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Vascular Smooth Muscle Cells Orchestrate the Assembly of Type I Collagen via α2β1 Integrin, RhoA, and Fibronectin Polymerization

2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)63464-5

1525-2191

Shaohua Li, Caroline Van Den Diepstraten, Sudhir J.A. D’Souza, Bosco M. C. Chan, J. Geoffrey Pickering,

Bone and Dental Protein Studies

Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-α2β1 integrin antibody and accelerated by an α2β1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-α5β1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition. Assembly of collagen into fibrils is widely studied as a spontaneous and entropy-driven process. To determine whether vascular smooth muscle cells (SMCs) impact the formation of collagen fibrils, we microscopically tracked the conversion of soluble to insoluble collagen in human SMC cultures, using fluorescent type I collagen at concentrations less than that which supported self-assembly. Collagen microaggregates were found to form on the cell surface, initially as punctate collections and then as an increasingly intricate network of fibrils. These fibrils displayed 67-nm periodicity and were found in membrane-delimited cellular invaginations. Fibril assembly was inhibited by an anti-α2β1 integrin antibody and accelerated by an α2β1 integrin antibody that stimulates a high-affinity binding state. Newly assembled collagen fibrils were also found to co-localize with newly assembled fibronectin fibrils. Moreover, inhibition of fibronectin assembly with an anti-α5β1 integrin antibody completely inhibited collagen assembly. Collagen fibril formation was also linked to the cytoskeleton. Fibrils formed on the stretched tails of SMCs, ran parallel to actin microfilament bundles, and formed poorly on SMCs transduced with retrovirus containing cDNA for dominant-negative RhoA and robustly on SMCs expressing constitutively active RhoA. Lysophosphatidic acid, which activates RhoA and stimulates fibronectin assembly, stimulated collagen fibril formation, establishing for the first time that collagen polymerization can be regulated by soluble agonists of cell function. Thus, collagen fibril formation is under close cellular control and is dynamically integrated with fibronectin assembly, opening new possibilities for modifying collagen deposition. The fundamental importance of type I collagen can be inferred by its presence in almost all human tissues and by the nonviability of embryos deficient in this extracellularmatrix (ECM) protein.1Jaenisch R Harbers K Schnieke A Lohler J Chumakov I Jahner D Grotkopp D Hoffmann E Germline integration of Moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death.Cell. 1983; 32: 209-216Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 2Breindl M Harbers K Jaenisch R Retrovirus-induced lethal mutation in collagen I gene of mice is associated with an altered chromatin structure.Cell. 1984; 38: 9-16Abstract Full Text PDF PubMed Scopus (64) Google Scholar Type I collagen exerts its roles, as a load-bearing structure and regulator of cell function, only after it has polymerized into fibrils. This assembly process proceeds after proteolytic removal of the globular termini of the secreted procollagen molecules. The resulting fibrils can vary considerably in size and organization. Fibril diameters can range from 20 to 500 nm and the fibrils themselves can organize into diverse patterns including bundles, weaves, and layers.3Kadler KE Holmes DF Trotter JA Chapman JA Collagen fibril formation.Biochem J. 1996; 316: 1-11Crossref PubMed Scopus (1104) Google Scholar, 4Trelstad RL Silver RH Matrix assembly.in: HED Cell Biology of the Extracellular Matrix. Plenum Publishing Corp., New York1981: 179-216Crossref Google Scholar This broad diversity in fibril size and topology has important implications for tissue function. For example, optical transparency in the cornea is conferred by collagen that has been assembled into thin fibrils in an orthogonal lattice, whereas the enormous tensile strength of tendon is because of thick collagen fibrils in parallel bundles. The precise manner in which collagen fibrils are assembled and spatially organized is thus critical to organ development and repair. Current understanding of collagen fibril assembly is based primarily on the long-standing recognition that molecules of type I collagen can self-assemble. It has been appreciated for more than 40 years that solubilized, tissue-extracted collagen will polymerize spontaneously when physiological pH, temperature, and