Elsevier

International Journal of Cardiology

Volume 168, Issue 2, 30 September 2013, Pages 1378-1385
International Journal of Cardiology

Regulation of autophagy and apoptosis in response to ox-LDL in vascular smooth muscle cells, and the modulatory effects of the microRNA hsa-let-7g

https://doi.org/10.1016/j.ijcard.2012.12.045Get rights and content

Abstract

Objectives

Regulation of autophagy and apoptosis during treatment of vascular smooth muscle cells (VSMCs) with pro-atherogenic stimuli, such as oxidized low density lipoprotein (ox-LDL), remains unclear.

Methods and results

We examined the expression of autophagy and apoptosis upon treatment of VSMCs with ox-LDL. Exposure to ox-LDL in modest amounts (10–40 μg/ml) enhanced autophagy (expression of beclin-1, LC3-II/LC3-1 ratio and Atg5) and apoptosis (expression of caspase-3, Bax, Bcl-2 and Bcl-xL); however, exposure to higher concentrations (≥ 60 μg/ml) induced high levels of apoptosis but autophagy declined. Pretreatment of VSMCs with the miRNA hsa-let-7g inhibited autophagy, as LOX-1 expression and apoptosis declined. Hsa-let-7g treatment also resulted in a decrease in intracellular ROS generation. Treatment with LOX-1 antibody had similar effects as hsa-let-7g. Next, we studied autophagy and apoptosis in aortic segments from wild-type and LOX-1 knockout mice fed a high cholesterol diet, and observed increased autophagy as well as apoptosis in lipid-rich sections of aortas from wild-type mice and LOX-1 knockout mice (vs. corresponding controls); however, both autophagy and apoptosis in lipid-rich areas in aortic sections of LOX-1 knockout mice were less than in WT mice. These in vivo data are in keeping with in vitro data showing enhanced autophagy and apoptosis of VSMCs exposed to modest amount of ox-LDL.

Conclusion

This study provides first set of data on the regulation of autophagy and apoptosis in ox-LDL-treated VSMCs. Our observations also suggest that hsa-let-7g acts as a critical regulator of autophagy and apoptosis by modulating LOX-1.

Introduction

Atherosclerosis is a chronic inflammatory disease of the arterial wall of large and medium-sized arteries, which is characterized by formation of an atherosclerotic plaque that can partially or totally occlude the vascular lumen [1], [2]. Oxidized-low density lipoproteins (ox-LDL) play a major role in atherogenesis [1], [2]. Lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) is one of the major receptors responsible for binding, internalizing and degrading ox-LDL [1], [2]. Activation of LOX-1 has been known to be related to many pathophysiological events, including endothelial cells and vascular smooth muscle cell (VSMC) proliferation, alteration in cell cycle signals and apoptosis [1], [2].

Autophagy is an evolutionarily conserved process involved in the degradation of long-lived proteins and excess or dysfunctional organelles, which becomes manifest during tissue remodeling and starvation when the cell needs amino acids and fatty acids from catabolism of proteins and lipids [3]. Under normal conditions in most vascular cells, autophagy is an important house-keeping process, and may be considered a cell survival program [4]. Although excessive autophagic activity leads to total collapse of all cellular functions and induction of autophagic death, moderately enhanced autophagy promotes cell survival [5]. There are no data in the literature on the effects of ox-LDL on autophagic response of VSMCs.

Apoptosis, on the other hand, represents programmed cell death meant to remove cells exposed to noxious stimuli, such as ox-LDL [1], [2]. The number of apoptotic cells in atherosclerotic regions is increased [6]. The regulation of autophagy and apoptosis in response to ox-LDL might be of interest in understanding VSMC biology in atherosclerotic regions where the concentrations of ox-LDL are high [6], [7], [8], [9].

MiRNAs are non-coding, single stranded molecules consisting of ≈ 22 nucleotides that regulate the expression of genes at post-transcriptional level [7]. MiRNAs have been associated with inflammation, oxidative stress and angiogenesis [8], [9], and have been shown to be critical modulators for cellular function [7]. MiRNAs let-7 family members were firstly observed in Canenorhabditis elegan, and nine members of the let-7 family have been found in humans [10]. Let-7 family expression is usually noted in tissues during embryonic stages and significantly increases towards maturity, which suggests that it plays a pivotal role in developmental processes [11]. Let-7 family has been shown to inhibit cancer cell proliferation through repression of oncogenes, including RAS and HMGA2, via binding to the 3′-UTR of their mRNAs, thus it has also been reported as a tumor suppressor family [12]. Let-7 family also plays a key role in cell proliferation and migration [14], and inflammation, steps that are critical in the evolution of atherosclerosis [13], [14], [15]. Let-7f can modulate angiogenesis, a key regulator of atherosclerosis and cancers, by targeting the angiogenesis inhibitor thrombospondin-1 [13]. Qin et al. [15] have suggested that let-7c contributes to endothelial cell apoptosis through suppression of Bcl-xl. Recently, Chen et al. [16]. described a negative feedback regulation between let-7g and LOX-1, and identified a let-7g binding site on the 3′-untranslated region of LOX-1 mRNA.

In the present study we show, for the first time, the regulatory effects of ox-LDL on autophagy and apoptosis responses in VSMCs. Further, we show that hsa-let-7g modulates both autophagy and apoptosis by inhibiting LOX-1 expression.

Section snippets

Cell culture and miRNAs transfection

Human primary aortic VSMCs were obtained from ATCC (Manassas, VA), and maintained in vascular cell basal medium supplemented with VSMC Growth Kit (ATCC). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. All experiments were conducted using VSMCs between passages 2 and 5. Cells were seeded into 6-well plates and grown to semi-confluent density (≈ 90%) before treatment.

Human hsa-let-7g mimic and inhibitor were synthesized by Applied Biosystems (Carlsbad, CA). VSMCs were

Ox-LDL, LOX-1, let-7g expression and VSMC viability

As observed previously in endothelial cells [6], LOX-1 expression in VSMCs increased in response to increasing ox-LDL concentration (10–60 μg/ml, incubation time 24 h), whereas n-LDL had no effect (Fig. 1A). LOX-1 expression in response to ox-LDL was inhibited by treatment of cells with LOX-1 Ab.

Treatment with ox-LDL (10 to 40 μg/ml) resulted in enhanced cell viability, and reached its highest value at 40 μg/ml. However, concentration beyond 60 μg/ml decreased cell viability (Fig. 1B). Pretreatment

Discussion

Autophagy is a well conserved intracellular degradation process by which cytoplasmic material, including soluble macromolecules and organelles, is delivered to lysosomes for degradation [23]. Hence, autophagy is considered a stress response that allows unicellular eukaryotic organisms to survive during harsh conditions, probably by regulating energy homeostasis and/or by protein and organelle degradation [19], [20]. Apoptosis, on the other hand, is a response to oxidant stress, and is mediated

Disclaimer

The opinions expressed in this manuscript do not necessarily represent those of the U.S. Food and Drug Administration.

Acknowledgments

This study was supported in part by funds from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, Washington, DC.

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