The roles of p62/SQSTM1 on regulation of matrix metalloproteinase-9 gene expression in response to oxLDL in atherosclerosis

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Abstract

Objective

Matrix metalloproteinase-9 (MMP-9) plays an important role in the remodeling of the extracellular matrix in atherosclerosis plaques. Autophagy protects macrophages against the processes of vascular disease. Our research explores how autophagy plays roles in macrophages to secret MMP-9.

Methods and results

In response to increased doses of oxLDL or CQ we monitored the autophagic flux. Our results revealed that oxLDL was dynamically associated with autophagy and 100 μg/ml oxLDL blocked autophagic flux in THP-1 cells. Moreover p62/SQSTM1 knocking down and CQ respectively inhibited and increased MMP-9 transcriptional expression. These effects were mediated by inhibition of NF-κB.

Conclusion

Abundant oxLDL blocked autophagic flux resulting in the aggregation of p62/SQSTM1. Then p62/SQSTM1 was involved in gene expression of MMP-9 via NF-κB-dependent signaling, and thus featuring novel plaque vulnerability properties of the atherosclerotic plaque. Understanding the mechanism that selectively modulates p62/SQSTM1 will provide a novel strategy for anti-atherogenesis.

Introduction

Autophagy is evolutionarily conserved cellular process through which long-live proteins and damaged organelles are recycled to maintain energy homeostasis. Autophagy occurs at basal levels in most cells to allow turnover of cytosolic compartments but it is also stimulated by environmental stress-relate signals, such as oxidative injury to recycle nutrient for maintenance of cell viability in unfavorable condition [1], [2], [3]. Autophagy is relevant to numerous physiological and pathological processes, including cell survival, cell metabolism [4], angiogenesis and calcification of the vessel walls [5]. The role of autophagy in atherosclerosis seems to be complex, both detrimental and protective effects. Therefore, homeostatic autophagy wipes out damaged cellular components and serves an important housekeeping function [6]. At physiological conditions, autophagic flux is blocked and induces aggregation of autophagy substrate p62/SQSTM1 (p62/sequestosome-1) [7]. Babak Razani shows characteristic of plaque is elevated levels of p62/SQSTM1 in atherosclerotic aortas [8]. As seta-interacting protein (ZIP) p62/SQSTM1 is a scaffold protein with multiple domains that functions in signal transduction, cell proliferation and oxidative stress response, especially in metabolic homeostasis and cancer [9], [10], [11]. Little is known about the role of p62/SQSTM1 in macrophage derived foam cell formation. Therefore our research discussed the role of p62/SQSTM1 in atherosclerotic pathophysiology.

Atherosclerotic plaque with a thin fibrous cap is prone to rupture that may lead to thromboembolism and subsequent ischemic stroke [12]. Collagen supplies the basic structural support for fibrous cap and sustains the plaque tension and stability [13]. Newby suggests that plaque growth and rupture depend on the expression of types of MMPs and stage of plaque development [14]. MMP-9 is first among them and cleaves ECM substrates (especially collagen) in the fibrous cap to attenuate thickness of fibrous cap [15]. The majority of MMP-9 are generated and secreted by macrophages and it is essential for the pathological process of the migration, vascular remodeling, plaque rupture and thrombopoiesis [16], [17]. In addition, Li Y in his recent study indicates MMP-9 is the independent risk factor for coronary heart diseases and increased MMP-9 in patients' serum prompts instability of atherosclerotic plaque [18]. Although oxLDL have been showed to induce autophagy, no definitive research has been done on the homeostatic regulation of the MMP-9 gene expression in terms of autophagic flux. Therefore, we sought to evaluate autophagic effects of macrophages on MMP-9 expression, and we investigated the underlying mechanisms.

Section snippets

Cells culture of THP-1 monocyte

THP-1 cells were obtained from the China Center for Type Culture Collection. 1 × 106 cells were cultured in 1640 medium supplemented with 2% fetal bovine serum (Hyclone, USA), 100 μg/mL penicillin and 100 μg/mL streptomycin at 37 °C in an incubator with 5% CO2. Thereafter, 100 ng/mL PMA was used to induce monocyte differentiation into macrophages. Then, cells were treated with according to requirement.

Oil red O staining

The THP-1 cells were measured for lipid accumulation through staining of oxLDL with oil red O.

Foam cell formation and apoptosis is dually regulated by oxLDL

In the coronary artery, the uptake of oxidized low-density lipoprotein (oxLDL) by macrophages leads to the formation of foam cells, thereby contributing to the generation of atherosclerotic plaques [19]. The volume of lipid droplets and lipid loading were significantly elevated in a doze-dependent way (Fig. 1). Quantitative data suggested that oxLDL promoted foam cell formation in THP-1 cells.

Monocyte apoptosis is associated with atherosclerotic plaque development and progression toward

Discussion

Autophagy is a physiological process in the routine turnover of cellular constituents and its self-digestion provides an alternative energy source, effectively to lipid homeostasis [25]. With low doses of oxLDL treatment autophagy activity was enhanced in order to protect against increased apoptosis. However with the aggregation of oxLDL, autophagy has never been a survived mechanism as we well recognized but oxLDL inhibited autophagosome elongation and sequestration. Although accumulated oxLDL

Disclosures

The authors have no conflict of interest.

Acknowledgments

The research was supported by the Postgraduate Innovation foundation of Harbin Medical University (YJSCX2014-28HYD).

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