This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, R. T. Articles by Hemler, M. E. Search for Related Content PubMed PubMed Citation Articles by Lee, R. T. Articles by Hemler, M. E.

(Circulation Research. 1995;76:209-214.)
© 1995 American Heart Association, Inc.

Articles

Integrin-Mediated Collagen Matrix Reorganization by Cultured Human Vascular Smooth Muscle Cells

Richard T. Lee, Fedor Berditchevski, George C. Cheng, Martin E. Hemler

From the Cardiovascular Division (R.T.L., G.C.C.), Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, and the Dana-Farber Cancer Institute (F.B., M.E.H.), Boston, Mass.

Correspondence to Richard T. Lee, MD, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Vascular smooth muscle cells perform the important function of modulation of vascular extracellular matrix. Because integrins mediate many cell-matrix interactions, the role of integrins in reorganization of collagen by cultured human vascular smooth muscle cells was studied. Immunoprecipitation demonstrated that human vascular smooth muscle cells express multiple ß₁ integrins. Monoclonal antibody A2-IIE10 (a blocking anti-² antibody) inhibited adhesion of smooth muscle cells to collagen by 31%. The blocking anti-¹ antibody 1B3.1 inhibited adhesion by 40%, whereas a blocking anti-³ antibody had no effect on adhesion. When 1B3.1 and A2-IIE10 were both used, a 79% reduction in adhesion was observed, indicating that active ¹ and ² integrins cooperatively mediate adhesion. The blocking anti-ß₁ antibody Mab13 abolished smooth muscle cell–mediated gel contraction, and the ²-blocking antibody A2-IIE10 had a dose-dependent partial inhibitory effect (37%). In contrast, blocking antibodies to ¹ and ³ had no effect. When anti-¹ (1B3.1) and anti-² (A2-IIE10) monoclonal antibodies were combined, no synergistic effect on inhibition of gel contraction was observed. Surprisingly, collagen gel contraction was inhibited by 46% by an anti-ß₁ antibody (TS2/16) known for its stimulatory effect on cell adhesion. Thus, whereas ¹ß₁ and ²ß₁ integrins both participate in adhesion of vascular smooth muscle cells to collagen, only ²ß₁ integrins mediate collagen reorganization. In addition, collagen reorganization appears to be a dynamic process, adversely affected by excessive adhesion strengthening.

Key Words: integrins • atherosclerosis • collagen • vascular smooth muscle • adhesion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular smooth muscle cells perform many functions that are critical for determining vascular structure during development, normal homeostasis, and disease states. In response to mechanical stimuli, smooth muscle cells change orientation, growth state, and extracellular matrix synthesis, permitting homeostatic adaptations to the pulsatile stresses of blood pressure.^{1 2} In addition, vascular smooth muscle cells migrate into the intima, proliferate, and secrete the extracellular matrix of atherosclerotic lesions.^{3 4} These lesions typically contain a collagen matrix that is relatively dense compared with the normal vessel. This collagenous matrix is, in part, responsible for changes in the biomechanical behavior of the vessel that can lead to instability and, ultimately, fracture of the lesion with thrombosis and occlusion.^{5 6}

Studies of fibroblasts, melanocytes, and other cells cultured in hydrated collagen lattices have demonstrated that mammalian cells will contract and reorganize collagen fibrils.^{7 8 9} This process of physically contracting collagen is analogous to the organization of collagen matrix that occurs in dermal wound healing and development of normal connective tissues and is mediated by integrins.^{7 9} Integrins are heterodimeric cell-surface receptors for extracellular matrix molecules that can transduce mechanical signals from the extracellular environment into the cell.^{10 11 12} The integrin family includes at least 15 subunits and 8 ß subunits that can form 21 different heterodimers, and three different subunits (¹, ², and ³) can form complexes with the ß₁ subunit and function as collagen receptors. In addition to ligand specificity, integrins may have cell type–specific differences in function. For example, the ²ß₁ integrin may function as a collagen receptor on fibroblasts or a collagen and laminin receptor on other cells.^{13 14} The ²ß₁ integrin appears to mediate collagen gel contraction, and this process has been implicated in the pathophysiology of vitreoretinal contraction and retinal detachment.^{15 16} Collagen gel contraction by ²ß₁ integrins can be abolished by exchange of the cytoplasmic domain of the ² subunit with that of the ⁴ subunit with no effect on adhesion to collagen.¹⁷ Thus, integrins function as more than adhesion receptors for extracellular matrix.

