Hcy regulates SIRT1-STAT3 phosphorylation by upregulating WWP2 expression and induce VSMCs proliferation, migration, and phenotypic switch.

Abstract

Abstract The proliferation, migration, and cellular morphology of vascular smooth muscle cells (VSMCs) play important roles in the pathogenesis of atherosclerosis (AS). Homocysteine (Hcy) is a sulfur-containing amino acid, which is an intermediate product of methionine metabolism. Hcy can induce proliferation, migration, and phenotypic switch of VSMCs, but details of these mechanisms are still unclear. NAD-dependent deacetylase sirtuin-1 (SIRT1-STAT3) signaling pathway is involved in various cellular functions. Here, we sought to determine if this multifunctional pathway played a role in Hcy-induced proliferation, migration, and phenotypic transformation of VSMCs, which has not been previously reported. NEDD4-like E3 ubiquitin-protein ligase WWP2 (WWP2) is involved in VSMCs phenotypic modulation and can be a potential target in the treatment of various cardiovascular diseases. In our study, Serum Hcy of the ApoE-/-mice fed 2% high-methionine diet was significantly higher than that of the normal diet group (P < 0.01), and the serum Hcy concentrations exceeded 20 µmol/L, indicating that the HHcy animal model was successfully replicated. ApoE-/-+HMD mouse showed changes in the expression level of aortic proteins, α-SMA and SM22α were decreased (P < 0.05) in aortic VSMCs, while osteopontin (OPN) increased (P < 0.05). Similarly, WWP2 mRNA and protein expression were increased (P < 0.01) in the aortic vasculature of ApoE-/-+HMD. In vitro, WWP2 mRNA and protein expressions were higher in the Hcy group (P < 0.01), and the results were consistent with the animal model. Overexpression of WWP2 promoted Hcy-induced VSMCs proliferation, migration, and transformation from contractile to synthetic phenotype, while interference with WWP2 had the opposite effect. The protein expression of SIRT1 in the Hcy group was decreased (P < 0.01) and p-STAT3 increased (P < 0.05). Overexpression of WWP2 significantly decreased (P < 0.05) SIRT1 and increased (P < 0.05) p-STAT3, while the inhibition of WWP2 had the opposite effect. Inhibition of SIRT1 increased (P < 0.01) p-STAT3 in contrast to the inhibition (P < 0.01) of p-STAT3 following SIRT1 stimulation in the Hcy group. Taken together, the results of the present study indicate that WWP2 promotes Hcy-induced VSMC proliferation, migration, and phenotype switch by regulating SIRT1-STAT3 phosphorylation.