Resveratrol attenuates monocyte-to-macrophage differentiation and associated inflammation via modulation of intracellular GSH homeostasis: Relevance in atherosclerosis

https://doi.org/10.1016/j.freeradbiomed.2016.05.003Get rights and content

Highlights

  • Resveratrol by improving GSH status, attenuate monocyte differentiation and inflammation.

  • AMPK activation by resveratrol is responsible for increased GSH levels.

  • Resveratrol attenuates Angiotensin-II-induced atherosclerosis.

Abstract

Monocyte-to-macrophage differentiation promotes an inflammatory environment within the arterial vessel wall that causes a mal-adaptive immune response, which contributes to the progression of atheromatous plaque formation. In the current study, we show that resveratrol, a well-known antioxidant, dose-dependently attenuated phorbol myristate acetate (PMA)-induced monocyte-to-macrophage differentiation, as measured by cell adhesion, increase in cell size, and scavenger receptor expression in THP-1 monocytes. Also, resveratrol significantly inhibited PMA-induced pro-inflammatory cytokine/chemokine and matrix metalloprotease (MMP-9) production. This inhibitory effect of resveratrol on monocyte differentiation results from its ability to restore intracellular glutathione (GSH) status, as resveratrol in the presence of buthionine sulfoximine (BSO) failed to affect monocyte differentiation. Furthermore, PMA-induced monocyte differentiation and inflammation was greatly inhibited when cells were co-treated with N-Acetyl-l-cysteine (NAC), a GSH precursor, while the presence of BSO aggravated these processes. These results also show that resveratrol mediated up-regulation of GSH is due to AMP-activated protein kinase (AMPK)-α activation, as compound C (AMPK inhibitor) treatment drastically depleted intracellular GSH and exacerbated PMA-induced monocyte differentiation and pro-inflammatory cytokine production. More importantly, chronic administration of resveratrol efficiently prevented monocyte infiltration and markedly diminished angiotensin (Ang)-II-induced atheromatous plaque formation in apolipoprotein-E knockout (ApoE−/−) mice. We conclude that, intracellular GSH status plays a critical role in regulating monocyte-to-macrophage differentiation and inflammation and resveratrol, by restoring GSH levels, inhibits these processes. Taken together, these results suggest that resveratrol can attenuate atherosclerosis, at least, in part, by inhibiting monocyte differentiation and pro-inflammatory cytokines production.

Introduction

Atherosclerosis is the leading cause of cardiovascular mortalities that is associated with clinical complications such as myocardial infarction, unstable angina, and stroke [1]. Atherosclerosis is a chronic degenerative condition of the vasculature, mainly due to an altered immune response, driven by the accumulation of macrophages in the intimal layers of arterial vessel walls [1], [2], [3]. Macrophages, the aggressive immune cells derived from the differentiation of monocyte precursors, represent the majority of the identified leukocytic infiltrates in tissue samples from coronary artery disease (CAD) patients [4]. In atherosclerotic lesions, macrophages engender the toxic environment by secreting a plethora of oxidants and pro-inflammatory cytokines, thereby maintaining a chronic inflammatory state that intrudes all stages of the disease [3]. These aforementioned toxic ingredients further promote the recruitment of monocytes as well as the proliferation of fibroblast and smooth muscle cells (SMCs), making the sub-endothelial space bulge and invade the inside of the arteries [5], [6]. Hence, current preventive strategies of atherosclerosis mainly focus on therapeutic interventions that ensue comprehensive blocking of monocyte infiltration and differentiation. Recently, we showed that metformin, a well-known anti-diabetic drug and an AMPK activator, inhibits PMA-induced monocyte-to-macrophage differentiation through the AMPK-STAT-3 axis, which also attenuated angiotensin (Ang)-II-induced atheromatous plaque formation in apolipoprotein-E knockout (ApoE−/−) mice [7].

