Coptisine inhibits neointimal hyperplasia through attenuating Pak1/Pak2 signaling in vascular smooth muscle cells without retardation of re-endothelialization

Abstract

Background and aims

Vascular injury-induced endothelium-denudation and profound vascular smooth muscle cells (VSMCs) proliferation and dis-regulated apoptosis lead to post-angioplasty restenosis. Coptisine (CTS), an isoquinoline alkaloid, has multiple beneficial effects on the cardiovascular system. Recent studies identified it selectively inhibits VSMCs proliferation. However, its effects on neointimal hyperplasia, re-endothelialization, and the underlying mechanisms are still unclear.

Methods

Cell viability was assayed by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and cell counting kit-8 (CCK-8). Cell proliferation and apoptosis were measured by flow cytometry and immunofluorescence of Ki67 and TUNEL. Quantitative phosphoproteomics (QPP) was employed to screen CTS-responsive phosphor-sites in the key regulators of cell proliferation and apoptosis. Neointimal hyperplasia was induced by balloon injury of rat left carotid artery (LCA). Adenoviral gene transfer was conducted in both cultured cells and LCA. Re-endothelialization was evaluated by Evan's blue staining of LCA.

Results

1) CTS had strong anti-proliferative and pro-apoptotic effects in cultured rat VSMCs, with the EC₅₀ 4∼10-folds lower than that in endothelial cells (ECs). 2) Rats administered with CTS, either locally to LCA's periadventitial space or orally, demonstrated a potently inhibited balloon injury-induced neointimal hyperplasia, but had no delaying effect on re-endothelialization. 3) The QPP results revealed that the phosphorylation levels of Pak1^S144/S203, Pak2^S20/S197, Erk1^T202/Y204, Erk2^T185/Y187, and Bad^S136 were significantly decreased in VSMCs by CTS. 4) Adenoviral expression of phosphomimetic mutants Pak1^D144/D203/Pak2^D20/D197 enhanced Pak1/2 activities, stimulated the downstream pErk1^T202/Y204/pErk2^T185/Y187/pErk3^S189/pBad^S136, attenuated CTS-mediated inhibition of VSMCs proliferation and promotion of apoptosis in vitro, and potentiated neointimal hyperplasia in vivo. 5) Adenoviral expression of phosphoresistant mutants Pak1^A144/A203/Pak2^A20/A197 inactivated Pak1/2 and totally simulated the inhibitory effects of CTS on platelet-derived growth factor (PDGF)-stimulated VSMCs proliferation and PDGF-inhibited apoptosis in vitro and neointimal hyperplasia in vivo. 6) LCA injury significantly enhanced the endogenous phosphorylation levels of all but pBad^S136. CTS markedly attenuated all the enhanced levels.

Conclusions

These results indicate that CTS is a promising medicine for prevention of post-angioplasty restenosis without adverse impact on re-endothelialization. CTS-directed suppression of pPak1^S144/S203/pPak2^S20/S197 and the subsequent effects on downstream pErk1^T202/Y204/pErk2^T185/Y187/pErk3^S189 and pBad^S136 underline its mechanisms of inhibition of VSMCs proliferation and stimulation of apoptosis. Therefore, the phosphor-sites of Pak1^S144/S203/Pak2^S20/S197 constitute a potential drug-screening target for fighting neointimal hyperplasia restenosis.