Angiotensin II-induced smooth muscle cell migration is mediated by LDL receptor-related protein 1 via regulation of matrix metalloproteinase 2 expression

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Abstract

Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.

Research highlights

► Ang II upregulates LRP1 expression and increases RAoSMCs migration. ► Ang II-induced RAoSMCs migration is mediated by LRP1. ► LRP1 regulates MMP2 expression in RAoSMCs.

Introduction

Vascular smooth muscle cell (VSMCs) migration clearly plays a critical role in the pathophysiology of several prominent cardiovascular disease states such as atherosclerosis and restenosis [1], [2]. Following migration, SMCs proliferate in the intima and secrete matrices and proteases that form atheromatous plaques under the influence of stimulatory cytokines. Many researchers have shown that the migration of SMCs is induced by various stimuli such as PDGF-BB and angiotensin II (Ang II) [3], [4]. Angiotensin II (Ang II), the effector molecule of the renin-angiotensin system, is a potent chemoattractant that induces VSMCs migration [4], [5]. These effects are not only hemodynamic in nature, but also comprise inflammation, thrombosis, and cell proliferation through stimulation of cytokine and growth factor production [6]. Recent studies with Ang II type 1 receptor (AT1R) blockers (ARBs) [5] and knockdown experiments have shown that intracellular signals involving activation of Rac1 are important for the Ang II-mediated hypertrophy of SMCs and neointimal formation in injured arteries [6].

Evidence that low-density lipoprotein receptor-related protein 1 (LRP1) plays important roles in cell migration as well as lipoprotein remnant catabolism, protease regulation, and signal transduction has been accumulating [7], [8], [9], [10], [11]. LRP1, a multifunctional large endocytic receptor that belongs to the LDLR family, is involved in the metabolism of various extracellular ligands, such as proteinases, that play critical roles in vascular wall integrity [12], [13]. LRP1 binds and endocytoses over 30 structurally and functionally distinct ligands including apolipoprotein E/lipoproteins, proteinases, proteinase–inhibitor complexes, and extracellular matrix proteins [13]. Internalization of apolipoprotein E, proteinase–inhibitor complexes, and α-defensin by LRP1 may alter vascular smooth muscle cell migration and contraction [14], [15]. In addition, Li et al. reported that silencing of LRP1 significantly inhibited PDGF-β-induced SMC migration [10]. Furthermore, a recent study reported that Ang II upregulates LRP1 expression in the vascular wall [16].

Nevertheless, the role of LRP1 in Ang II-induced SMC migration still remains to be elucidated. Because matrix metalloproteinases (MMP) 2 and 9 play key roles in VSMCs migration [17] and LRP1 promotes cancer cell migration through upregulation of MMP2 and MMP9 [11], we hypothesized that the Ang II-induced increase in LRP1 expression would enhance SMC migration via altering the expression profiles of extracellular matrix (ECM) degradation-related proteins.

Section snippets

Cell culture and reagents

Rat aortic SMCs (RAoSMCs) were isolated from 6- to 8-week-old Sprague-Dawley rats. The thoracic aortas were removed and transferred on ice to serum-free DMEM. The aorta was freed from connective tissue, transferred into a Petri dish containing 5 ml of an enzyme dissociation mixture containing DMEM with 1 mg/ml of collagenase type I (Sigma) and 0.5 mg/ml elastase (USB), and incubated for 30 min at 37 °C. The aorta was then transferred into DMEM and the adventitia was stripped off with forceps under a

Ang II upregulates LRP1 and increases RAoSMCs migration

We investigated the effects of Ang II on LRP1 expression and migration of RAoSMCs. We observed that treatment of RAoSMCs for 12 h with Ang II increased LRP1 expression in a dose-dependent manner (Fig. 1A). We also found that LRP1 transcript levels were dose-dependently and significantly increased by Ang II based on RT-PCR analysis (Fig. 1B). To investigate whether Ang II affects RAoSMCs migration, we conducted two-dimensional cell migration assays. As shown in Fig. 1C, Ang II significantly

Discussion

In this study, we showed that LRP1 silencing led to a significant decrease in Ang II-induced RAoSMCs migration, which is a key event in the early progression of atherosclerosis [20], and provided evidence that LRP1 regulates the transcriptional levels of MMP2 in RAoSMCs. Our finding that LRP1 silencing resulted in a significant decrease in Ang II-induced RAoSMCs migration is consistent with a previous report that LRP1 is essential to human vascular smooth muscle cell migration [10]. Moreover,

Acknowledgments

This research was supported by Seoul St. Mary’s Clinical Medicine Research Program year of 2009 through the Catholic University of Korea. Anti-LRP1 IgG, human recombinant receptor-associated protein (RAP), minireceptor of LRP1 mLRP4, and MMP2 and MMP9 promoter luciferase vectors were kindly provided by Dr. Heesang Song.

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