Silencing of PCK1 mitigates the proliferation and migration of vascular smooth muscle cells and vascular intimal hyperplasia by suppressing STAT3/DRP1-mediated mitochondrial fission

Abstract

The pathological proliferation and migration of vascular smooth muscle cells (VSMCs) are key processes during vascular neointimal hyperplasia (NIH) and restenosis. Phosphoenolpyruvate carboxy kinase 1 (PCK1) is closely related to a variety of malignant proliferative diseases. However, the role of PCK1 in VSMCs has rarely been investigated. This study aims to examine the role of PCK1 in the proliferation and migration of VSMCs and vascular NIH after injury. In vivo, extensive NIH and increased expression of PCK1 within the neointima are observed in injured arteries. Interestingly, the administration of adeno-associated virus-9 (AAV-9) carrying Pck1 short hairpin RNA (shPck1) significantly attenuates NIH and stenosis of the vascular lumen. In vitro, Pck1 small interfering RNA (siPck1)-induced PCK1 silencing inhibits VSMC proliferation and migration. Additionally, silencing of PCK1 leads to reduced expression of dynamin-related protein 1 (DRP1) and attenuated mitochondrial fission. Lentivirus-mediated DRP1 overexpression markedly reverses the inhibitory effects of PCK1 silencing on VSMC proliferation, migration, and mitochondrial fission. Finally, PCK1 inhibition attenuates the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Activation of STAT3 abolishes the suppressive effects of PCK1 silencing on DRP1 expression, mitochondrial fission, proliferation, and migration in VSMCs. In conclusion, PCK1 inhibition attenuates the mitochondrial fission, proliferation, and migration of VSMCs by inhibiting the STAT3/DRP1 axis, thereby suppressing vascular NIH and restenosis.

1. Introduction

Cardiovascular diseases (CVDs) are the leading cause of death worldwide [1]. As one of the most important treatment methods, percutaneous coronary intervention (PCI) significantly reduces the mortality of coronary heart disease (CHD) [2]. However, mechanical injury initiates a series of pathological processes, causing the development of neointimal hyperplasia (NIH) and ultimately leading to vascular restenosis [3]. The main pathophysiological processes of NIH include endothelial cell damage and dysfunction, inflammatory cell infiltration, the subsequent migration and proliferation of VSMCs and vascular adventitial fibroblasts, and the accumulation of the extracellular matrix (ECM) [4–6]. The abnormal proliferation and migration of VSMCs, the major component of vessels, is one of the most critical pathophysiological mechanisms during NIH [7]. Drug-eluting stents (DESs) that target cell proliferation significantly reduce the rates of vascular restenosis and revascularization. However, its incidence remains as high as 4%–8% [8]. DES not only inhibits cell proliferation but also inhibits vascular re-endothelialization, resulting in a longer duration of DES-related NIH than of bare metal stent (BMS)-related NIH [9]. Therefore, identifying new therapeutic targets and developing new drug stents or balloons are crucial for preventing restenosis.

PCK1 was originally identified as a key rate-limiting enzyme of gluconeogenesis. Furthermore, PCK1 is highly enriched in the liver, kidney, intestine, and adipose tissue and regulates the production of glucose, fatty acid re-esterification and citric acid cyclic anions [10–12]. It is closely related to the development of diabetes, obesity, cardiovascular disease, and tumors [12,13]. In addition, PCK1 is involved in the biosynthesis of serine, an amino acid that is the major substrate for one-carbon metabolism; is vital for nucleotide synthesis, methylation, and antioxidation; and is associated with the migration and proliferation of tumor cells [14,15]. Moreover, PCK1 also promotes protein kinase activity to activate sterol regulatory element binding proteins (SREBPs), resulting in the transcription of lipogenesis genes and promoting the rapid proliferation of tumor cells [16]. Therefore, PCK1 can promote the development of various malignant proliferative diseases, such as non-small cell lung cancer, breast cancer, colon cancer, and hepatocellular carcinoma [11,17,18]. Moreover, PCK1 is involved in vascular endothelial function. In a recent study, PCK1 shRNA was found to inhibit the proliferation and migration and decrease the number of tube-like structures and average sprouting length in human umbilical vein endothelial cells. These characteristics indicate that silencing of PCK1 results in antiangiogenic activity through mediating AKT inactivation and subsequent Gαi3 downregulation [19]. Additionally, in vivo knockdown of PCK1 disru