A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: Enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway

Abstract

Objective: Arsenic trioxide (ATO or As₂O₃) has beneficial effects on suppressing neointimal hyperplasia and restenosis, but the mechanism is still unclear. The goal of this study is to further understand the mechanism of ATO's inhibitory effect on vascular smooth muscle cells (VSMCs).

Methods and results: Through in vitro cell culture and in vivo stent implanting into the carotid arteries of rabbit, a synthetic-to-contractile phenotypic transition was induced and the proliferation of VSMCs was inhibited by ATO. F-actin filaments were clustered and the elasticity modulus was increased within the phenotypic modulation of VSMCs induced by ATO in vitro. Meanwhile, Yes-associated protein (YAP) nuclear translocation was inhibited by ATO both in vivo and in vitro. It was found that ROCK inhibitor or YAP inactivator could partially mask the phenotype modulation of ATO on VSMCs.

Conclusions: The interaction of YAP with the ROCK pathway through ATO seems to mediate the contractile phenotype of VSMCs. This provides an indication of the clinical therapeutic mechanism for the beneficial bioactive effect of ATO-drug eluting stent (AES) on in-stent restenosis (ISR).

Keywords: Arsenic trioxide (ATO); Bioactive; In-stent restenosis (ISR); Yes-associated protein (YAP).

Graphical abstract

Fig. 1

In vivo, AES inhibits in-stent restenosis and induces differentiation of smooth muscle cells in neointima. (A) Hematoxylin and eosin (HE) staining of vascular after stent implantation for 1 mo, scar bar = 200 μm. (B) The statistical results of neointima/media area ratio for HE staining after stent implantation for 1 mo, n = 3. (C) Immunofluorescent staining with α-SMA (green) after stent implantation for 1 mo, scar bar = 100 μm. (D) The statistical results of mean fluorescence intensity of α-SMA after stent implantation for 1 mo in neointima, n = 6. (E) Immunofluorescent staining with α-SMA (green) after stent implantation for and 3 mo, scar bar = 50 μm. (F) The statistical results of cell aspect ratio (cell long/short axis ratio) of VSMCs after stent implantation for 3 mo in neointima, n = 30. “L” for the lumen. BMS (bare metal stent), PMS (polymer coating-metal stent), and AES (arsenic trioxide-drug eluting stent); 1 mo (1 month) and 3 mo (3 months), “ns” means no significance, and P values < 0.01 (**), <0.001 (***) and <0.0001 (****).

Fig. 2

In vitro, arsenic trioxide induces phenotype modulation, and inhibits cell viability of synthetic phenotype VSMCs. (A) α-SMA immunostaining of A7r5 with or without ATO treating for 1 d. (B) The statistical results of mean fluorescence intensity of α-SMA in (A). (C) The statistical results of cell aspect ratio (cell long/short axis ratio) of A7r5 with or without ATO treating for 1 d. (D) and (E), Protein levels of SM22α and α-SMA were determined by WB after ATO treating for 1 d. (F) The extraction and identification of primary porcine coronary artery smooth muscle cells (PCASMCs), scar bar = 100 μm. (G) The proliferation profile of PCASMCs, scar bar = 100 μm (H) The schematic diagram for the phenotype induction of PCASMCs. (I) The viability of contractile- and synthetic-phenotype PCASMCs on different concentration of ATO for 1 and 3 d. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μM of ATO, respectively. “ns” means no significance, P values < 0.01 (**), <0.001 (***) and <0.0001 (****).

Fig. 3

RNA genome sequencing analysis of contractile and synthetic PCASMCs with or without ATO treating for 8 h, and qPCR validation of genes and signals. (A) The schematic diagram for the phenotype induction and ATO treating of PCSMCs. (B) Venn diagram. (C) Gene Ontology (GO) enrichment analysis. (D) and (E) are the statistical tables of changes in ATO treatment, related cell processes (D) and main gene expression (E) of synthetic PCASMCs (SH2 vs SH0). (F) qPCR of α-SMA, Calponin, RhoA, ROCK and YAP for A7r5. SS0: contractile PCASMCs without ATO treatment; SS2: contractile PCASMCs treated with 2 μM ATO for 8 h; SH0: synthetic PCASMCs without ATO treatment; and SH2: synthetic PCASMCs treated with 2 μM ATO for 8 h. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μM of ATO, respectively.

Fig. 4

ATO induces actin cytoskeleton organization and mechanical changes in VSMCs in vitro. (A) Immunostaining for cytoskeleton F-actin of A7r5 with or without ATO treating for 1 d, scar bar = 50 μm. (B) The statistical analysis of cell area. (C) Immunostaining for F-actin of A7r5 with or without Y27632 treating for 4 h, then treating ATO for 1 d, scar bar = 200 μm. The statistical analysis of fluorescence intensity (D) and cell area (E). (F) Young's modulus determined by atomic force microscope for A7r5 treating ATO for 1 d. (G) Young's modulus determined by atomic force microscope for A7r5 with Y27632 treating for 4 h, then treating ATO for 1 d. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μM of ATO respectively; “ns” means no significance, P values < 0.05 (*) and <0.0001 (****).

Fig. 5

ATO induces VSMCs phenotype modulation. (A) Immunostaining of YAP after ATO treating A7r5 for 1 d, scar bar = 20 μm. (B) Immunostaining for YAP in cross-sections of carotid arteries post-implanting stent for 1 week. (C) Protein expression levels of YAP, SM22α and Osteopontin (OPN) were determined by WB after ATO treating for 1 d. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μM of ATO respectively; BMS (bare metal stent), PMS (polymer coating-metal stent), and AES (arsenic trioxide-drug eluting stent); P values < 0.05 (*), <0.01 (**) and <0.001 (***).

Fig. 6

The activity of YAP is associated with ATO regulating the phenotype transformation of VSMCs in vitro. (A) Immunofluorescence co-staining of F-actin and p-YAP after ATO treating A7r5 for 1 d, scar bar = 20 μm. (B) The fluorescence intensity of F-actin and p-YAP. (C) VSMCs (A7r5 cell lines) were treated with control or ATO for 1 d with or without ROCK inhibitor Y27632 and WB experiments for indicated the protein levels of p-YAP and SM22α were performed. (D) VSMCs (A7r5 cell lines) were treated with control or ATO for 1 d with or without YAP inactivator verteporfin and WB experiments for indicated the protein levels of p-YAP and SM22α were performed. (E) Working hypothesis which elaborates role of YAP and ROCK in ATO modulation the differentiation of VSMC. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μM of ATO respectively; “ns” means no significance, P values < 0.05 (*), <0.01 (**), <0.001 (***) and <0.001 (****).