Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

Yubing Yu,Yong Li,Huixin Peng,Qixue Song,Xingwen Da,Hui Li,Zuhan He,Xiang Ren,Chengqi Xu,Yufeng Yao,Qing K Wang

doi:10.1016/j.jbc.2022.101759

Abstract

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.

Highlights

Coronary artery disease (CAD) is the leading cause of death in the world, accounting for 15.9%of all types of death (1)
We previously reported that AGGF1 inhibited neointimal formation and restenosis after vascular injury by promoting phenotypic switching of vascular smooth muscle cells (VSMCs) from a synthetic state to the contractile state
Quantitative RT-PCR analysis showed that the expression of MYH11, ACTA2 and TAGLN was significantly upregulated in MOVAS-1 by treatment with wild type (WT) AGGF1 and deletion mutants AGGF1-C1 and AGGF1-C2 compared with control PBS, the effect got lost by deletion mutants AGGF1-C3 to AGGF1-C13 (Fig. S2A-S2C)

Summary

Introduction

Coronary artery disease (CAD) is the leading cause of death in the world, accounting for 15.9%. The restenosis is caused by neointimal formation after vascular injury, which is associated with the proliferation and migration of vascular smooth muscle cells (VSMCs) (4). The domain responsible for VSMC functions and neointimal formation as well as MEK, ERK1/2, and ELK signaling remains to be identified. We used deletion analysis to define an AGGF1 domain critical to VSMC phenotypic switching between amino acids 574-614, which contains a RDDAPAS motif with sequence homology to the RGD binding motif for integrins. We showed that AGGF1 used integrin 7, but not 5 or 8, as a receptor on VSMCs, and suppressed neointimal formation after vascular injury via interacting with integrin α7 and regulating the phenotypic switching, proliferation and migration of VSMCs through the MEK-ERK1/2-ELK signaling pathway. The results provide fundamental understanding of a novel therapy based on AGGF1 to block neointimal formation and restenosis after vascular injury

Results

DISCUSSION

Experimental procedures Plasmids