BACKGROUND

Endothelial-to-mesenchymal transition (EndMT) is the process by which endothelial cells lose their specific markers and acquire mesenchymal or myofibroblastic phenotypes. Studies have demonstrated the importance of endothelial-derived vascular smooth muscle cells (VSMCs) through EndMT in neointimal hyperplasia. Histone deacetylases (HDACs) are epigenetic modification enzymes involved in the epigenetic control of important cellular functions. Recent studies found that HDAC3, a class I HDAC, causes posttranslational modifications, including deacetylation and decrotonylation. However, the effect of HDAC3 on EndMT in neointimal hyperplasia via posttranslational modifications remains to be seen. Therefore, we investigated the effects of HDAC3 on EndMT in carotid artery-ligated mice and human umbilical vein endothelial cells (HUVECs) and the underlying posttranslational modifications.

METHODS

HUVECs were treated with transforming growth factor (TGF)-β1 or the inflammatory cytokine tumor necrosis factor (TNF)-α at different concentrations and durations. In HUVECs, HDAC3 expression, the expression of endothelial and mesenchymal markers, and posttranslational modifications were analyzed with Western blotting, quantitative real-time polymerase chain reaction (PCR), and immunofluorescence. C57BL/6 mice underwent left carotid artery ligation. Mice were treated with the HDAC3-selective inhibitor RGFP966 (10 mg/kg, i.p.) from 1 day before to 14 days after ligation. Then, the sections of the carotid arteries were examined histologically using hematoxylin and eosin (HE) and immunofluorescence staining. The carotid arteries from other mice were examined for the expression of EndMT markers and inflammatory cytokines. Furthermore, the acetylation and crotonylation of carotid arteries were immunostained in mice.

RESULTS

In HUVECs, TGF-β1 and TNF-α induced EndMT by decreasing CD31 expression and increasing α-smooth muscle actin expression. TGF-β1 and TNF-α also upregulated HDAC3 expression in HUVECs. The study in mice indicated that RGFP966 significantly alleviated neointimal hyperplasia of the carotid artery compared with vehicle treatment. Furthermore, RGFP966 suppressed EndMT and the inflammatory response in carotid artery-ligated mice. Further investigation revealed that HDAC3 regulated EndMT by posttranslational modifications of deacetylation and decrotonylation.

CONCLUSIONS

These results suggest that HDAC3 regulates EndMT in neointimal hyperplasia through posttranslational modifications.