A variety of integrins on cultured vascular smooth muscle cells have been described. Clyman and colleagues^{18 19} described several subunits associated with ß₁ subunits that mediated adhesion of rat vascular smooth muscle cells to fibronectin, laminin, and collagen. Lamb ductus arteriosus smooth muscle cells have ß₁ integrins that appear to mediate adhesion to fibronectin, laminin, and collagen types I and IV, whereas migration of these cells on these substrates is heavily dependent on ^vß₃ integrins.²⁰ Because integrins may play an important role in smooth muscle cell regulation of vascular structure, we studied integrin-mediated extracellular matrix reorganization by cultured human vascular smooth muscle cells. We found that ¹ß₁ and ²ß₁ integrins both participated in mediating adhesion to collagen, whereas ²ß₁ integrins mediated collagen gel contraction, demonstrating specificity of subunit functions in human vascular smooth muscle cells. In addition, we report that collagen gel contraction can be inhibited by an anti–ß₁ integrin antibody that usually stimulates adhesion, suggesting that dynamic conformational changes in ß₁ integrins are necessary for collagen reorganization.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monoclonal Antibodies
Monoclonal antibodies used included Mab13 (anti-ß₁) and Mab16 (anti-⁵),²¹ 1B3.1 (anti-¹),²² 12F1 (anti-²),²³ P3 (nonspecific control),²⁴ TS2/7 (anti-¹),²⁵ B5G10 (anti-⁴),²⁶ TS2/16 (anti-ß₁),^{27 28 29} and P1H5 (anti-²)⁹ (see Table). Additional monoclonal antibodies used that were developed and characterized in the laboratory of M. Hemler include A2-IIE10 and A2-3E9 (both anti-²), A5-PUJ2 (anti-⁵), and A6-ELE (anti-⁶). A6-ELE was raised by injection of human mammary epithelial cells into RBF/DnJ mice. Monoclonal antibody IA3 (anti-³) was developed and characterized by F. Berditchevski and J. Taylor-Papadimitriou.

View this table: [in this window] [in a new window]   Table 1. Monoclonal Antibodies Against Integrins

Cell Preparation and Culture
Smooth muscle cells were cultured by explant outgrowth from unused portions of human saphenous veins from coronary bypass surgery by a protocol approved by the Human Research Committee of Brigham and Women's Hospital. The cell cultures were grown in Dulbecco's modified Eagle's medium (M.A. Bioproducts) with 10% fetal calf serum. These conditions are selective for growth of smooth muscle cells over endothelial cells.³⁰ The explant and culture technique was identical to the protocol used in previous studies of cultured vascular smooth muscle cells.^{31 32 33} All tissue culture constituents were selected for low endotoxin levels (<40 pg/mL) by Limulus amebocyte lysate assay (QCL 1000, M.A. Bioproducts). Experiments were performed at passage 4 or 5 after harvesting.

Immunoprecipitation
Cultured human smooth muscle cells were detached from tissue culture plastic with 2 mmol/L EDTA and surface-labeled with sodium ¹²⁵I by using lactoperoxidase and glucose oxidase as previously described.³⁴ Cellular proteins were solubilized in the immunoprecipitation buffer (1% of Nonidet P-40, 50 mmol/L Tris HCl, pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl₂, 2 mmol/L phenylmethylsulfonyl fluoride, 20 µg/mL aprotinin, and 20 µg/mL leupeptin) for 1 hour at 4°C, and the protein extract was incubated with appropriate monoclonal antibodies for 1 hour at 4°C. Immune complexes were recovered on protein A–Sepharose beads preabsorbed with rabbit anti-mouse polyclonal antisera and washed five times with immunoprecipitation buffer. Immunoprecipitated proteins were eluted from the protein A–Sepharose beads in Laemmli loading buffer and resolved in 8.5% sodium dodecyl sulfate–polyacrylamide gels. The dried gels were exposed for 24 hours at -70°C with X-OMAT film (Kodak).

Cell Adhesion Assays
Ninety-six–well plates were coated overnight with a solution of collagen type I (5 µg/mL), blocked with 0.1% heat-denatured bovine serum albumin (hdBSA) for 45 minutes at 37°C, and washed twice with phosphate-buffered saline. Cells were detached from the tissue culture plastic with 2 mmol/L EDTA, washed with phosphate-buffered saline, and labeled with the fluorescent dye BCECF-AM (Molecular Probes, Inc) for 30 minutes at 37°C. After labeling, the cells were washed twice with phosphate-buffered saline, resuspended in 0.1% hdBSA/RPMI, and preincubated with appropriate antibodies for 30 minutes at 4°C before addition to the matrix-coated plate (50 000 cells per well). After incubation for 25 to 30 minutes at 37°C, the plates were washed three times with RPMI to remove nonadherent cells. The fluorescence before and after washes was evaluated with a CytoFluor 2300 fluorescent analyzer machine (Millipore Co). Every measurement point was performed in triplicate, and adhesion was estimated as the number of attached cells per square millimeter.

