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To provide a theoretical basis for the prevention and treatment of atherosclerosis (AS), the current study aimed to investigate the mechanism underlying the effect of homocysteine (Hcy) on regulating the proliferation, migration and phenotypic transformation of vascular smooth muscle cells (VSMC) via sirtuin-1 (SIRT1)/signal transducer and activator of transcription 3 (STAT3) through Nedd4-like E3 ubiquitin-protein ligase WWP2 (WWP2). Here, Based on the establishment of ApoE-/- mouse models of high Hcy As and the model of Hcy stimulation of VSMC in vitro to observe the interaction between WWP2 and STAT3 and its effect on the proliferation, migration, and phenotypic transformation of Hcy-induced VSMC, which has not been previously reported. This study revealed that WWP2 could promote the proliferation, migration, and phenotype switch of Hcy-induced VSMC by up-regulating the phosphorylation of SIRT1/STAT3 signaling. Furthermore, Hcy might up-regulate WWP2 expression by inhibiting histone H3K27me3 expression through up-regulated UTX. These data suggest that WWP2 is a novel and important regulator of Hcy-induced VSMC proliferation, migration, and phenotypic transformation.</div

WWP2 inhibited SIRT1 and up-regulated STAT3 phosphorylation.

A. Immunofluorescence staining. The photographs are representative of three separate experiments. Blue fluorescence is the nucleus of the vascular smooth muscle cell (DAPI), and green and red fluorescence is the target protein (40×). B. Co-localization of α-SMA with SIRT1 and p-STAT3 in mice aortic VSMC, respectively. C. SIRT1 mRNA expression in mice aortic VSMC. D. SIRT1 protein expression in mice aortic VSMC. E. p-STAT3 protein expression in mice aortic VSMC. F. SIRT1 protein expression in Hcy-induced VSMC. G. p-STAT3 and STAT3 protein expression in Hcy-induced VSMC. H. The effects of the Ex527 and SRT1720 on p-STAT3 and STAT3 protein expression in Hcy-induced VSMC. The experiment was performed in triplicate, and the representative images are shown.*PP#P<0.05. compared with the Hcy group.</p

Hcy increased the expression of WWP2 in VSMC <i>in vivo and in vitro</i>.

A. WWP2 mRNA and protein expression in mice aortic VSMC. B. WWP2 mRNA and protein expression in VSMC. The experiment was performed in triplicate, and the representative images are shown.**P-/-+NC group and control group.</p

Sequence information of primers used in RT-PCR.

To provide a theoretical basis for the prevention and treatment of atherosclerosis (AS), the current study aimed to investigate the mechanism underlying the effect of homocysteine (Hcy) on regulating the proliferation, migration and phenotypic transformation of vascular smooth muscle cells (VSMC) via sirtuin-1 (SIRT1)/signal transducer and activator of transcription 3 (STAT3) through Nedd4-like E3 ubiquitin-protein ligase WWP2 (WWP2). Here, Based on the establishment of ApoE-/- mouse models of high Hcy As and the model of Hcy stimulation of VSMC in vitro to observe the interaction between WWP2 and STAT3 and its effect on the proliferation, migration, and phenotypic transformation of Hcy-induced VSMC, which has not been previously reported. This study revealed that WWP2 could promote the proliferation, migration, and phenotype switch of Hcy-induced VSMC by up-regulating the phosphorylation of SIRT1/STAT3 signaling. Furthermore, Hcy might up-regulate WWP2 expression by inhibiting histone H3K27me3 expression through up-regulated UTX. These data suggest that WWP2 is a novel and important regulator of Hcy-induced VSMC proliferation, migration, and phenotypic transformation.</div

Hcy up-regulates WWP2 expression by inhibiting histone H3K27me3 via up-regulated UTX.

A. UTX mRNA expression in mice aortic VSMC. B. UTX protein expression in mice aortic VSMC. C. H3K27me3 protein expression in mice aortic VSMC. D. UTX protein expression in Hcy-induced VSMC. E. H3K27me3 protein expression in Hcy-induced VSMC. The experiment was performed in triplicate, and the representative images are shown.*P#P%P&P<0.05 compared with the Hcy+WWP2 group.</p

Overexpression of WWP2 promoted Hcy-induced proliferation, migration, and phenotypic transformation of VSMC.

A. The transfection efficiency of WWP2 overexpression and interference plasmid was determined by western blot. B. Cell viability assessed using MTT assay. C. Quantification of results from EdU assays. D. Results of the scratch test. E. EdU staining diagram. Red fluorescence refers to nuclei in the proliferative period and blue fluorescence refers to nuclei in the non-proliferative period. The cell proliferation rate for each group was calculated by dividing the number of red nuclei by blue nuclei. F. Cell scratch test at 0h and 48h. G. qRT-PCR was used to detect the mRNA expressions of a-SMA, SM22a and OPN. H. Western blot was used to detect the protein expressions of a-SMA, SM22a and OPN. The experiment was performed in triplicate, and the representative images are shown.*PP# P## P<0.01 compared with the Hcy group.</p

Hcy promoted the transformation of mice aortic VSMC from contractile to secretory phenotype.

A-C. Changes of serum Hcy, TC and TG levels. D. Immunofluorescence staining methods were used to detect the α-SMA protein levels in ApoE-/- mice aortic root frozen sections. E-F. Co-localization of α-SMA with SM22a and OPN in mice aortic VSMC, respectively. G. Immunofluorescence staining. The photographs are representative of three separate experiments. Blue fluorescence is the nucleus of the vascular smooth muscle cell (DAPI), and green and red fluorescence is the target protein (40×). H-J. Western blot analysis was used to detect the α-SMA, SM22a and OPN protein levels in ApoE-/- mice aortic VSMC. The experiment was performed in triplicate, and the representative images are shown. *PP-/-+NC group.</p