cellular processes such as adhesion, migration and proliferation. Furthermore, in atherosclerosis, the inability of the cells within the lesion to produce a mechanically stable matrix may lead to plaque rupture. In this immunohistochemical study of atherosclerotic mice aorta, we have reviewed the presence of ECM components with roles in maintaining tissue structure and function. These components include osteopontin and COMP as well as the leucine rich repeats proteins decorin, PRELP, and fibromodulin. Immunohistochemistry demonstrated presence of osteopontin, COMP, decorin, PRELP and fibromodulin in lesion areas of ApoE/LDLr deficient mice. Some advanced lesions exhibited areas of cartilage-like morphology and were shown to represent cartilage by their content of the cartilage specific proteins collagen II and aggrecan. The results suggest that cartilage-associated cell/collagen binding ECM proteins may be involved in the pathogenesis of atherosclerosis.     

Effects of basic fibroblast growth factor on human microvessel endothelial cell migration on collagen I correlates inversely with adhesion and is cell density dependent

Angiogenesis, new vessel growth from existing vessels, is critical to tissue development and healing. Much is known about the molecular and cellular elements of angiogenesis, such as the effects of growth factors and matrix molecules on proliferation and migration. However, it is not clear how these elements are coordinated to produce specific microvascular beds. To address this, the effects of basic fibroblast growth factor (bFGF) on β1 integrin-mediated adhesion relative to migration in human microvessel endothelial cells (HMVEC) was examined. Using two assays of migration that differ in the density of cells being examined, bFGF stimulated single cell migration and reduced cell migration from a confluent monolayer on collagen I. Adhesion to collagen I of HMVEC treated at low density (2−4 × 10⁴ cells/cm²) with bFGF for 22 h was reduced, while bFGF increased cell adhesion of HMVEC treated at high density (6−8 × 10⁴ cells/cm²). Adhesion of both bFGF-treated and untreated HMVEC was mediated by the β1 integrin matrix receptor. Basic FGF treatment did not significantly alter surface expression of the β1 integrin subunit. Reduction in bFGF-mediated adhesion correlated with delayed cell spreading and altered organization of β1 integrin into substrate contacts. Thus, integrin-mediated cell adhesion in microvessel endothelial cells is sensitive to regulation by a growth factor. Furthermore, the nature of the response to this signal depends on another cell regulator, cell density. In addition, modulation of cell adhesion by a growth factor may be a central regulatory feature in controlling endothelial cell migration. © 1996 Wiley-Liss, Inc.     

Integrins,Cell Matrix Interactions and Cell Migration Strategies: Fundamental Differences in Leukocytes and Tumor Cells

The principles determining the migration of different cell types may result from their differences in origin, size and shape, function of adhesion receptors, and environmental factors, including the extracellular matrix. Polarized leukocytes (T lymphocytes and dendritic cells) migrating in three-dimensional collagen lattices are small developing a highly dynamic leading edge and a trailing uropod, whereas invasive melanoma cells are larger, highly polarized and less dynamic. In contrast to leukocytes, tumor cells may additionally develop migrating cell clusters maintaining intense cell-cell interaction and cluster polarity. Leukocytes show a speed-oriented, oscillating and directionally unpredictable path profile strongly guided by matrix fibers, while melanoma cells and migrating cell clusters exhibit slow yet highly directional migration. Whereas leukocytes form short-lived interactions with collagen fibers in complete absence of tissue remodeling, melanoma cells and neoplastic cell clusters reorganize the matrix via profound pulling at attachment sites, limited fiber disruption upon detachment, and the shedding of cell surface determinants. Using blocking anti-integrin antibodies, tumor cell migration and migration-associated matrix reorganization were shown to be dependent on β integrin-mediated adhesion, whereas migrating T cells cannot be inhibited by a panel of anti-β1-, β2-, β3-, and α-integrin antibodies, either alone or in combination. Consequently, migrating melanoma cells use focal adhesions of integrins coclustered with cytoskeletal components at contacts with collagen fibers. T cells, however, lack typical focal adhesions, redistribute β1 integrins to the uropod and the focal adhesion kinase to the leading edge. In conclusion, an adhesion-dependent and reorganizing migration type employed by melanoma cells may be distinct from largely integrinindependent and non-reorganizing migration strategies used by leukocytes.     