Collagen Gel Contraction
Cell culture in hydrated collagen gels was performed with Vitrogen 100 collagen (Collagen Corp) as previously described.⁷ Vascular smooth muscle cells were preincubated with monoclonal antibodies at various concentrations for 30 minutes and then diluted 10-fold to yield final gel concentrations. The preincubated cells were then added to neutralized collagen (2.5 mg/mL) at a concentration of 2x10⁵ cells per milliliter. The collagen-cell suspensions (1.5 mL each) were then incubated in 24-well plates (Costar) at 37°C for 1 hour to polymerize the collagen, and the gel was then gently cut away from the sides of the well and lifted off the bottom. At selected time points, the diameter of the hydrated gels was measured by use of an inverted microscope.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunoprecipitation and Adhesion to Collagen
To study the role of integrins in reorganization of collagenous matrix by human vascular smooth muscle cells, we first analyzed their integrin profile by immunoprecipitation using specific monoclonal antibodies (Fig 1). Vascular smooth muscle cells express abundant levels of several integrins of the ß₁ family (lanes 1 through 6), including ¹ß₁, ²ß₁, ³ß₁, and ⁵ß₁, whereas the cell surface expression of ⁴ß₁ was low, and ⁶ß₁ was almost undetectable. An anti-ß₁ antibody precipitated all of the ß₁-containing integrins at once (lane 7).

View larger version (38K): [in this window] [in a new window]   Figure 1. Integrin expression in cultured human vascular smooth muscle cells. Cells were surface-labeled with sodium 125I, lysed in 1% Brij 96 extraction buffer, and immunoprecipitated with specific monoclonal antibodies against different integrin subunits. Immunoprecipitates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradiography. The anti-integrin antibodies were as follows: lane 1, TS2/7 (anti-1); lane 2, A2-IIE10 (anti-2); lane 3, IA3 (anti-3); lane 4, B5G10 (anti-4); lane 5, Mab16 (anti-5); lane 6, A6-ELE (anti-6); lane 7, Mab13 (anti-ß1); and lane 8, P3 (nonspecific control).

To explore the role of these integrins in adhesion of vascular smooth muscle cells to collagen type I, adhesion experiments were performed in the presence of specific blocking monoclonal antibodies (Fig 2). Monoclonal antibodies P1H5 and A2-IIE10 (which block ²-mediated adhesion) reduced adhesion by 34% and 31%, respectively, and the anti-¹–blocking antibody 1B3.1 reduced adhesion by 40%. However, when 1B3.1 and A2-IIE10 were both used, adhesion was reduced by 79%, indicating that these cells use both ¹ß₁ and ²ß₁ integrins to interact with collagen. Accordingly, monoclonal antibody Mab13 (which blocks the function of all ß₁ integrins) reduced adhesion to collagen by 95%. In parallel experiments, blocking anti-³ß₁ or nonblocking anti-¹ß₁ and anti-²ß₁ monoclonal antibodies did not block adhesion (data not shown). Notably, adhesion to collagen was not increased by TS2/16, presumably because adhesion is already at a near maximal level.

View larger version (15K): [in this window] [in a new window]   Figure 2. Bar graph showing the effect of specific anti-integrin monoclonal antibodies on the adhesion of the human smooth muscle cells to collagen type 1. The plastic surface was coated with collagen type I (5 µg/mL) and blocked with heat-denaturated bovine serum albumin. Cells labeled with fluorescent dye were preincubated with antibodies for 30 minutes at 4°C and allowed to attach to the adhesion surface for 30 minutes at 37°C. Nonadherent cells were removed in three consecutive washes, and the attachment was analyzed by use of CytoFluor 2300. P3 is a control nonintegrin antibody; 1B3.1, an inhibitory anti-1ß1 antibody; P1H5 and A2-IIE10 (IIE10), inhibitory anti-2ß1 antibodies; Mab13, an inhibitory anti-ß1 antibody; and TS2/16, an anti-ß1 antibody known to stimulate adhesion. Error bars denote one standard deviation.

Collagen Gel Contraction
When human vascular smooth muscle cells were cultured in floating hydrated collagen gels, the gels contracted in a highly reproducible and symmetrical manner. Significant reduction in gel diameter occurred within the first 24 hours, and contraction was essentially complete after 72 hours. To evaluate the role of collagen-binding integrins in this process, collagen gel contraction experiments were performed in the presence of blocking anti-integrin monoclonal antibodies. The anti-²–blocking antibody A2-IIE10 had a dose-dependent partial inhibitory effect on gel contraction (37% at 72 hours, Fig 3A), whereas the nonblocking anti-² A2-3E9 antibody had no inhibitory effect (Fig 3B). The anti-²–blocking antibody P1H5 also inhibited gel contraction by 40% at 72 hours (data not shown). In contrast, the presence of blocking antibodies to ¹ (Fig 4A) and ³ (Fig 4B) had no effect on collagen gel contraction. Moreover, in contrast with the results of the adhesion experiments, when anti-¹ (1B3.1) and anti-² (A2-IIE10) antibodies were combined, no synergistic effect on inhibition of gel contraction was observed (Fig 5).

