Cyclic strain induces vascular smooth muscle cell differentiation from murine embryonic mesenchymal progenitor cells

Abstract

Background: Hemodynamic forces play a crucial role in regulating vascular cell phenotype. However, the underlying molecular mechanisms are largely unknown. The objective of this study was to test our hypothesis that cyclic strain could affect smooth muscle cell (SMC) differentiation.

Methods: A murine embryonic mesenchymal progenitor cell line (C3H/10T1/2) was cultured with or without cyclic strain for 6 days. Changes in cell morphology were studied with fluorescence dye Calcein-AM staining. Expression of specific SMC markers, smooth muscle specific alpha-actin (alpha-SMA), and smooth muscle myosin heavy chain (SMMHC), was determined by real-time polymerase chain reaction (PCR) and Western blot. Transforming growth factor- beta (TGF-beta) was used as a positive control.

Results: With cyclic strain, CH3/10T1/2 cells demonstrated spindle-shaped morphology and parallel alignment. Cells exposed to cyclic strain illustrated significantly increased mRNA levels of alpha-SMA and SMMHC by 3- and 2-fold, respectively, compared with static cells (P<.05). In addition, cells cultured under cyclic strain with TGF-beta (2 ng/ml) supplementation demonstrated increased mRNA levels of alpha-SMA and SMMHC by 10- and 2-fold, respectively, compared with static cells (P<.05). Furthermore, protein levels of alpha-SMA and SMMHC were also significantly increased by more than 3-fold in cyclic strain-treated cells compared with static cultures (P<.05). TGF-beta synergistically enhanced the effect of cyclic strain on alpha-SMA mRNA expression in CH3/10T1/2 cells.

Conclusions: This is the first study to demonstrate that cyclic strain significantly induces expression of two of the most important SMC markers in a murine embryonic mesenchymal progenitor cell line. Cyclic strain and TGF-beta have a synergistic effect on alpha-SMA mRNA expression.