MFN2 Prevents Neointimal Hyperplasia in Vein Grafts via Destabilizing PFK1

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.

An Overview: The Diversified Role of Mitochondria in Cancer Metabolism

“…Overexpression of NNMT and knockdown of DNMT1 change OXPHOS-inhibition sensitive cell line to be resistant ones, indicating their energy resource reliance shifted from OXPHOS to glycolysis 61 . In the process of energy metabolic switch, the M2 isoform of pyruvate kinase (PKM2, a rate-limiting enzyme in glycolysis) interacts with the key regulator of mitochondrial fusion protein MFN2 to promote the mitochondria fusion and OXPHOS, meanwhile attenuate glycolysis 62 , consistently, the activation of MFN2 interacting with PFK1 mediate PFK1 degradation and therefore suppresses glycolysis 63 . In Hepatocellular carcinoma (HCC), inhibition of lysophosphatidic acid receptor 6 (LPAR6) overcomes sorafenib resistance by switching glycolysis into OXPHOS 64 .…”

Role of RhoA and Rho-associated kinase in phenotypic switching of vascular smooth muscle cells: Implications for vascular function

“…Likewise, SMCs were found to show distinct metabolic patterns when subjected to venous versus arterial mechanical stretches [186]. Indeed, mechanical stretch activates the Rho/ROCK signaling in venous SMCs, which is then followed by phosphorylation and activation of JNK.…”

LDHA maintains the growth and migration of vascular smooth muscle cells and promotes neointima formation via crosstalk between K5 crotonylation and K76 mono-ubiquitination

“…Since PTMs of major glycolytic enzymes were reported to play pivotal roles in coordinating glycolysis and mitochondrial metabolism, [24][25][26] we next set up to investigate whether PTMs of LDHA is involved in this effect. We first evaluated the relationship between LDHA and mitochondrial through immunofluorescence staining.…”

“…25 Mitofusin 2 (MFN2) prevents neointimal hyperplasia in vein grafts via decreasing phosphofructokinase 1 (PFK1) ubiquitination, which increases glycolysis and decreases oxidative phosphorylation. 26 Previous observation that targeting LDHA by shRNA stimulated oxidative phosphorylation of cancer cells provides a direct link between glycolysis and oxidative phosphorylation that involves LDHA. 27 Phosphorylation, acetylation and succinylation of LDHA were also demonstrated to be involved in the regulation of aerobic glycolysis in different type of tumors.…”