View larger version (15K): [in this window] [in a new window]   Figure 3. Graphs showing the effect of inhibition of 2ß1 function on contraction of collagen matrices by vascular smooth muscle cells. A, Effect of antibody A2-IIE10. B, Effect of antibody A2-3E9. indicates presence of Mab13 (1 µg/mL); , absence of antibody; , antibody diluted 1:500; , antibody diluted 1:100; , antibody diluted 1:20; and , antibody diluted 1:10. Points are average of duplicate matrices.

View larger version (14K): [in this window] [in a new window]   Figure 4. Graphs showing the effect of inhibition of 1ß1 or 3ß1 function on contraction of collagen matrices by vascular smooth muscle cells. indicates presence of Mab13 (1 µg/mL); , absence of antibody. A, Effect of antibody 1B3.1 in concentrations of 1:2500 (), 1:500 (), and 1:250 () as dilutions of ascites fluid. B, Effect of antibody IA3 in concentrations of 0.2 (), 2.0 (), and 20 () µg/mL. Points are average of duplicate matrices.

View larger version (14K): [in this window] [in a new window]   Figure 5. Graph showing the effect of inhibition of 1ß1 and 2ß1 function on contraction of collagen matrices by vascular smooth muscle cells. indicates absence of antibody; , 1:100 dilution of antibody 1B3.1 ascites fluid; , 1:20 dilution of antibody A2-IIE10 culture supernatant; and , presence of both 1B3.1 and A2-IIE10. Mean±SD values of triplicates are shown.

The ß₁-blocking antibody Mab13 abolished smooth muscle cell–mediated gel contraction (Fig 3A and 3B), but in all experiments, A2-IIE10 and P1H5 failed to inhibit gel contraction as effectively as Mab13. Although the anti-ß₁ antibody TS2/16 had no inhibitory effect on cell adhesion, it strongly inhibited collagen gel contraction in a dose-dependent manner, with 46% inhibition (at 72 hours) for the highest dose (Fig 6). The maximal inhibitory effect of TS2/16 was less than that observed with Mab13 (86%) but similar to the inhibitory effects of blocking anti-² antibodies (37% to 40%).

View larger version (15K): [in this window] [in a new window]   Figure 6. Graph showing the effect of stimulating anti-ß1 antibody TS2/16 on contraction of collagen matrices by vascular smooth muscle cells. indicates presence of Mab13 (1 µg/mL); , absence of antibody; , 1:5000 dilution of ascites fluid; , 1:2000 dilutions of ascites fluid; and , 1:500 dilution of ascites fluid. Points are average of triplicate matrices.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we found that function-blocking antibodies to individual subunits of collagen-binding integrins partially blocked vascular smooth muscle cell adhesion, whereas the combination of antibodies to both ¹ and ² subunits markedly inhibited adhesion. It is not surprising that a cell responsible for regulating structure in a collagenous matrix both acutely through active contraction and chronically through organization of matrix should have a redundant system for adhering to collagen. On the other hand, the addition of antibodies blocking ¹ function did not lead to additional inhibition of collagen gel contraction over the ²-blocking antibody, indicating that in the collagen reorganization process, integrins function as more than simply collagen adhesion receptors. At this time, there are few other clear examples of multiple integrins adhering to the same ligand and translating adhesive information into diverse subsequent post–ligand binding events. In this regard, it was previously shown that replacement of the ² cytoplasmic domain with that of ⁴ did not alter adhesion to collagen but did abolish gel contraction.¹⁷

The complexity of the collagen reorganization process was further demonstrated by the effects of the stimulatory monoclonal antibody TS2/16. The TS2/16 antibody can increase adhesion in cells with ß₁ integrins that are not fully active, presumably by inducing a conformational change that causes increased ligand binding. The results of the present study indicate that ß₁ integrins of cultured vascular smooth muscle cells are already in a highly active state, since adhesion was not increased by TS2/16. Although the stimulatory antibody had minimal effect on adhesion, collagen gel contraction was markedly inhibited. One potential explanation for this finding is a dynamic mechanism of collagen reorganization that requires (1) integrin-mediated adhesion to collagen, (2) contraction through cytoskeletal force generation, and (3) subsequent release of collagen from the integrin. The integrin could then bind to new collagen ligands, and the contraction process would begin again. This dynamic adhesion and release mechanism may be similar to proposed mechanisms of integrin-mediated cell migration.³⁵ Indeed, it was recently demonstrated that the stimulatory antibody 8A2 inhibited migration, presumably by freezing eosinophil ß₁ integrins in the high-avidity adhesion state.³⁶

Previous studies have identified ²ß₁ integrin as the primary mediator of the gel contraction phenomenon.^{7 9} However, as seen in previous studies,⁹ antibodies blocking ² function failed to achieve the magnitude of inhibition of collagen gel contraction of Mab13 in the present study. Although we observed this incomplete inhibition with two different ² function–blocking antibodies (A2-IIE10 and P1H5), it is possible that Mab13 has a much higher potency for blocking integrin function than the other antibodies.