Resveratrol (trans-3,5,4-Trihydroxystilbene) is a naturally occurring polyphenolic component of grapes, berries, and red wine that has been well documented to reduce cardiovascular and ischemic injuries as well as enhance stress resistance and extend lifespan [8], [9], [10]. The World Health Organization's MONItoring trends and determinants in CArdiovascular diseases (MONICA) study from individuals of 26 countries implied an inverse correlation between dietary saturated fat intake and the risk of coronary heart diseases, commonly referred to as “French paradox” [11]. This effect is attributed to the consumption of wine in the French and Swiss population, more specifically; resveratrol is thought to be responsible for the beneficial effects of red wine consumption. This important observation has made it possible to further study the beneficial effects of resveratrol on the risk parameters that are considered as hallmarks of cardiovascular diseases [9], [12]. In fact, clinical trials in patients with stable CAD demonstrated that oral intake of resveratrol capsules for 3 months was able to prevent platelet-aggregation and drastically improved flow-mediated vasodilation, an indicator of endothelial function [13]. Another study showed that resveratrol treatment enhances re-endothelialization and reduces neointima formation after endothelial injury [14]. In addition, resveratrol supplementation has been shown to significantly decrease plasma total cholesterol, low-density lipoprotein (LDL) and triglyceride contents; and significantly increased HDL levels in ApoE/− mice fed an atherogenic diet [15]. In vitro experiments further revealed that the cardiovascular protective effects of resveratrol might occur through a number of mechanisms. Resveratrol inhibits the proliferation of SMCs, oxidation of LDL-cholesterol, and synthesis of lipids and eicosanoids that promote inflammation and atherosclerosis [16], [17]. Furthermore, the production of pro-inflammatory cytokines, including IL-1β, IL-8, and TNF-α, were reduced in resveratrol treated peripheral blood mononuclear cells [18], [19].

Although several specific cellular and molecular pathways are involved in propagating the process of atherosclerosis, impaired intracellular redox status due to marked decrease of antioxidant levels, has been implicated as a major contributing factor. Among the array of antioxidant defense systems, reduced glutathione (GSH) acts as a key endogenous antioxidant of the cell that coordinates multiple cellular pathways, including cell proliferation, growth, and differentiation, besides the maintenance of cellular redox status [20]. There have been few clinical studies that have unanimously reported that intracoronary infusion of GSH could alleviate the constriction of epicardial arterial diameter in subjects with coronary risk factors [21]. In fact, GSH treatment significantly reduced the thrombotic cardiovascular events as well as the progression of atherogenesis, by reversing endothelial dysfunction via improving nitric oxide bioavailability [22]. Despite the cumulative data showing the potential role of GSH in maintaining vascular tone by improving endothelial function, the effects of GSH status in the context of monocyte-to-macrophage differentiation and its associated inflammation is still incompletely understood. Nevertheless, it was previously shown that reduced intracellular GSH levels are associated with osteoclast differentiation in response to M-CSF and RANKL stimulation [23].

In the present study, we document that resveratrol inhibits PMA-induced monocyte-to-macrophage differentiation and the associated inflammation by enhancing AMPK activation and restoring intracellular GSH. Furthermore, we show that resveratrol significantly attenuated Ang-II-induced plaque formation in ApoE−/− mice, possibly by impairing monocyte recruitment and its differentiation into macrophages in the arterial vessel wall.

Section snippets

Cell culture and treatment

THP-1 monocytes were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 containing 10% FBS, glucose (11 mmol/L), l-glutamine (4 mmol/L), penicillin (100 U/ml) and streptomycin (100 μg/ml) and incubated at 37 °C containing 5% CO2. For all experiments, THP-1 cells were seeded at a density of 2×105/ml of media and differentiated to macrophages by stimulating with PMA (0–200 nmol/L).

Cytokine analysis

THP-1 cells were seeded in 12 wells and following the treatments, TNF-α,

Resveratrol inhibits PMA-induced monocyte-to-macrophage differentiation

Resveratrol, a natural phenolic compound, is known to perform its biochemical effects via modulation of cellular antioxidant status. In addition, PMA-induced THP-1 monocyte differentiation is a well-accepted in vitro model for studying the monocyte-to-macrophage differentiation process [29]. Therefore, to study the effect of resveratrol on monocyte differentiation, THP-1 monocytes were treated with PMA (100 nmol/L) for 48 h in the presence or absence of resveratrol (0–100 µmol/L). Morphological

Discussion

Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall characterized by the thickening of the intima, primarily due to sub-endothelial accumulation of macrophages, which represent the chief participants in the initiation and progression of atherosclerotic lesion development [1], [2], [3]. By releasing numerous pro-inflammatory cytokines, MMPs, and ROS, macrophages promote atherosclerosis via accelerating inflammatory cell recruitment, vascular SMC migration/proliferation,

Conflict of interest

None declared.

Acknowledgements

This work was supported by grants from the Department of Science and Technology (SR/SO/HS-110/2008), Department of Biotechnology (BT/PR14613/BRB/10/859/2010, BT/PR4477/MED/30/692/2011) and CSIR, India under 12th Five Year Plan projects SMiLE (CSC-0111) and EpiHeD (BSC-0118). S.B.V. and R.G. acknowledge CSIR, P.N.G acknowledges UGC and S.Ka. acknowledges ICMR, New Delhi, India for the award of research fellowships. We thank Holly C. Cappelli, Northeast Ohio Medical University for critical

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