Liuwei Dihuang, a traditional Chinese medicinal formula, inhibits proliferation and migration of vascular smooth muscle cells via modulation of estrogen receptors

Abstract

The phenotypic modulation of vascular smooth muscle cells (VSMC s) serves an important role in atherosclerosis-induced vascular alterations, including vascular remodeling. However, the precise mechanisms underlying VSMC phenotypic modulation remain to be elucidated. Our previous study demonstrated that Liuwei Dihuang formula (LWDHF) could improve menopausal atherosclerosis by upregulating the expression of estrogen receptors (ER s). The present study examined the role of ER s in the effects of LWDHF on VSMC phenotypic modulation. VSMC proliferation and cell cycle progression were examined by MTT assay and flow cytometry, respectively. The expression levels of α-smooth muscle actin, osteopontin and ER s were determined using reverse transcription-quantitative polymerase chain reaction (RT- qPCR ) and western blot analysis. Cell ultrastructure was observed under an electron microscope. F- actin polymerization was detected by fluorescein isothiocyanate-phalloidin staining using fluorescence microscopy. A modified Boyden chamber assay was employed to assess VSMC s migration. Small interfering (si)RNA technology was used to examine the role of ER α in the effects of LWDHF on phenotypic modulation. The results indicated that LWDHF (3-12 μg/ml) inhibited proliferation and induced a cell cycle arrest in VSMC s treated with angiotensin II (Ang II ; 100 nM) in a concentration-dependent manner. In addition, Ang II- stimulated migration of VSMC s and reorganization of actin were markedly inhibited by treatment with 12 μg/ml LWDHF. Results of RT- qPCR and western blotting demonstrated that LWDHF markedly stimulated transcription and expression of ER α and ER β, and inhibited VSMC synthetic phenotype. Furthermore, LWDHF- induced inhibition of phenotypic switching was partially suppressed by tamoxifen, and transfection with ER α siRNA markedly abolished the effects of LWDHF on VSMC phenotypic switching. In conclusion, these results revealed that ER α served an important role in LWDHF- induced regulation of the VSMC phenotype, including proliferation and migration.