Matrine blocks AGEs- induced HCSMCs phenotypic conversion via suppressing Dll4-Notch pathway

Abstract

Vascular smooth muscle cells (VSMCs) contractile- synthetic phenotypic conversion takes responsibility in the atherosclerotic plaque formation by abnormal synthesis, secretion and deposition of extracellular matrix (ECM). Matrine exerts therapeutic effects on both cardiovascular diseases and organ fibrosis. In this study, we investigated matrine's inhibitory effect and mechanisms on AGEs- induced VSMC contractile- synthetic phenotypic conversion. Cultured human coronary smooth muscle cells (HCSMCs) were exposed to AGEs. Matrine at serially diluted concentrations were used to treat the cells. HCSMCs phenotype was identified by immunofluorescent staining of contractile phenotypic markers including mooth muscle myosin heavy chain (MYH11) and smooth muscle α-actin (ACTA2). Sircol collagen assay was used to assess the collagen secretion level. Notch signaling activation was determined by luciferase assay. Western blotting was used to evaluate expression levels of collagen I, collagen VIII, Delta-like (Dll)1, Dll3, Dll4, Jagged1, Jagged2, Notch intracellular domain (NICD)1 and Hes family basic helix-loop-helix (bHLH) transcription factor1 (HES1). Matrine pre-treatment recovered the AGEs- induced contractile- synthetic phenotypic conversion by increasing MYH11 and ACTA2 in HCSMCs. Matrine reduced AGEs- mediated activation of Notch signaling, down-regulated expression levels of NICD1, HES1, collagen I and collagen VIII and collagen secretion contents in HCSMCs. Matrine inhibited expression level of Dll4 without affecting other Notch ligands including Dll1, Dll3, Jagged1 and Jagged2 in HCSMCs exposed to AGEs. These results suggested that AGEs exposure facilitated the contractile- synthetic phenotypic conversion of HCSMCs. Matrine blocked this phenotypic conversion by suppressing Dll4- Notch signaling pathway activation.

Introduction

Coronary artery heart disease is highly associated with diabetes, which is characterized by formation and progression of atherosclerotic plaques (Harper et al., 2016). Under circumstance of hyperglycemia, set of high-molecular weight protein and fluorescent entities are produced after non-enzymatic glycation reactions, referred as advanced glycation end products (AGEs)(Vlassara and Uribarri, 2014). It was believed that AGEs was an indicator of adverse outcomes of atherosclerosis (Ahmad et al., 2017; Falcone et al., 2013). Previous investigations have proved that AGEs induced pathological changes of atherosclerosis such as proliferation and migration in arterial and vascular smooth muscle cells (VSMCs)(San Martin et al., 2007).

As a component of arterial vessel, dysfunctions of VSMCs are involved in the occurrence and progression of atherosclerosis (Bennett et al., 2016). VSMCs behave a range of phenotypes (Thyberg, 2002). Under normal physiological conditions, the VSMCs are at their quiescent status which is also referred as contractile phenotype (Tang et al., 2017), maintaining blood vessel tone by performing constriction and relaxation (Neuman et al., 2009). This phenotype is marked by specific contractile proteins including smooth muscle myosin heavy chain (MYH11) and smooth muscle α-actin (ACTA2)(Owens et al., 2004). When encountering harmful stimuli, the phenotype conversion takes place (Rensen et al., 2007). VSMCs lose expressions of the contractile proteins and produce excessive extracellular matrix (ECM) proteins, participating in vessel stenosis and atherosclerosis.

Notch receptors are highly conserved, playing critical roles in many cellular biological processes (Yamamoto et al., 2014). Delta-like (Dll1, Dll3 and Dll4) and Serrate (Jagged1, Jagged2) are recognized as the ligands of Notch receptors (Hori et al., 2013). These ligands would result in cleavage of Notch receptors and release of Notch intracellular domain (NICD) in to plasma (Tagami et al., 2008) which further initiates expression of its targeted genes (Gokulan and Halagowder, 2014). Notch signaling pathway was proposed to govern the phenotypic modulation of VSMCs (Lin and Lilly, 2014). Another previous study pointed out that the activation of Notch signaling lead to synthesis of ECM (Dees et al., 2011). Moreover, the blockage of Dll4 by specific antibody attenuated atherosclerosis (Fukuda et al., 2012). These above information raised our interests in investigating the possible involvement of Dll4- Notch signaling in the contractile- synthetic phenotype conversion of VSMCs.

Matrine (C₁₅H₂₄N₂O) (Fig. 1A) is one of the bio-active molecules of Sophora alpecuroides L. have been attracting researchers’ attention due to its wide spectrum of biological activities and bio-safety. In our previous studies, we observed matrine attenuated cardiac fibrosis in experimental diabetic cardiomyopathy. A recently published study showed that parathyroid hormone, a Notch signaling pathway activator, impaired matrine's anti-fibrotic activity (Yang et al., 2016). Thus, we hypothesize that matrine participates in VSMC phenotypic conversion by regulating Notch signaling. In the current study, human coronary smooth muscle cells (HCSMCs) were exposed to AGEs. The involvement of Notch signaling pathway in HCSMCs phenotypic conversion was investigated. The anti-fibrotic effect of matrine on AGEs- exposed HCSMCs was also studied. We believe that results from our study would not only add current knowledge of diabetic atherogenesis, but also provide novel theoretical basis for clinical application of matrine.