Abstract
Vascular endothelial cell growth factor A (VEGF-A) signaling promotes the endothelial cell proliferation, macrophage infiltration and foam cell formation, which play pivotal roles in the pathogenesis of atherosclerosis (AS). However, the role of alternative splicing of VEGF here is not known. Here, ApoE (-/-) mice supplied high-fat diet (HFD mice) were used to generate AS, while ApoE (-/-) mice supplied with normal diet (NOR mice) were used as a control. Aortic endothelial cells (AECs) and infiltrated macrophages were purified and quantified by flow cytometry. Alternative splicing of VEGF and the regulator of VEGF splicing, SRPK1, were assessed by RT-qPCR and immunoblotting in both AECs and aortic macrophages. We found that HFD mice developed AS in 12 weeks, while the NOR did not. Compared to NOR mice, HFD mice possessed significantly more AECs and AEC proliferation, and had significantly more aortic infiltrated macrophages and more apoptosis of them. Significant shift of VEGF-A splicing to pro-angiogenic VEGF165 was detected in both AECs and macrophages from HFD mice, seemingly through upregulation of SRPK1. In vitro, SRPK1 overexpression significantly increased EC proliferation and macrophage apoptosis. Thus, our data suggest that alternative splicing of VEGF-A to pro-angiogenic VEGF165 may contribute to the development of AS.
Highlights
As the primary cause of heart disease and stroke, atherosclerosis (AS) is characterized with a gradual deposition of lipids and fibrous elements in the arterial wall, due to a chronic inflammatory response to the intramural retention of cholesterol-rich, apolipoprotein B‐containing lipoproteins [1]
Our results suggest that alternative splicing of Vascular endothelial growth factor A (VEGF-A) to pro-angiogenic VEGF165 may contribute to the development of AS
Apolipoprotein E (ApoE) (-/-) mice were fed with High-fat diet (HFD; simplified as High fat diet (HFD) mice) for 12 weeks to induce experimental AS, which was confirmed by increases in plaque area (Figure 1A), by increases in circumferential plaque extension (Figure 1B) and by increases in maximal plaque thickness (Figure 1C), compared to the control littermate ApoE (-/-) mice that had received normal diet (NOR)
Summary
As the primary cause of heart disease and stroke, atherosclerosis (AS) is characterized with a gradual deposition of lipids and fibrous elements in the arterial wall, due to a chronic inflammatory response to the intramural retention of cholesterol-rich, apolipoprotein B‐containing lipoproteins [1]. It is well accepted that VEGF-A signaling regulates the endothelial cell proliferation, macrophage infiltration and foam cell formation, all of which play pivotal roles in the AS pathogenesis, the details of the molecular regulation may be complicated due to complexity of the regulation of VEGF-A signaling. Proximal splice site selection in exon 8 of VEGFA produces pro-angiogenic VEGF165, whereas distal splice site selection in exon 8 of VEGF-A generates anti-angiogenic, cytoprotective VEGF165b [5]. The balance of endogenous pro/anti-angiogenic VEGF-A splice isoforms is regulated by serine/ arginine-rich splicing factor protein kinase 1 (SRPK1) [9,10,11]. SRPK1 phosphorylates serine/arginine-rich splicing factor 1 (SRSF1), resulting in its binding to the VEGF-A mRNA and production of VEGF165 [11]. A switch in VEGF-A mRNA splicing to VEGF165 has been associated with cancer-associated neovascularization and metastasis [12,13,14], a role of alternative splicing of VEGF-A in the pathogenesis of AS is not known
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