Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha2beta1 integrin, RhoA, and fibronectin polymerization

Abstract

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-alpha2beta1 integrin antibody and accelerated by an alpha2beta1 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-alpha5beta1 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.

Figure 1.

SMCs stimulate collagen fibril assembly. In vitro collagen assembly was performed by incubating Texas Red-labeled, acid-solubilized collagen at 37°C in culture medium (M199 plus 10% fetal bovine serum, pH 7.4). Fibrils that formed and attached to the substrate were fixed in paraformaldehyde and imaged microscopically. a: Spontaneously assembled fibrils that formed and adhered to a fibronectin-coated coverslip after incubating 10 μg/ml of Texas Red-Vitrogen collagen in cell-free culture media for 3 hours at 37°C. b: Fibrils are not evident when 2 μg/ml of Texas Red-collagen is incubated with culture medium, indicating this concentration to be below the critical threshold for self-assembly under these conditions. c and d: Confocal microscopic images acquired 1 hour (c) and 3 hours (d) after addition of 1 μg/ml of Texas Red-labeled Vitrogen collagen to human SMCs. Punctate accumulations of fluorescence are evident initially, followed by small fibrils on the surface of SMCs. e: Lower magnification image (nonconfocal) 24 hours after addition of soluble collagen to SMCs, showing extensive fibril formation. f: Cell-associated fibrils also formed after the addition of 1 μg/ml of Texas Red-labeled rat-tail collagen to SMC cultures. Nuclei were counterstained with Hoechst 33258. Scale bars, 50 μm.

Figure 2.

Native collagen fibrils assemble in human SMC cultures. Electron micrographs of human SMCs, cultured in ascorbate-deficient media, 24 hours after the addition of soluble, bovine skin collagen. Cross-striated collagen fibrils are apparent near the cell surface (a and at higher magnification in inset). Fibrils can also be seen in membrane-delimited cellular invaginations (arrows). b: Collection of fibrils seen in cross-section within a cell-surface depression at a zone of apposition with an adjacent cell. Scale bar, 500 nm (a).

Figure 3.

SMC-associated collagen assembly is mediated by α2β1 integrin. Human SMCs were incubated for 48 hours with solubilized FITC-labeled collagen (2 μg/ml) and either control IgG (a), an anti-α1β1 integrin antibody (5E8D9, 20 μg/ml) (b), or an anti-α2β1 integrin antibody (BHA2.1, 20 μg/ml) (c). Antibodies were added 30 minutes before addition of FITC-collagen monomers. Fibril assembly was also evaluated in cultures preincubated for 30 minutes with anti-α2β1 integrin-stimulating antibody (JBS2, 1:50) and then incubated with Texas Red-labeled collagen (1 μg/ml) for 6 hours. Compared to control conditions (d), the fibers in JBS2-treated cultures were more abundant and straighter (e). Washed cultures were fixed in paraformaldehyde and nuclei were stained with Hoechst 33258. Scale bars, 30 μm.

Figure 4.

SMC-directed collagen assembly is integrated with α5β1 integrin-mediated fibronectin assembly. A: Human SMCs co-incubated for 16 hours with Oregon Green-labeled fibronectin and Texas Red-labeled bovine skin collagen showing co-localization of assembling fibrils. B: SMCs co-incubated with Oregon Green-labeled fibronectin and Texas Red-labeled collagen together with control IgG, anti-α5β1 integrin antibody (JBS5, 20 μg/ml), or anti-α2β1 integrin antibody (BHA2.1, 20 μg/ml). Nuclei were stained with Hoechst 33258. Scale bar, 40 μm.

Figure 5.

SMC-directed collagen assembly is dependent on the actin cytoskeleton. a: Collagen fibrils formed over the stretched tail of human SMCs (arrow) 6 hours after addition of soluble, Texas Red-labeled rat tail collagen (1 μg/ml) to cultures (overlay of phase contrast with fluorescence image). b: Six hours after addition of soluble, FITC-labeled bovine skin collagen (2 μg/ml) to cultures, early fibrils can be seen oriented parallel to the actin microfilament bundles, the latter stained using Texas Red-labeled phalloidin. c and d: Collagen assembly was evaluated in SMC cultures incubated for 24 hours with FITC-collagen and either DMSO (c) or cytochalasin D (10 μmol/L) (d), both added 1 hour before addition of solubilized collagen. Nuclei were counterstained with Hoechst 33258. Scale bars, 30 μm.

Figure 6.

SMC-directed collagen assembly is mediated by RhoA. Human arterial SMCs were infected with retrovirus containing empty vector (pLNCX, a), cDNA for dominant-negative RhoA (pLNCX-T19N RhoA, b), or cDNA for constitutively active RhoA (pLNCX-N63L RhoA, c). Transductants were selected by G418 treatment and then incubated with FITC-labeled collagen (2 μg/ml) for 24 hours. Collagen fibrils are reduced on SMCs expressing dominant-negative RhoA and extensive on SMCs expressing constitutively active RhoA. Nuclei were counterstained with Hoechst 33258. Scale bars, 50 μm.

Figure 7.

LPA stimulates SMC-directed collagen assembly. SMCs were incubated for 12 hours with 2 μg/ml of FITC-labeled soluble collagen and either vehicle (a, b) or LPA (20 μmol/L) (c, d), added 30 minutes before addition of collagen. Cultures were fixed in 4% paraformaldehyde and nuclei stained with Hoechst 33258. Cultures were also stained with Texas Red-labeled phalloidin to show actin microfilament bundles (b and d, control and LPA-treated, respectively). The Texas Red fluorescence images were acquired with identical exposure times. Scale bars, 50 μm.