Bi-directional signal transduction by integrin receptors

The integrin family of cell surface glycoproteins functions primarily as receptors for extracellular matrix ligands. There are now many well characterized integrin-ligand interactions which are known to influence many aspects of cell behaviour including cell morphology, cell adhesion, cell migration as well as cellular proliferation and differentiation. However, in fulfilling these functions, integrins are not simple adhesion receptors that physically mediate connections across the plasma membrane. Rather, integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within cells. It is these intracellular interactions which allow integrin function to effect many biochemical signalling pathways and therefore to impinge upon complex cellular activities. Recently, much research has focused on elucidating the molecular mechanisms which control integrin function and the molecular processes which transduce integrin-mediated signalling events. In this review, we discuss progress in the field of integrin signal transduction including, where applicable, potential therapeutic applications arising from the research.     

胶原样分子与肿瘤关系研究进展

细胞外基质不仅维持着体内细胞微环境的稳定,还在细胞的正常生长、增殖以及细胞之间的信号传导中起着重要作用。肿瘤发生时,基质中的分子组分发生了改变,这些改变朝着有利于肿瘤细胞生长侵袭的方向发展。在这个过程中,细胞外基质的主要成分在合成和分解上发生巨大变化,胶原分子便是其中之一,胶原分子作为细胞外基质中的主要成分,对细胞的黏附、运动、迁移等活动起着重要作用。随着研究的深入,发现越来越多的胶原分子参与了肿瘤的发生发展。基质中还存在着一些分子,它们在结构上和胶原蛋白一样含有三螺旋胶原结构域,在肿瘤的发生发展过程中同样发挥着重要作用。本文就包括胶原分子在内的含有胶原结构的分子在肿瘤中的作用做一综述。     

Modulation of smooth muscle cell behaviour by platelet-derived factors and the extracellular matrix

We have studied the combined effects of platelet-derived soluble factors and three types of macromolecular substrata on the proliferation and migration of smooth muscle cells in vitro. Bovine aortic smooth muscle cells were plated onto three-dimensional gels of type I collagen or onto cell-free extracellular matrices deposited on such gels by either bovine aortic endothelial cells or smooth muscle cells. The cells were cultured in the presence of whole-blood serum (WBS) or platelet-poor plasma (PPP). Smooth muscle cell proliferation on type I collagen gels was dependent on the presence of platelet-derived factors, i.e. the cells proliferated in the presence of WBS but not in PPP. In contrast, cell proliferation on the extracellular matrices occurred at the same rate in PPP and WBS. Smooth muscle cells plated onto collagen gels rapidly migrated down into the gel matrix; the percentage of cells migrating was inversely proportional to cell density. The presence of extracellular matrices did not alter the rate of cell migration into the underlying gel matrix. Irrespective of the substratum used, smooth muscle cell migration was independent of platelet-derived or plasma factors and occurred in the absence of proliferation. These results indicate that possible chemotactic, chemokinetic, and/or mitogenic factors produced by the vascular cells and deposited within the extracellular matrix may play an important role in modulating smooth muscle cell behaviour in the vascular wall.     

Focal adhesion kinase mediates defects in the force-dependent reinforcement of initial integrin-cytoskeleton linkages in metastatic colon cancer cell lines

Micro-environmental clues, including the biophysical interpretation of the extracellular matrix, are critical to proliferation, apoptosis and migration. Here, we show that metastatic human colon cancer cell lines display altered matrix interaction. Interaction of colon cancer cells with collagen I depends on integrins (mainly alpha(1)/beta(1)) but metastatic cells display delayed spreading and reduced extension of lamellipodia. In addition, cells show defective strengthening of integrin-cytoskeleton linkages upon mechanical stimulation, as determined by laser trapping experiments and binding of large beads to the cell surface. However, adhesion to pliable surfaces is ameliorated in metastatic variants. These changes are caused by constitutive activation of focal adhesion kinase (FAK) and can be modulated by changing expression and/or activity of FAK via RNA-interference or expression of inhibitory constructs, respectively. In addition, consistent with defective strengthening of integrin-cytoskeleton linkages, metastatic cell lines show reduced random motility. Taken together these data suggest that constitutive activation of FAK causes defects in spreading, reinforcement of integrin-cytoskeleton linkages and migration and at the same time could ameliorate the adhesion of metastatic cells to suboptimal surfaces.     

Extra-cellular matrix in vascular networks

The vascular network is a series of linked conduits of blood vessels composed of the endothelium, a monolayer of cells that adorn the vessel lumen and surrounding layer(s) of mesenchymal cells (vascular smooth muscle, pericytes and fibroblasts). In addition to providing structural support, the mesenchymal cells are essential for vessel contractility. The extracellular matrix is a major constituent of blood vessels and provides a framework in which these various cell types are attached and embedded. The composition and organization of vascular extracellular matrix is primarily controlled by the mesenchymal cells, and is also responsible for the mechanical properties of the vessel wall, forming complex networks of structural proteins which are highly regulated. The extracellular matrix also plays a central role in cellular adhesion, differentiation and proliferation. This review examines the cellular and extracellular matrix components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.     