The ability of cells to contract and organize collagen is fundamental in the wound-healing process. For example, this generation of physical forces allows myofibroblasts of the dermis to close a skin wound and increase the strength of the loose granulation tissue, ultimately forming a strong scar. Atherosclerosis resembles the wound-healing process in many ways.³⁷ In response to a variety of stimuli, smooth muscle cells migrate into the intima and secrete extracellular matrix rich in collagen and proteoglycans.⁶ It is likely that smooth muscle cells participate in organizing the collagen in this newly synthesized matrix to form the dense network of collagen that is often seen in the advanced atheroma. The organized collagen network is one reason that atherosclerotic tissue is several times stiffer than the normal vessel wall.³⁸ Studies of vascular mechanics suggest that this difference in stiffness may predispose the vessel to plaque rupture and thrombosis by establishing regions of high stress near the junction of the stiffer plaque with the more normal vessel.³⁹

It is important to recognize that cultured human vascular smooth muscle cells are phenotypically different from smooth muscle cells in vivo.^{40 41} Previous reports have suggested that the profile of integrins on vascular smooth muscle cells changes in cell culture. Koteliansky et al⁴² found that expression of ¹ integrins by human aortic smooth muscle cells decreased during subculturing; in addition, smooth muscle cells from thickened intima of human adult aorta express five times less ¹ than cells from adult aortic media.⁴³ Our studies focused on cultured smooth muscle cells from human saphenous veins, although contraction of human aortic smooth muscle cells was also inhibited by anti–ß₁ integrin antibodies. Because the distribution and activity of integrins throughout the circulatory system has not been fully characterized, these data should not be directly extrapolated to the pathophysiology of human arterial disease. In one experiment with cultured human aortic smooth muscle cells, aortic smooth muscle cells contracted collagen gels by 30% at 2 days in culture (data not shown). Similar to the experiments with saphenous vein smooth muscle cells, the ß₁-blocking antibody Mab13 inhibited aortic smooth muscle cell–mediated gel contraction by 92% at 2 days, and the anti-ß₁ antibody TS2/16 inhibited gel contraction by 60%. Vascular smooth muscle cells in vivo may have different morphologies, and the "synthetic" phenotype is associated with enhanced migration and proliferation in experimental studies.⁴¹ It will be interesting to determine if changes in integrin expression and activity are associated with changes in smooth muscle cell phenotype.

Vascular smooth muscle cells cultured in collagen gels have some differences compared with cells grown on plastic; eg, smooth muscle cells reduce collagen synthesis and are less responsive to growth factors.^{44 45} Although we have not observed changes in integrin profile between smooth muscle cells grown on plastic or grown in collagen gels (preliminary studies not shown), Klein et al⁷ have found that ²ß₁ integrins in some cells are upregulated by growth in collagen gels. It is also possible that the integrin activation state could be changed by culture in the collagen lattice.

In conclusion, the present study serves to identify a potential mechanism used by vascular smooth muscle cells in vivo to organize extracellular matrix and opens the way for further studies to determine how modulating ²ß₁ function might change vessel wall structure. In addition, these experiments indicate that the process of collagen reorganization by cells has stringent requirements; it does not occur if adhesion to collagen is mediated by the "wrong" integrin (ie, ¹ß₁) or if adhesion is excessively strong (ie, in the presence of TS2/16).

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid from the American Heart Association, Massachusetts Affiliate, Inc (Dr Lee) and National Institutes of Health grant GM-38903 (Dr Hemler). The authors thank Dr Maria Muszynski and Smruti Parikh for their expert assistance in preparing the vascular smooth muscle cells and Dr Peter Libby for his advice and review of the manuscript.

