Elsevier

Cardiovascular Pathology

Volume 34, May–June 2018, Pages 1-8
Cardiovascular Pathology

Original Article
Blocking Wnt5a signaling decreases CD36 expression and foam cell formation in atherosclerosis

https://doi.org/10.1016/j.carpath.2018.01.008Get rights and content

Highlights

  • Noncanonical Wnt5a receptors, Fz5 and Ror2, are expressed in atherosclerosis.

  • Wnt5a regulates CD36 expression and lipid accumulation in macrophages.

  • Wnt5a is an important player in initiation of foam cell development.

  • Blocking Wnt5a/Fz5 signaling is sufficient to prevent foam cell formation.

  • Wnt5a signaling is an attractive therapeutic to target in atherosclerosis.

Abstract

Background and aims

Wnt5a is a highly studied member of the Wnt family and recently has been implicated in the pathogenesis of atherosclerosis, but its precise role is unknown. Foam cell development is a critical process to atherosclerotic plaque formation. In the present study, we investigated the role of noncanonical Wnt5a signaling in the development of foam cells.

Methods

Human carotid atherosclerotic tissue and THP-1-derived macrophages were used to investigate the contribution of Wnt5a signaling in the formation of foam cells. Immunohistochemistry was used to evaluate protein expression of scavenger receptors and noncanonical Wnt5a receptors [frizzled 5 (Fz5) and receptor tyrosine kinase-like orphan receptor 2 (Ror2)] in human atherosclerotic macrophages/foam cells. Changes in protein expression in response to Wnt5a stimulation/inhibition were determined by Western blot, and lipid accumulation was evaluated by fluorescent lipid droplet staining.

Results

Wnt5a (P<.05), Fz5 (P<.01), and Ror2 (P<.01) were significantly expressed in advanced atherosclerotic lesions compared to less advanced lesions (N=10). Wnt5a, Fz5, and Ror2 were expressed in macrophages/foam cells within the plaque. In vitro studies revealed that Wnt5a significantly increased the expression of the lipid uptake receptor CD36 (P<.05) but not the lipid efflux receptor ATP-binding cassette transporter (P>.05). rWnt5a also significantly increased lipid accumulation in THP-1 macrophages (P<.05). Furthermore, inhibition of Wnt5a signaling with Box5 prevented lipid accumulation (P<.01) and prevented CD36 up-regulation (P<.01).

Conclusions

These results suggest a direct role for Wnt5a signaling in the pathogenesis of atherosclerosis, specifically the accumulation of lipid in macrophages and the formation of foam cells.

Introduction

Atherosclerosis is a chronic vascular disease and described as “silent” because of the absence of precipitating clinical symptoms in most patients. Importantly, the manifestation of atherosclerosis can be either acute myocardial infarction or stroke which both potentially have a high morbidity and mortality, notwithstanding the significant cost to an already challenged health care system. Despite concerted efforts in lifestyle modification and pharmaceutical developments, cardiovascular disease continues to be the number one cause of death in the United States [1].

Progression and development of the atherosclerotic plaque begin with an initial endothelial injury, induction of an inflammatory response, and accumulation of lipid in the subintimal space. Initial lesions, termed fatty streaks, are acquired as children, and some of these streaks progress to form mature atheromatous plaques in adulthood [2], [3]. Lipid particles within the vessel intima recruit blood monocytes which differentiate into macrophages and internalize the extracellular lipid. Accumulation of lipid within macrophages results in lipid-laden macrophages termed foam cells due to their fluffy/foamy appearance with histological staining. Macrophages/foam cells are the main component of atherosclerotic plaques and contribute significantly to intraplaque volume [4]. Rapidly growing plaques are more vulnerable to spontaneous plaque rupture and, among other parameters, are associated with large pools of foam cells [5], [6], [7]. The mechanisms of foam cell initiation and development at an early stage are still unknown. Providing early diagnosis and treatment is critical to preventing plaque rupture; however, given the lack of clinical symptoms and current limitations in detection, novel biomarkers and therapeutics are needed.

The Wnt proteins are important in homeostatic processes, both in embryological development and in nadulthood [8], [9]. There are two major Wnt signaling pathways: the canonical (Wnt/β-catenin) pathway involving β-catenin and the noncanonical pathway. The latter consists of two subgroups: the Wnt/Ca2+ and the Planar Cell Polarity pathway (PCP) [8], [9]. The specific Wnt/receptor/co-receptor combinations are particularly important in dictating the resulting downstream signaling effects. For example, receptor tyrosine kinase-like orphan receptor 2 (Ror2) is critical for activation of the PCP signaling pathway by Wnt5a [10], [11]. Interestingly, Wnt5a has also been demonstrated to block canonical signaling through the frizzled-2 (Fz2) receptor [12], [13]. Wnt5a is an extensively studied noncanonical member and is highly expressed in atherosclerosis, and Wnt5a mRNA is associated specifically with the lipid core and shoulder regions of the plaque [14], [15], [16]. However, the precise functional role of Wnt5a in the initiation, progression, and development of the atherosclerotic plaque is not yet clear.

In the present study, we hypothesized that Wnt5a signaling contributes to the accumulation of lipid within macrophages and subsequent foam cell formation. The results from the current study provide novel evidence that Wnt5a induces foam cell formation via up-regulating scavenger receptor CD36. Importantly, blocking Wnt5a signaling is sufficient to prevent lipid accumulation in macrophages/foam cells. These findings suggest that Wnt5a signaling plays a prominent role in the development and progression of atherosclerosis.

Section snippets

Source and preparation of human atherosclerotic tissue

Human atherosclerotic plaque material was obtained during elective carotid endarterectomy and carotid stenting procedures performed at Riverside Methodist Hospital, Columbus, OH. Consent was obtained, and human samples were used in compliance with the Institutional Review Board Committee at Ohio University (#15E348). In total, 10 patients were recruited for the study. Tissue sections were stained with hematoxylin & eosin (H&E), and the lesions were characterized histologically according to

Noncanonical Wnt5a receptors, Fz5 and Ror2, are expressed in advanced atherosclerosis

Human atherosclerotic plaque materials obtained during elective carotid endarterectomy and carotid stenting procedures were characterized using H&E and classified as described by the American Heart Association [15], [17], [18]. Fig. 1A shows representative advanced and less advanced human carotid artery tissue sections at low and high magnification. Important structural characteristics of the artery, such as the lumen and adventitial side of the artery, are highlighted [Fig. 1A(a, b)]. Advanced

Discussion

In the present study, we demonstrate a novel functional role of noncanonical Wnt5a signaling in the initiation and progression of the atherosclerotic plaque. Using human atherosclerotic plaques, we found that advanced atherosclerotic lesions express Wnt5a and the receptors (Fz5 and Ror2) implicated in the PCP pathway. On the other hand, using a human monocyte/macrophage cell line, we provide evidence that Wnt5a signaling up-regulates CD36 expression and lipid accumulation, both indicators of

Acknowledgments

The authors would like to thank Dr. Karen Coschigano for her critical review of the manuscript. We also acknowledge the valuable technological assistance that Gunjan Saxena provided for this study.

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