ionic strength are restored.5Gross J Kirk D Heat precipitation of collagen from neutral salt solutions: some rate regulating factors.J Biol Chem. 1958; 233: 355-360Abstract Full Text PDF PubMed Google Scholar, 6Wood GC The precipitation of collagen fibers from solution.Int Rev Connect Tissue Res. 1964; 2: 1-31Crossref PubMed Google Scholar, 7Williams BR Gelman RA Poppke DC Piez KA Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results.J Biol Chem. 1978; 253: 6578-6585Abstract Full Text PDF PubMed Google Scholar Collagen fibrils can also be generated in vitro by subjecting soluble type I procollagen to sequential cleavage of its propeptide termini by procollagen metalloproteinases. This action reduces the solubility of the protein and initiates the entropy-driven self-assembly process.8Miyahara M Njieha FK Prockop DJ Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.J Biol Chem. 1982; 257: 8442-8448Abstract Full Text PDF PubMed Google Scholar, 9Kadler KE Hojima Y Prockop DJ Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropy-driven process.J Biol Chem. 1987; 262: 15696-15701Abstract Full Text PDF PubMed Google Scholar Although only the latter approach involves the physiologically relevant step of procollagen cleavage, both in vitro systems yield early collagen fibrils with characteristics similar to those found in developing tissues.3Kadler KE Holmes DF Trotter JA Chapman JA Collagen fibril formation.Biochem J. 1996; 316: 1-11Crossref PubMed Scopus (1104) Google Scholar, 10Holmes DF Lowe MP Chapman JA Vertebrate (chick) collagen fibrils formed in vivo can exhibit a reversal in molecular polarity.J Mol Biol. 1994; 235: 80-83Crossref PubMed Scopus (53) Google Scholar Moreover, both approaches have been valuable for elucidating conditions for collagen self-assembly and in defining controlling elements for this within the collagen molecule.7Williams BR Gelman RA Poppke DC Piez KA Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results.J Biol Chem. 1978; 253: 6578-6585Abstract Full Text PDF PubMed Google Scholar, 11Prockop DJ Fertala A Inhibition of the self-assembly of collagen I into fibrils with synthetic peptides. Demonstration that assembly is driven by specific binding sites on the monomers.J Biol Chem. 1998; 273: 15598-15604Crossref PubMed Scopus (119) Google Scholar Self-assembly however cannot by itself explain the diverse morphology of collagen fibrils found in tissues, and determinants other than those intrinsic to the collagen molecule are likely required. In this regard, collagen-associating ECM molecules, including decorin, fibromodulin, and lumican, have been found to impact the size and architecture of type I collagen fibrils.12Danielson KG Baribault H Holmes DF Graham H Kadler KE Iozzo RV Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.J Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1192) Google Scholar, 13Chakravarti S Magnuson T Lass JH Jepsen KJ LaMantia C Carroll H Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican.J Cell Biol. 1998; 141: 1277-1286Crossref PubMed Scopus (588) Google Scholar, 14Svensson L Aszodi A Reinholt FP Fassler R Heinegard D Oldberg A Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.J Biol Chem. 1999; 274: 9636-9647Crossref PubMed Scopus (381) Google Scholar Recently, the elaboration of collagen structures by mouse embryonic cells has been shown to require the assembly of fibronectin fibrils.15Sottile J Hocking DC Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions.Mol Biol Cell. 2002; 13: 3546-3559Crossref PubMed Scopus (457) Google Scholar Therefore, in the context of a cellular environment, interactions between collagen and noncollagen molecules seem to be important for collagen fibril formation and organization. It remains difficult however, in a cell-based system, to experimentally separate the process of collagen assembly from the cellular production and secretion of collagen. No study to date has specifically examined the assembly of collagen (ie, the conversion of soluble collagen to an insoluble fibril) in the presence of cells. Likewise, in the presence of cells it is a challenge to distinguish the phenomenon of collagen self-assembly, in which only collagen-collagen interactions are at play, from interactions between collagen and other proteins that might drive assembly. This includes potential interactions with cell-surface ECM receptors and with other ECM fibrils, such as fibronectin, that depend on the cell for polymerization.16Wu C Keivens VM O'Toole TE McDonald J Ginsberg MH Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix.Cell. 