Retinal capillary endothelial cells prefer different substrates for growth and migration

Recent studies have shown that the extracellular matrix modifies the behaviour of endothelial cells. We have studied the effects of extracellular matrix components on retinal capillary endothelial cell migration and proliferation. Bovine retinal capillary endothelial cells were selectively cultured from collagenase-digested microvessel fragments. In a filter system for the assessment of migration, endothelial cells responded to substrate-bound fibronectin but not to soluble fibronectin. Cell migration on collagen- or gelatin-coated filters was minimal, and these cells failed to adopt a spread morphology, remaining instead as round cells. Cell replication was quantified using a protein dye binding assay for adherent cells in 96 well plates. Serum was essential for growth irrespective of the substrate. Cells harvested from microvessel cultures proliferated more rapidly on collagen- and gelatin-coated plastic than on fibronectin and were unaffected by additions to the medium such as endothelial cell conditioned medium, whereas cells proliferating directly from the microvessels grew at a faster rate on fibronectin and also responded to conditioned medium supplement. When cultured on collagen gels, initial microvessel cells and harvested cells required surface fibronectin in order to adopt a cobblestone morphology. These results show that fibronectin is a requirement for bovine retinal capillary endothelial cell migration, but proliferation of these cells can be supported, with slight differences, by both fibronectin and collagen provided serum growth factors are present. These findings are relevant to the early phase of angiogenesis in which migration and proliferation of endothelial cells occurs.     

CCN4 regulates vascular smooth muscle cell migration and proliferation

The migration and proliferation of vascular smooth muscle cells (VSMCs) are essential elements during the development of atherosclerosis and restenosis. An increasing number of studies have reported that extracellular matrix (ECM) proteins, including the CCN protein family, play a significant role in VSMC migration and proliferation. CCN4 is a member of the CCN protein family, which controls cell development and survival in multiple systems of the body. Here, we sought to determine whether CCN4 is involved in VSMC migration and proliferation. We examined the effect of CCN4 using rat cultured VSMCs. In cultured VSMCs, CCN4 stimulated the adhesion and migration of VSMCs in a dose-dependent manner, and this effect was blocked by an antibody for integrin α5β1. CCN4 expression was enhanced by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Furthermore, knockdown of CCN4 by siRNA significantly inhibited the VSMC proliferation. CCN4 also could up-regulate the expression level of marker proteins of the VSMCs phenotype. Taken together, these results suggest that CCN4 is involved in the migration and proliferation of VSMCs. Inhibition of CCN4 may provide a promising strategy for the prevention of restenosis after vascular interventions.     

Glycated collagen and altered glucose increase endothelial cell adhesion strength

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen‐coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm² in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength α_vβ₃ integrins to bind to glycated collagen instead of the typical α₂β₁ integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm² higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis. J. Cell. Physiol. 228: 1727–1736, 2013. © 2013 Wiley Periodicals, Inc.     

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by alphaVbeta3 integrin in human breast cancer cells.

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.     

Non-collagenous ECM proteins in blood vessel morphogenesis and cancer

Background

The extracellular matrix (ECM) is constituted by diverse composite structures, which determine the specific to each organ, histological architecture and provides cells with biological information, mechanical support and a scaffold for adhesion and migration. The pleiotropic effects of the ECM stem from the dynamic changes in its molecular composition and the ability to remodel in order to effectively regulate biological outcomes. Besides collagens, fibronectin and laminin are two major fiber-forming constituents of various ECM structures.

Scope of review

This review will focus on the properties and the biological functions of non-collagenous extracellular matrix especially on laminin and fibronectin that are currently emerging as important regulators of blood vessel formation and function in health and disease.

Major conclusions

The ECM is a fundamental component of the microenvironment of blood vessels, with activities extending beyond providing a vascular scaffold; extremely versatile it directly or indirectly modulates all essential cellular functions crucial for angiogenesis, including cell adhesion, migration, proliferation, differentiation and lumen formation. Specifically, fibronectin and laminins play decisive roles in blood vessel morphogenesis both during embryonic development and in pathological conditions, such as cancer.

General significance

Emerging evidence demonstrates the importance of ECM function during embryonic development, organ formation and tissue homeostasis. A wealth of data also illustrates the crucial role of the ECM in several human pathophysiological processes, including fibrosis, skeletal diseases, vascular pathologies and cancer. Notably, several ECM components have been identified as potential therapeutic targets for various diseases, including cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.