Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells

Abstract

Abstract —We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II–generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0.2% calf serum for 48 hours. Immunofluorescence and protein levels of α-smooth muscle (SM) actin and expression of SM22α mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 μg/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II–like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II–generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-β1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.