1995; 83: 715-724Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 17Schwarzbauer JE Sechler JL Fibronectin fibrillogenesis: a paradigm for extracellular matrix assembly.Curr Opin Cell Biol. 1999; 11: 622-627Crossref PubMed Scopus (251) Google Scholar An important context for collagen fibril assembly is the blood vessel wall. The manner in which collagen fibrils are assembled in the vasculature is critical to the mechanical properties of both the normal and diseased artery. The major source of type I collagen in the vasculature is vascular smooth muscle cells (SMCs). SMCs can influence higher levels of fibril organization, as illustrated by their ability to contract a preformed collagen fibril lattice.18Lee RT Berditchevski F Cheng GC Hemler ME Integrin-mediated collagen matrix reorganization by cultured human vascular smooth muscle cells.Circ Res. 1995; 76: 209-214Crossref PubMed Scopus (115) Google Scholar However, it is not known whether SMCs have control over the process by which a collagen fibril is generated from its soluble precursors. To determine whether SMCs play a meaningful role in the de novo assembly of type I collagen, we developed a cell culture system in which fluorescence-labeled collagen, in a soluble, unpolymerized state, is applied to SMC cultures. This allowed us to microscopically track the conversion of soluble to insoluble collagen on the cell surface. As such, cell-associated collagen assembly process could be distinguished from collagen production and also be compared with collagen assembly in the absence of the cell. The findings established that SMCs have a pronounced effect on the assembly of collagen fibrils through a mechanism that involves α2β1 integrin and RhoA-mediated signaling. Moreover, using differentially labeled precursors we established that collagen polymerization was intimately related to fibronectin polymerization. Finally, we observed that collagen polymerization could be regulated by exogenous mediators of cell behavior. The results thus identify a central role for SMCs in orchestrating the formation of collagen fibrils, implying that cellular information drives collagen assembly in the vasculature. Primary cultures of human arterial SMCs were initiated by explant outgrowth from segments of internal thoracic artery retrieved at the time of coronary artery bypass surgery.19Pickering JG Bacha P Weir L Jekanowski J Nichols JC Isner JM Prevention of smooth muscle cell outgrowth from human atherosclerotic plaque by a recombinant fusion protein specific for the epidermal growth factor receptor.J Clin Invest. 1993; 91: 724-729Crossref PubMed Scopus (35) Google Scholar The identity of vascular SMCs was confirmed morphologically and by immunostaining with a monoclonal antibody to smooth muscle α-actin (1A4; DAKO, Mississauga, Canada). Cells were grown in M199 (Life Technologies, Inc., Gaithersburg, MD), supplemented with 10% fetal bovine serum in the absence of ascorbate. Experiments were performed using SMCs in the third to seventh subculture, cultured on glass coverslips precoated with 100 μg/ml of human fibronectin. Solubilized collagen was obtained either from bovine skin, after pepsin digestion and solubilization in HCl (Vitrogen; Cohesion Technologies, Palo Alto, CA), or from rat tail, solubilized in acetic acid.20Bell E Ivarsson B Merrill C Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.Proc Natl Acad Sci USA. 1979; 76: 1274-1278Crossref PubMed Scopus (1806) Google Scholar The collagen preparations were dialyzed against borate-buffered saline (170 mmol/L boric acid, 170 mmol/L sodium tetraborate, 75 mmol/L NaCl, pH 9.3) at 4°C overnight and then labeled with either fluorescein isothiocyanate (FITC) or Texas Red (Molecular Probes, Eugene, OR) by transferring the dialysate to a solution of borate-buffered saline containing 30 mg/ml of the fluorochrome and mixing in the dark at 4°C for 6 hours. Acidic pH was restored and unbound FITC or Texas Red was removed by dialysis against 0.1% acetic acid at 4°C for 4 days. The concentration of labeled collagen was measured spectrophotometrically by measuring absorption at 280 nm and either 493 nm or 595 nm wavelengths. To assay collagen assembly, labeled soluble collagen (500 μg/ml) was mixed with ice-cold M199 with 10% fetal bovine serum to the final designated concentration (0.2 to 10 μg/ml). This was added to SMC cultures that were maintained on ice for 2 minutes, at room temperature for 2 minutes, and then at 37°C for designated intervals. Cultures were then washed in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. Cells