	Footnotes

 
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received May 25, 1994; accepted October 3, 1994.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Sumpio BE, Banes AJ, Link WG, Johnson G Jr. Enhanced collagen production by smooth muscle cells during repetitive mechanical stretching. Arch Surg. 1988;123:1233-1236. [Abstract]
2. Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE. Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J Cell Biol. 1993;123:741-747. [Abstract/Free Full Text]
3. Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol. 1990;15:1667-1687. [Abstract]
4. Casscells W. Migration of smooth muscle and endothelial cells. Circulation. 1992;86:723-729. [Free Full Text]
5. Davies MJ. A macro and micro view of coronary vascular insult in ischemic heart disease. Circulation. 1990;82(suppl II):II-38-II-46.
6. Fuster V, Badimon L, Badimon JJ, Chesebro JH: The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992;326:242-250. [Medline] [Order article via Infotrieve]
7. Klein CE, Dressel D, Steinmayer T, Mauch C, Eckes B, Krieg T, Bankert RB, Weber L. Integrin ₂ß₁ is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils. J Cell Biol. 1991;115:1427-1436. [Abstract/Free Full Text]
8. Nakagawa S, Pawelek P, Grinnell F. Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity. J Invest Dermatol. 1989;93:792-798. [Medline] [Order article via Infotrieve]
9. Schiro JA, Chan BMC, Roswit WT, Kassner PD, Pentland AP, Hemler ME, Eisen AZ, Kupper TS. Integrin ²ß₁ (VLA-2) mediates reorganization and contraction of collagen matrices by human cells. Cell. 1991;67:403-410. [Medline] [Order article via Infotrieve]
10. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11-25. [Medline] [Order article via Infotrieve]
11. Hemler ME. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol. 1990;8:365-400. [Medline] [Order article via Infotrieve]
12. Ingber DE. The riddle of morphogenesis: a question of solution chemistry or molecular cell engineering? Cell. 1993;75:1249-1252. [Medline] [Order article via Infotrieve]
13. Kirchofer D, Languino LR, Ruoslahti E, Pierschbacher MD. ₂ß₁ integrins from different cell types show different binding specificities. J Biol Chem. 1990;265:615-618. [Abstract/Free Full Text]
14. Elices MJ, Hemler ME. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others. Proc Natl Acad Sci U S A. 1989;86:9906-9910. [Abstract/Free Full Text]
15. Kupper TS, Ferguson TA. A potential pathophysiologic role for ₂ß₁ integrin in human eye diseases involving vitreoretinal traction. FASEB J. 1993;7:1401-1406.[Abstract]
16. Hunt RC, Pakalanis VA, Choudhury P, Black EP. Cytokines and serum cause ₂ß₁ integrin-mediated contraction of collagen gels by cultured retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1994;35:955-963. [Abstract/Free Full Text]
17. Chan BMC, Kassner PD, Schiro JA, Byers HR, Kupper TS, Hemler ME. Distinct cellular functions mediated by different VLA integrin subunit domains. Cell. 1992;68:1051-1060. [Medline] [Order article via Infotrieve]
18. Clyman RI, McDonald KA, Kramer RH. Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen. Circ Res. 1990;67:175-186. [Abstract/Free Full Text]
19. Clyman RI, Turner DC, Kramer RH. An ₁/ß₁-like integrin receptor on rat aortic smooth muscle cells mediates adhesion to laminin and collagen types I and IV. Arteriosclerosis. 1990;10:402-409. [Abstract/Free Full Text]
20. Clyman RI, Mauray F, Kramer RH. ß₁ and ß₃ integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix. Exp Cell Res. 1992;200:272-284. [Medline] [Order article via Infotrieve]
21. Akiyama SK, Yamada SS, Chen WT, Yamada KM. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J Cell Biol. 1989;109:863-875. [Abstract/Free Full Text]
22. Bank I, Hemler M, Brenner MB, Cohen D, Levy V, Belko J, Crouse C, Chess L. A novel monoclonal antibody, 1B3.1, binds to a new epitope of the VLA-1 molecule. Cell Immunol. 1989;122:416-423. [Medline] [Order article via Infotrieve]
23. Pischel KD, Hemler ME, Huang C, Bluestein HG, Woods VL. Use of the monoclonal antibody 12F1 to characterize the differentiation antigen VLA-2. J Immunol. 1987;138:226-233. [Abstract]
24. Lemke H, Hammerling GJ, Hohmann C, Rajewsky K. Hybrid cell lines secreting monoclonal antibody specific for major histocompatibility antigens of the mouse. Nature. 1978;271:249-251. [Medline] [Order article via Infotrieve]
25. Hemler ME, Sanchez-Madrid F, Flotte TJ, Krensky AM, Burakoff SJ, Bhan AK, Springer TA, Strominger JL. Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines. J Immunol. 1984;132:3011-3018. [Abstract]
26. Hemler ME, Huang C, Takada Y, Schwarz L, Strominger JL, Clabby ML. Characterization of the cell surface heterodimer VLA-4 and related peptides. J Biol Chem. 1987;262:11478-11485. [Abstract/Free Full Text]
27. Masumoto A, Hemler ME. Multiple activation states of VLA-4. J Biol Chem. 1993;268:228-234. [Abstract/Free Full Text]
28. Chan BMC, Hemler ME. Multiple functional forms of the integrin VLA-2 can be derived from a single ² cDNA clone: interconversion of forms induced by an anti-ß₁ antibody. J Cell Biol. 1993;120:537-543. [Abstract/Free Full Text]
29. Kawaguchi S, Hemler ME. Role of the subunit cytoplasmic domain in regulation of adhesive activity mediated by the integrin VLA-2. J Biol Chem. 1993;268:16279-16285. [Abstract/Free Full Text]
30. Gimbrone MA, Cotran RS. Human vascular smooth muscle in culture. Lab Invest. 1975;33:16-27. [Medline] [Order article via Infotrieve]
31. Libby P, Warner SK, Friedman GB. Interleukin-1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest. 1988;81:487-498.
32. Amento EP, Ehsani N, Palmer H, Libby P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler Thromb. 1991;11:1223-1230. [Abstract/Free Full Text]
33. Karas RH, Petterson BL, Mendelsohn ME. Human vascular smooth muscle cells contain functional estrogen receptor. Circulation. 1994;89:1943-1950. [Abstract/Free Full Text]
34. Hemler ME, Huang C, Schwarz L. The VLA protein family: characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight ß subunit. J Biol Chem. 1987;262:3300-3309. [Abstract/Free Full Text]
35. Schmidt CE, Horwitz AF, Lauffenburger DA, Sheetz MP. Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated. J Cell Biol. 1993;123:977-991. [Abstract/Free Full Text]
36. Kuijpers TW, Mul EPJ, Blom M, Kovach NL, Gaeta FCA, Tollefson V, Elices MJ, Harlan JM. Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration. J Exp Med. 1993;178:279-284. [Abstract/Free Full Text]
37. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;262:801-809.
38. Loree HM, Grodzinsky AJ, Park SY, Gibson LJ, Lee RT. Static circumferential tangential modulus of human atherosclerotic tissue. J Biomech. 1994;27:195-204. [Medline] [Order article via Infotrieve]
39. Cheng GC, Loree HM, Kamm RD, Fishbein MC, Lee RT: Distribution of circumferential stress in ruptured and stable atherosclerotic lesions. Circulation. 1993;87:1179-1187. [Abstract/Free Full Text]
40. Campbell JH, Campbell GR. Culture techniques and their applications to studies of vascular smooth muscle. Clin Sci. 1993;85:501-513. [Medline] [Order article via Infotrieve]
41. Dilley RJ, McGeachie JK, Prendergast FJ. A review of the proliferative behavior, morphology and phenotypes of vascular smooth muscle. Atherosclerosis. 1987;63:99-107. [Medline] [Order article via Infotrieve]
42. Koteliansky VE, Belkin AM, Frid MG, Glukhova MA. Developmental changes in expression of adhesion-mediating proteins in human aortic smooth muscle. Biochem Soc Trans. 1991;19:1072-1076. [Medline] [Order article via Infotrieve]
43. Belkin VM, Belkin AM, Koteliansky VE. Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development. J Cell Biol. 1990;111:2159-2170. [Abstract/Free Full Text]
44. Redecker-Beuke B, Thie M, Rauterberg J, Robenek H. Aortic smooth muscle cells in a three-dimensional collagen lattice culture. Arterioscler Thromb. 1993;13:1572-1579. [Abstract/Free Full Text]
45. Thie M, Harrach B, Schonherr E, Kresse H, Robenek H, Rauterberg J. Responsiveness of aortic smooth muscle cells to soluble growth mediators is influenced by cell-matrix contact. Arterioscler Thromb. 1993;13:994-1004.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. N. Popova, M. Barczyk, C.-F. Tiger, W. Beertsen, P. Zigrino, A. Aszodi, N. Miosge, E. Forsberg, and D. Gullberg {alpha}11{beta}1 Integrin-Dependent Regulation of Periodontal Ligament Function in the Erupting Mouse Incisor Mol. Cell. Biol., June 15, 2007; 27(12): 4306 - 4316. [Abstract] [Full Text] [PDF]

