Myocardin suppression increases lipid retention and atherosclerosis via downregulation of ABCA1 in vascular smooth muscle cells

Highlights

• MYOCD and ABCA1 expressions are decreased in aortic atherosclerosis patients.

• MYOCD regulates ABCA1 expression in an SRF-dependent manner in VSMCs.

• MYOCD suppression exacerbates atherosclerosis in rodent model.

Abstract

Myocardin (MYOCD) plays an important role in cardiovascular disease. However, its underlying impact on atherosclerosis remains to be elucidated. ATP binding cassette transporter A1 (ABCA1), a key membrane-associated lipid transporter which maintains intracellular lipid homeostasis, has a protective function in atherosclerosis progress. The purpose of this study was to investigate whether and how the effect of MYOCD on atherosclerosis is associated with ABCA1 in vascular smooth muscle cells (VSMCs). We found both MYOCD and ABCA1 expression were dramatically decreased in atherosclerotic patient aortas compared to control. MYOCD knockdown inhibited ABCA1 expression in human aortic vascular smooth muscle cells (HAVSMCs), leading to reduced cholesterol efflux and increased intracellular cholesterol contents. MYOCD overexpression exerted the opposite effect. Mechanistically, MYOCD regulates ABCA1 expression in an SRF-dependent manner. Consistently, apolipoprotein E-deficient mice treated with MYOCD shRNA developed more plaques in the aortic sinus, which is associated with reduced ABCA1 expression, increased cholesterol retention in the aorta, and decreased high-density lipoprotein cholesterol levels in the plasma. Our data suggest that MYOCD deficiency exacerbates atherosclerosis by downregulating ABCA1 dependent cholesterol efflux from VSMCs, thereby providing a novel strategy for the therapeutic treatment of atherosclerotic cardiovascular disease.

Introduction

Atherosclerosis is the main pathological basis of cardiovascular disease (CVD), the leading cause of morbidity and mortality worldwide. Disorder in lipid metabolism is a major risk factor for atherogenesis, including uncontrolled lipid uptake and impairment of mechanisms associated with lipid efflux pathways [[1], [2], [3]]. ATP binding cassette transporter A1 (ABCA1) is an ATP hydrolysis driven “pump” that mediates the transfer of intracellular phospholipids and free cholesterol to apolipoprotein A1 (apoA-1), the main component of high-density lipoprotein cholesterol (HDL-c) [4]. Studies have documented that upregulating the expression of ABCA1 may promote cholesterol efflux and ameliorate atherosclerosis in mouse models [5,6]. Conversely, downregulating ABCA1 expression disturbs the balance between lipid uptake and efflux, promoting foam cell formation and atherosclerosis [7,8]. In support, foam cells in human advanced coronary lesions have a selective reduction in ABCA1 expression [2]. This suggests that ABCA1 plays a critical role in maintaining intracellular lipid homeostasis and preventing foam cell formation. Macrophages and vascular smooth muscle cells (VSMCs) are the main sources of foam cells. Among them VSMC-derived foam cells comprise over 50% of atherogenic foam cells in human plaques [2,9] and 70% in mouse models [10,11]. However, there is a striking lack of knowledge regarding the mechanisms that regulate ABCA1 expression and cholesterol metabolism in arterial VSMCs.

Myocardin (MYOCD), a smooth muscle-specific transcriptional coactivator of serum response factor (SRF), plays a central, but not exclusive, role in controlling biological functions of the VSMCs via a MYOCD/SRF regulatory module [12]. By binding with SRF onto a specific DNA sequence termed the CArG box, MYOCD regulates diverse downstream gene expression and determines the VSMCs' destiny, such as cell growth, differentiation, and phenotypic switch. MYOCD closely relates to cardiovascular diseases, including congenital heart disease, hypertrophic cardiomyopathy, heart failure, and essential hypertension [13]. Additionally, growing evidence has unveiled unexpected roles of MYOCD in lipid metabolism by elucidating the full spectrum of these CArG elements in the genome (i.e. the CArGome) [[14], [15], [16]]. For example, MYOCD drives the formation of caveolae, the Ω shaped and lipid-rich membrane pits that sculpt the membrane through multiple interactions with membrane lipids [17,18]. Deficiency and dysfunction of caveolae are related to lipodystrophy, dyslipidemia, and various other diseases [19,20]. Additionally, MYOCD expression inversely correlates to levels of oxidized low-density lipoprotein receptor 1 (OLR-1) in VSMCs and macrophage-like cells [21]. Moreover, the compulsive expression of MYOCD can ameliorate plaque formation in murine models [[22], [23], [24], [25]]. However, whether and how MYOCD affects lipid metabolism in VSMCs and transduces its effects into atherosclerotic progression remains an enigma.

Here, we found the protein levels of both MYOCD and ABCA1 to be dramatically decreased, along with increased lipid retention, in the atherosclerotic patient aortas. In vitro, suppressing the expression of MYOCD resulted in decreased ABCA1 mRNA and protein levels in human aortic VSMCs (HAVSMCs), leading to decreased cholesterol efflux and increased intracellular lipid content. The overexpression of MYOCD resulted in an opposite effect. Mechanistically, MYOCD regulated ABCA1 expression in an SRF dependent manner. MYOCD and SRF co-transfection facilitated the binding of SRF onto the ABCA1 promoter and significantly increased its gene expression. In addition, apolipoprotein E-deficient (apoE^−/−) mice treated with an adenovirus vector expressing MYOCD shRNA displayed a dramatic reduction of ABCA1 expression in aortic vessel walls. This resulted in decreased high-density lipoprotein cholesterol (HDL-c) in the plasma, increased cholesterol in the aorta, and increased plaque size in the aortic sinus. Thus, our data suggest that MYOCD deficiency accelerates lipid retention likely through downregulating ABCA1 in VSMCs, and the novel role of MYOCD in VSMCs' lipid metabolism may offer a new insight into the atherosclerotic CVD.