were then stained with Hoechst 33258 (Molecular Probes), mounted in glycerol/PBS (9:1), and collagen fibril formation was evaluated by fluorescence microscopy (Axiovert S100; Zeiss). To assess for collagen self-assembly under these conditions, the identical procedure was performed without cells. The effect of collagen-binding integrins on SMC-directed assembly was assessed using an α1β1 integrin-specific blocking antibody (5E8D9; Upstate Biotechnology Inc., Lake Placid, NY), an α2β1 integrin-specific blocking antibody (BHA2.1),21Hangan D Uniyal S Morris V MacDonald I von Ballestrem C Chau T Schmidt E Chambers A Groom A Chan BMC Integrin VLA-2 (α2β1) function in post-extravasation movement of human rhabdomyosarcoma cells in the liver.Cancer Res. 1996; 56: 3142-3149PubMed Google Scholar and an α2β1 integrin-specific stimulating antibody (JBS2),22Ho WC Heinemann C Hangan D Uniyal S Morris VL Chan BM Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver.Mol Biol Cell. 1997; 8: 1863-1875Crossref PubMed Scopus (41) Google Scholar each added 30 minutes before addition of fluorescent collagen. Solubilized collagen was added to SMCs cultured on plastic coverslips (Therminox; Nalge-Nunc) in ascorbate-free medium. At designated times the cultures were fixed with 2% phosphate-buffered glutaraldehyde, postfixed in 1% osmium tetroxide, and then embedded in Polybed/araldite epoxy. Coverslips with cells and collagen were cross-sectioned followed by sequential staining with 2% uranyl acetate in 70% ethanol and Reynolds aqueous lead citrate. Specimens were viewed with a transmission electron microscope (Phillips 410) at 60 kV. Fibronectin assembly by human SMCs was evaluated as we have described previously,23Pickering JG Chow LH Li S Rogers KA Rocnik EF Zhong R Chan BM α5β1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.Am J Pathol. 2000; 156: 453-465Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar using fluorescently labeled, plasma-derived human fibronectin. Fibronectin protomers were labeled with Oregon Green conjugated to an amine-reactive succinimidyl ester (Molecular Probes). Labeled soluble fibronectin (20 μg/ml) was added to SMCs cultured in M199, supplemented with fibronectin-free fetal bovine serum, together with Texas Red-labeled soluble collagen. In some experiments, labeled collagen was added to cultures after they had been incubated with labeled fibronectin protomers for 1 hour and then extensively washed. cDNA fragments encoding dominant-negative (T19N RhoA) or constitutively active (Q63L RhoA) RhoA, kindly provided by Dr. Gary Bokoch (Scripps Research Institute, La Jolla, CA),24Zhang S Han J Sells MA Chernoff J Knaus UG Ulevitch RJ Bokoch GM Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1.J Biol Chem. 1995; 270: 23934-23936Crossref PubMed Scopus (653) Google Scholar were excised from pRK5 Myc and cloned into the retroviral gene delivery vector pLNCX (BD Biosciences Clontech, Palo Alto, CA). Retroviral delivery constructs were introduced into the Phoenix-amphotropic retrovirus packaging/producing cell line (American Type Culture Collection, Rockville, MD)25Pear WS Nolan GP Scott ML Baltimore D Production of high-titer helper-free retroviruses by transient transfection.Proc Natl Acad Sci USA. 1993; 90: 8392-8396Crossref PubMed Scopus (2307) Google Scholar by CaCl2-mediated transfection. The virus-containing culture supernatant was harvested 48 to 72 hours later and, after centrifugation and filtration (0.45-μm pore filters), was added to proliferating human SMCs for 48 hours. To obtain stably expressing cells, transductants were selected in M199 containing 500 μg/ml of G418. Cells were maintained in G418-containing medium for two passages before use in the fibril assembly assay. Paraformaldehyde-fixed SMCs were permeabilized in cold acetone for 15 seconds, nonspecific binding sites were blocked with 5% normal goat serum, and cells were incubated with Texas Red-labeled phalloidin (1:100 dilution, Molecular Probes) for 1 hour. To evaluate the assembly of collagen in SMC cultures, independent of collagen production, we labeled tissue-extracted, soluble collagen with FITC or Texas Red and microscopically tracked fibril formation after subjecting the collagen solution to physiological temperature and pH. This was undertaken in both cellular and noncellular environments. Nonfibrillar precipitation of the collagen was minimized by adding the labeled precursors to precooled culture medium that was then gradually warmed to 37°C. Two different collagen preparations were used, pepsin-digested bovine skin collagen