				N. E. Winterwood, A. Varzavand, M. N. Meland, L. K. Ashman, and C. S. Stipp A Critical Role for Tetraspanin CD151 in {alpha}3beta1 and {alpha}6beta4 Integrin-dependent Tumor Cell Functions on Laminin-5 Mol. Biol. Cell, June 1, 2006; 17(6): 2707 - 2721. [Abstract] [Full Text] [PDF]

				C. Franco, B. Ho, D. Mulholland, G. Hou, M. Islam, K. Donaldson, and M. P. Bendeck Doxycycline Alters Vascular Smooth Muscle Cell Adhesion, Migration, and Reorganization of Fibrillar Collagen Matrices Am. J. Pathol., May 1, 2006; 168(5): 1697 - 1709. [Abstract] [Full Text] [PDF]

				R. G. Leyh, M. Wilhelmi, P. Rebe, S. Ciboutari, A. Haverich, and H. Mertsching Tissue Engineering of Viable Pulmonary Arteries for Surgical Correction of Congenital Heart Defects Ann. Thorac. Surg., April 1, 2006; 81(4): 1466 - 1471. [Abstract] [Full Text] [PDF]

				O. D. Defawe, R. D. Kenagy, C. Choi, S. Y.C. Wan, C. Deroanne, B. Nusgens, N. Sakalihasan, A. Colige, and A. W. Clowes MMP-9 regulates both positively and negatively collagen gel contraction: A nonproteolytic function of MMP-9 Cardiovasc Res, May 1, 2005; 66(2): 402 - 409. [Abstract] [Full Text] [PDF]

				B. He, L. Liu, G. A. Cook, S. Grgurevich, L. K. Jennings, and X. A. Zhang Tetraspanin CD82 Attenuates Cellular Morphogenesis through Down-regulating Integrin {alpha}6-Mediated Cell Adhesion J. Biol. Chem., February 4, 2005; 280(5): 3346 - 3354. [Abstract] [Full Text] [PDF]

