MFN2 Prevents Neointimal Hyperplasia in Vein Grafts via Destabilizing PFK1

Yuanjun Tang, Yiting Jia, Linwei Fan, Han Liu, Yuan Zhou, Miao Wang, Yuefeng Liu, Juanjuan Zhu, Wei Pang, and Jing Zhou

Volume 130, Number 11

Abstract

Background:

Mechanical forces play crucial roles in neointimal hyperplasia after vein grafting; yet, our understanding of their influences on vascular smooth muscle cell (VSMC) activation remains rudimentary.

Methods:

A cuff mouse model was used to study vein graft hyperplasia. Fifteen percent to 1 Hz uniaxial cyclic stretch (arterial strain), 5% to 1 Hz uniaxial cyclic stretch or a static condition (venous strain) were applied to the cultured VSMCs. Metabolomics analysis, cell proliferation and migration assays, immunoblotting, co-immunoprecipitation, mutagenesis, pull-down and surface plasmon resonance assays were employed to elucidate the potential molecular mechanisms.

Results:

RNA-sequencing in vein grafts and the controls identified changes in metabolic pathways and downregulation of mitochondrial protein MFN2 (mitofusin 2) in the vein grafts. Exposure of VSMCs to 15% stretch resulted in MFN2 downregulation, mitochondrial fragmentation, metabolic shift from mitochondrial oxidative phosphorylation to glycolysis, and cell proliferation and migration, as compared with that to a static condition or 5% stretch. Metabolomics analysis indicated an increased generation of fructose 1,6-bisphosphate, an intermediate in the glycolytic pathway converted by PFK1 (phosphofructokinase 1) from fructose-6-phosphate, in cells exposed to 15% stretch. Mechanistic study revealed that MFN2 physically interacts through its C-terminus with PFK1. MFN2 knockdown or exposure of cells to 15% stretch promoted stabilization of PFK1, likely through interfering the association between PFK1 and the E3 ubiquitin ligase TRIM21 (E3 ubiquitin ligase tripartite motif [TRIM]-containing protein 21), thus, decreasing the ubiquitin-protease-dependent PFK1 degradation. In addition, study of mechanotransduction utilizing pharmaceutical inhibition indicated that the MFN2 downregulation by 15% stretch was dependent on inactivation of the SP1 (specificity protein 1) and activation of the JNK (c-Jun N-terminal kinase) and ROCK (Rho-associated protein kinase). Adenovirus-mediated MFN2 overexpression or pharmaceutical inhibition of PFK1 suppressed the 15% stretch-induced VSMC proliferation and migration and alleviated neointimal hyperplasia in vein grafts.

Conclusions:

MFN2 is a mechanoresponsive protein that interacts with PFK1 to mediate PFK1 degradation and therefore suppresses glycolysis in VSMCs.

Meet the First Author, see p 1645

Coronary artery bypass grafting that restores normal blood flow to the heart by creating vascular access around the blocked arteries has been the gold standard for the treatment of 3-vessel or left main coronary artery disease.¹ Autologous saphenous vein is commonly used for grafting; however, it undergoes adverse remodeling within days after implantation and develops obliterative neointimal hyperplasia that ultimately leads to vein graft (VG) occlusion.² Vascular smooth muscle cell (VSMC) activation in the form of excessive proliferation, migration, and apoptosis has been demonstrated in occluded VGs.^3–5 One prominent factor that causes VG VSMC activation is the arterial blood pressure-induced mechanical cyclic stretch,^6–9 which is defined as repetitive deformation of vascular wall rhythmical distending and relaxing during the cardiac cycle.¹⁰ Once implanted into arterial pressures reaching values >120 mm Hg, the VGs derived from veins with pressures ranging from 0 to 30 mm Hg are subjected to sudden increases in cyclic stretch, which has been hypothesized and demonstrated to induce behavioral changes of VSMC.^11,12