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.