solubilized in HCl (Vitrogen collagen) and acetic acid-solubilized rat tail collagen. The two preparations were studied recognizing that the method of collagen preparation might influence its subsequent polymerization. We first established the threshold concentration below which collagen self-assembly was not apparent, by adding precursor to culture media in the absence of cells. This threshold proved to be 2 μg/ml for Vitrogen collagen and 5 μg/ml for rat tail collagen. At higher concentrations, fibrils 6 to 12 μm in length and similar to self-assembled fibrils previously described7Williams BR Gelman RA Poppke DC Piez KA Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results.J Biol Chem. 1978; 253: 6578-6585Abstract Full Text PDF PubMed Google Scholar, 8Miyahara M Njieha FK Prockop DJ Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.J Biol Chem. 1982; 257: 8442-8448Abstract Full Text PDF PubMed Google Scholar settled onto the fibronectin-coated coverslips (Figure 1a). At concentrations below these thresholds, fibrils were not detected on the substrate or suspended in the media (Figure 1b). However, when a subthreshold concentration of collagen was added to media bathing a monolayer of human SMCs, an insoluble collagen network of formed on the cells. As illustrated in Figure 1c, within 1 hour of adding Vitrogen collagen, there was a finely punctate distribution of fluorescent collagen on the cell surface (Figure 1c). Fibril-like structures were generally not evident at this time but in the ensuing 2 hours, short fibrils formed on the apical surface of cells (Figure 1d). By 24 hours, an intricate pericellular network developed (Figure 1; c to e). This network was seen with precursor concentrations as low as 0.2 μg/ml. The network that formed using rat tail collagen was similar to that using Vitrogen collagen, with somewhat less cell-to-cell bridging of fibrils (Figure 1f). In neither case were fibrils found in regions devoid of cells. Fibril formation was not influenced by the substrate on which SMCs were grown, with no discernable difference in fibrils seen for SMCs on fibronectin, vitronectin, or rat tail collagen (data not shown). When collagen precipitates from solution it can potentially assume nonfibrillar forms that are not relevant to in vivo circumstances, or abnormal fibril variants that are only rarely observed in vivo.26Paige MF Rainey JK Goh MC Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.Biophys J. 1998; 74: 3211-3216Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar To elucidate the ultrastructure of collagen polymerizing on the SMCs, cultures were studied by transmission electron microscopy. SMCs were cultured in ascorbate-free medium to minimize endogenous collagen production. Under these conditions, and in the absence of exogenous collagen precursor, no fibrils were evident by electron microscopy 36 hours after cell plating. Likewise, when soluble collagen was added in the absence of SMCs, only scattered amorphous aggregates were found on the substrate. However, addition of soluble collagen to SMC cultures yielded cross-banded fibrils. The banding displayed 67-nm periodicity, confirming the structures to be native collagen fibrils (Figure 2a). It was also noteworthy that assembled collagen fibrils were found in association with distinct morphological conformations of the cell surface. Some fibrils were found within plasma membrane-delimited spaces. These spaces were much larger than uptake vesicles, with a greater degree of clarity. They were thus consistent with recesses and invaginations of the cell surface. Newly assembled fibrils were also found adjacent to discrete cell surface depressions and at sites of plasma membrane apposition between cells (Figure 2b). These observations suggest that participation of the cell surface is a feature of collagen assembly. We considered that the basis of cell-associated collagen polymerization could be the provision of a local microenvironment favorable to self-assembly or a more direct interaction that involved surface receptors. SMCs express α1β1 and α2β2 integrin-type collagen receptors.27Pickering JG Uniyal S Ford C Chau T Laurin MA Chow LH Ellis CG Fish J Chan BMC Fibroblast growth factor-2 potentiates vascular smooth muscle cell migration to platelet-derived growth factor: upregulation of α2β1 integrin and disassembly of actin filaments.Circ Res. 1997; 80: 627-637Crossref PubMed Scopus (70) Google Scholar Therefore, to test if SMC-associated collagen assembly was mediated by either of these collagen receptors, fluorescent collagen monomers