Activation of MMP8 and MMP13 by angiotensin II correlates to severe intra-plaque hemorrhages and collagen breakdown in atherosclerotic lesions with a vulnerable phenotype

Abstract

Angiotensin II (ATII)-mediated hypertension increases the risk for acute coronary events, which may be caused by augmented collagen degradation. Interstitial fibers of collagen type I in the plaque can be degraded by MMP8 and MMP13 specifically. Indeed high MMP8 levels have been correlated with ruptured plaques in patients. To study the contribution of ATII in plaque rupture, we evaluated its effect on MMP8 and MMP13 activity on the vulnerable lesions using an extravascular device that induces regions of pro-atherogenic shear stress in the carotid arteries of ApoE KO mice. This triggers the growth of lesions with a “vulnerable” macrophage-rich phenotype (referred to as upstream lesions) and lesions with a “stable” fibrotic phenotype (referred to as downstream lesions).

ATII administration increased mean blood pressure, and increased the incidence of intra-plaque hemorrhages (IPH) from 30% to 73% of the animals in the upstream segments. The area of IPH was also increased by 5-fold. No IPHs were observed in the downstream lesions of the control group or the ATII group. In addition, ATII treatment doubled the size of upstream and downstream lesions. Upstream lesions in the ATII group were decreased in collagen content by 3-fold, contained 2-fold higher MMP8 and MMP13 levels, with a 2- and 3-fold increase in collagen type I degradation by MMP8 and MMP13 respectively compared to the upstream lesions in the control group. Gene expression analysis showed general increase in procollagens and TIMPs expression in response to ATII. However, ATII also decreased procollagen 5α3 expression in downstream lesions and decreased TIMP4 expression in upstream lesions.

These data show that ATII promotes a “stable” fibrotic phenotype by inducing severe intra-plaque hemorrhages, characterized by increased degradation of interstitial collagen I via an MMP-mediated (MMP8 and MMP13) mechanism.

Introduction

Rupture of atherosclerotic plaques with subsequent thrombus formation is the most significant mechanism underlying clinically important ischaemic events. Atherosclerotic lesions vulnerable to rupture typically consist of a large lipid core covered by a thin fibrous cap with severe infiltration of macrophages in the shoulder region [1], [2]. Plaque rupture itself occurs as the result of bio-mechanically induced tears in the weak spots of the vulnerable plaque, located in either the thin fibrous cap or the shoulder region. Macrophages secrete a large number of different matrix metalloproteinases (MMPs), a group of zinc-containing endopeptidases with overlapping specificities that processes different extracellular matrix (ECM) components. The process of proteolysis of the ECM is crucial for the migration of mononuclear leukocytes and medial vascular smooth muscle cells (VSMCs) into the intimal area, and in addition, provides a stimulus for the VSMCs to proliferate. These functions suggest that MMPs contribute to atherosclerotic plaque growth and fibrous cap formation [3]. However, increased activity of MMPs could also play an important role in plaque destabilization and plaque rupture. Specific localization of elevated mRNA, protein, and activity levels of MMPs within atherosclerotic lesions, particularly at the shoulder regions of the fibrous cap [4], [5], imply that MMPs play a role in fibrous cap weakening by degrading extracellular matrix components, such as interstitial collagens. Raised intra-plaque levels of active MMP8/collagenase-8 were observed in patients with plaques showing histological evidence of rupture [6]. MMP8 belongs to the group of interstitial collagenases of the MMP family, which also includes MMP1/collagenase-1, and MMP13/collagenase-3. Together with MMP14 (a membrane type metalloproteinase), they are the only metalloproteinases capable to cleave native collagen types I and III, the major structural components of the fibrous plaque cap [7]. The contribution of these collagenases to plaque vulnerability has not been evaluated, largely because an appropriate in vivo animal model for atherosclerotic lesions with a vulnerable plaque phenotype was not available. Our laboratory has recently developed a device (referred to as the cast) that modifies the shear stress, i.e. the drag force (per unit area) acting on the endothelium as a result of blood flow, in vivo. The normal, physiological level of shear stress in carotid arteries of mice is altered by cast placement, inducing low shear stress upstream, and oscillatory shear stress with vortices and flow reversal downstream from the cast. In addition, a region with a gradual increase in shear stress is created within the cast [8].

In the apolipoprotein E-knockout (apoE −/−) mouse model of cholesterol induced atherosclerosis, these shear stress alterations induce atherosclerotic lesions with distinct phenotypes: in the low shear stress region upstream, plaques develop with histological characteristics reminiscent of those of human vulnerable plaques, including severe lipid and macrophage accumulation, low numbers of VSMCs and a low level of collagen deposition, a large necrotic core, and a thin fibrous cap. In contrast, in the oscillatory shear stress region downstream, plaques show histological characteristics reminiscent of those of human stable atherosclerotic plaques, including a low level of macrophages and lipid accumulation, and a high level of VSMCs and collagen deposition, combined with a small to absent necrotic core, and a thick fibrous cap. In addition these low shear stress induced “vulnerable” macrophage-rich lesions (referred to in the texts as upstream lesions) show high incidence of spontaneous intra-plaque hemorrhages (IPH), whereas the oscillatory shear stress induced “stable” fibrotic lesions (referred to in the text as downstream lesions) remain free of IPHs. Infusion of angiotensin II (ATII) increased the incidence and the size of IPHs in the vulnerable lesions exclusively [9]. In the last decade, strong evidence from experimental and clinical studies suggests a role for this vasoactive hormone in the pathogenesis of atherosclerosis (reviewed in [10]). Tissue culture and animal experiments demonstrate a variety of mechanisms by which ATII may promote atherogenesis. Several large clinical trials have showed a reduction in the re-infarction rate in patients treated with inhibitors of the angiotensin-converting enzyme (ACE) after myocardial infarction [10], and increased ACE activity has been observed in culprit coronary lesions of patients with an acute coronary syndrome [11]. Nevertheless, the effect of ATII on the expression and activation of MMPs in vulnerable plaque remains unexplored. In the current paper, we used our cast-induced atherosclerosis model to study the effect of ATII infusion on the stabilization of the “vulnerable” macrophage-rich and “stable” fibrotic plaque. We test the hypothesis that ATII destabilization of the vulnerable plaque is mediated by increased collagen type I breakdown, facilitated by increased MMP8 and MMP13 expression and activation.