				K. D. Little, M. E. Hemler, and C. S. Stipp Dynamic Regulation of a GPCR-Tetraspanin-G Protein Complex on Intact Cells: Central Role of CD81 in Facilitating GPR56-G{alpha}q/11 Association Mol. Biol. Cell, May 1, 2004; 15(5): 2375 - 2387. [Abstract] [Full Text] [PDF]

				N. Chattopadhyay, Z. Wang, L. K. Ashman, S. M. Brady-Kalnay, and J. A. Kreidberg {alpha}3{beta}1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTP{micro} expression and cell-cell adhesion J. Cell Biol., December 22, 2003; 163(6): 1351 - 1362. [Abstract] [Full Text] [PDF]

				D. Rizzoni, E. Porteri, G. E.M. Boari, C. De Ciuceis, I. Sleiman, M. L. Muiesan, M. Castellano, M. Miclini, and E. Agabiti-Rosei Prognostic Significance of Small-Artery Structure in Hypertension Circulation, November 4, 2003; 108(18): 2230 - 2235. [Abstract] [Full Text] [PDF]

				H. M. ROSENFELDT, Y. AMRANI, K. R. WATTERSON, K. S. MURTHY, R. A. PANETTIERI JR, and S. SPIEGEL Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells FASEB J, October 1, 2003; 17(13): 1789 - 1799. [Abstract] [Full Text] [PDF]

				S. Li, C. Van Den Diepstraten, S. J. D'Souza, B. M. C. Chan, and J. G. Pickering Vascular Smooth Muscle Cells Orchestrate the Assembly of Type I Collagen via {alpha}2{beta}1 Integrin, RhoA, and Fibronectin Polymerization Am. J. Pathol., September 1, 2003; 163(3): 1045 - 1056. [Abstract] [Full Text] [PDF]

				X. A. Zhang, W. S. Lane, S. Charrin, E. Rubinstein, and L. Liu EWI2/PGRL Associates with the Metastasis Suppressor KAI1/CD82 and Inhibits the Migration of Prostate Cancer Cells Cancer Res., May 15, 2003; 63(10): 2665 - 2674. [Abstract] [Full Text] [PDF]

				A. R. Kazarov, X. Yang, C. S. Stipp, B. Sehgal, and M. E. Hemler An extracellular site on tetraspanin CD151 determines {alpha}3 and {alpha}6 integrin-dependent cellular morphology J. Cell Biol., September 29, 2002; 158(7): 1299 - 1309. [Abstract] [Full Text] [PDF]

				X. Yang, C. Claas, S.-K. Kraeft, L. B. Chen, Z. Wang, J. A. Kreidberg, and M. E. Hemler Palmitoylation of Tetraspanin Proteins: Modulation of CD151 Lateral Interactions, Subcellular Distribution, and Integrin-dependent Cell Morphology Mol. Biol. Cell, March 1, 2002; 13(3): 767 - 781. [Abstract] [Full Text] [PDF]

				E. P. Moiseeva Adhesion receptors of vascular smooth muscle cells and their functions Cardiovasc Res, December 1, 2001; 52(3): 372 - 386. [Abstract] [Full Text] [PDF]

				H. Huang, R. D. Kamm, P. T.C. So, and R. T. Lee Receptor-Based Differences in Human Aortic Smooth Muscle Cell Membrane Stiffness Hypertension, November 1, 2001; 38(5): 1158 - 1161. [Abstract] [Full Text] [PDF]

				X. A. Zhang, A. L. Bontrager, C. S. Stipp, S.-K. Kraeft, G. Bazzoni, L. B. Chen, and M. E. Hemler Phosphorylation of a Conserved Integrin {alpha}3 QPSXXE Motif Regulates Signaling, Motility, and Cytoskeletal Engagement Mol. Biol. Cell, February 1, 2001; 12(2): 351 - 365. [Abstract] [Full Text]

				M. M. Medhora Retinoic acid upregulates beta 1-integrin in vascular smooth muscle cells and alters adhesion to fibronectin Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H382 - H387. [Abstract] [Full Text] [PDF]

				K. J. Bayless, R. Salazar, and G. E. Davis RGD-Dependent Vacuolation and Lumen Formation Observed during Endothelial Cell Morphogenesis in Three-Dimensional Fibrin Matrices Involves the {alpha}v{beta}3 and {alpha}5{beta}1 Integrins Am. J. Pathol., May 1, 2000; 156(5): 1673 - 1683. [Abstract] [Full Text] [PDF]

R. L. Yauch, A. R. Kazarov, B. Desai, R. T. Lee, and M. E. Hemler Direct Extracellular Contact between Integrin alpha 3beta 1 and TM4SF Protein CD151 J. Biol. Chem., March 24, 2000; 275(13): 9230 - 9238. [Abstract]