were added to SMC cultures in the presence of specific anti-integrin antibodies. As illustrated in Figure 3, the extensive collagen fibril network that formed by adding Vitrogen to SMCs for 48 hours was reduced by BHA2.1, an anti-α2β1 integrin antibody. Similar inhibition was observed for rat tail collagen (data not shown). Fibril formation was however unaffected by a function-blocking antibody against α1β1 integrin. We reasoned that if α2β1 integrin mediates collagen fibril assembly, stimulating its function should enhance the process. This in fact was observed. Cultures incubated with JBS2, an α2β1 integrin-activating antibody that stimulates a high-affinity binding state and augments attachment to collagen,22Ho WC Heinemann C Hangan D Uniyal S Morris VL Chan BM Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver.Mol Biol Cell. 1997; 8: 1863-1875Crossref PubMed Scopus (41) Google Scholar showed greater fibril assembly. This accentuation was most apparent during early assembly (eg, 6 hours) (Figure 3, d and e). Another binding partner for collagen is fibronectin, and it has been recognized for several years that collagen fibrils can co-localize with fibronectin fibrils.28Furcht LT Smith D Wendelschafer-Crabb G Mosher DF Foidart JM Fibronectin presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization.J Histochem Cytochem. 1980; 28: 1319-1333Crossref PubMed Scopus (53) Google Scholar It is also known that fibronectin polymerization is dependent on interaction with cell-surface receptors.29Wu C Bauer JS Juliano RL McDonald JA The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.J Biol Chem. 1993; 268: 21883-21888Abstract Full Text PDF PubMed Google Scholar Therefore, to determine whether there was a relationship between the assembly of collagen on SMCs and assembly of fibronectin, we co-incubated SMC cultures with Texas Red-labeled soluble collagen and Oregon Green-labeled soluble fibronectin. As shown in Figure 4A, newly assembled collagen fibrils co-localized with newly assembled fibronectin fibrils. To ensure that this finding was not because of association of collagen and fibronectin precursors in solution, before contacting the cell surface, we performed experiments in which fibronectin was incubated with the cells for 1 hour, the cultures were extensively washed, and then labeled collagen was added for another 12 hours. Under these circumstances, fibril co-localization was consistently evident, although the fluorescent collagen fibrils tended to extend beyond the fluorescent regions of the fibronectin fibrils, in keeping with the brief incubation period of the latter. We also found that the early fibronectin formations were always in the form of linear fibrils, whereas the earliest collagen formations could be found as fine punctate accumulations (Figure 1c). We have previously shown that the assembly of fibronectin by human SMCs is dependent on α5β1 integrin.23Pickering JG Chow LH Li S Rogers KA Rocnik EF Zhong R Chan BM α5β1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.Am J Pathol. 2000; 156: 453-465Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Given the close physical association of actively assembling collagen and fibronectin fibrils, we sought to determine whether disrupting the α5β1 integrin-fibronectin interaction influenced collagen assembly. For this, we incubated SMCs with JBS5, an anti-α5β1 integrin antibody, together with fluorescent fibronectin and fluorescent collagen precursors. As shown in Figure 4b, JBS5 completed inhibited fibronectin assembly. Importantly, it also fully inhibited collagen fibril assembly. In some cells, a fine punctate collagen fluorescence could be seen, but fibril formation was not. We next determined if fibronectin assembly was influenced by α2β1 integrin, by incubating cultures with BHA2.1. Under these conditions, collagen fibril formation was reduced but fibronectin assembly was unaffected (Figure 4B). We observed that newly assembled collagen fibrils tended to orient along the long axis of the cell. As well, at lower cell densities human SMCs display a more irregular shape with thin projections, and in these circumstances collagen fibrils were found running along the length of these projections (Figure 5a). The relationship between collagen assembly and cell shape was further evaluated by staining the actin cytoskeleton with Texas Red phalloidin, 6 hours after incubating cells with FITC-labeled collagen monomers. This revealed newly assembled collagen fibrils that generally co-aligned with the underlying actin microfilament bundles (Figure 5b).