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RGS1 Regulates Myeloid Cell Accumulation in Atherosclerosis and Aortic Aneurysm Rupture Through Altered Chemokine Signalling

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RGS1 Regulates Myeloid Cell Accumulation in Atherosclerosis and Aortic Aneurysm Rupture Through Altered Chemokine Signalling ARTICLE Received 16 May 2014 | Accepted 12 Feb 2015 | Published 18 Mar 2015 DOI: 10.1038/ncomms7614 OPEN RGS1 regulates myeloid cell accumulation in atherosclerosis and aortic aneurysm rupture through altered chemokine signalling Jyoti Patel1,2, Eileen McNeill1,2, Gillian Douglas1,2, Ashley B. Hale1,2, Joseph de Bono1,2, Regent Lee1,2, Asif J. Iqbal3, Daniel Regan-Komito3, Elena Stylianou4, David R. Greaves3 & Keith M. Channon1,2 Chemokine signalling drives monocyte recruitment in atherosclerosis and aortic aneurysms. The mechanisms that lead to retention and accumulation of macrophages in the vascular wall remain unclear. Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation. Here we show that Rgs1 is upregulated in atherosclerotic plaque and aortic aneurysms. Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling. In early atherosclerotic lesions, Rgs1 regulates macrophage accumulation and is required for the formation and rupture of Angiotensin II-induced aortic aneurysms, through effects on leukocyte retention. Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease. 1 Division of Cardiovascular Medicine, British Heart Foundation Centre of Research Excellence, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK. 2 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. 3 Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK. 4 Jenner Institute, University of Oxford, Oxford OX3 7DQ, UK. Correspondence and requests for materials should be addressed to J.P. (email: [email protected]) or to K.M.C. (email: [email protected]). NATURE COMMUNICATIONS | 6:6614 | DOI: 10.1038/ncomms7614 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7614 hemokine signalling plays a key role in leukocyte 5 ** 8 16 week ApoE –/– trafficking in the pathogenesis of vascular inflammation, ** 8 week ApoE –/– ) 4 ) the underlying cause of cardiovascular diseases such as Rgs1 6 16 week C57 Ct C Ct atherosclerosis and abdominal aortic aneurysms (AAA). Leuko- Δ 3 ( ΔΔ cyte activation and chemotaxis is mediated by chemokines 4 2 binding to multiple G-protein-coupled receptors (GPCRs). Cd68 mRNA ( of 2 Genetic or pharmacological inhibition of chemokines or 1 r 2 = 0.8987 Relative expression Relative chemokine receptors results in reduced atherosclerotic plaque induction of Fold 1–4 5,6 0 0 formation and inhibits AAA formation . Deletion of either WT 8 weeks 16 weeks 0 100 200 300 400 Ccr2 or Ccr5 decreases plaque formation in mouse models of ApoE –/– atherosclerosis that is accompanied by reduced macrophages1,2, Relative expression of Rgs1 (ΔCt) and deletion of Ccr2 inhibits aortic aneurysm formation6,7. Although the roles for chemokines and chemokine receptors are 0.006 0.3 well defined in the recruitment of leukocytes to the vascular wall ) Ct) Ct Δ 8 Δ in driving disease progression , it remains less clear how recruited 0.004 0.2 leukocytes accumulate or emigrate in the presence of ongoing chemokine stimulation. mRNA ( mRNA ( Elucidating these cellular mechanisms would provide strategies 0.002 0.1 Rgs1 targeted at reducing macrophage accumulation or promoting Rgs1 ND ND macrophage emigration in the inflamed vasculature. Chemokine 0.000 0.0 φ φ receptors couple to Gai subunits that have an intrinsic GTPase EC VSMC BM M S BC Mo M SV IMA OA AAA activity, which can be enhanced by RGS proteins, leading to 0.009 25 GPCR desensitization. Of the RGS proteins, Regulator of ** M1 ) M0 20 Ct G-Protein Signalling-1 (RGS1) accelerates Gai GTPase activity Rgs1 Δ 0.006 Ct) and acts to downregulate the response to sustained chemokine 15 activation9,10. Genome-wide association studies have speculated a ΔΔ 10 link between Rgs1 and polymorphisms associated with the risk of mRNA ( 0.003 several chronic inflammatory diseases such as celiac disease, mRNA ( 5 11–13 Rgs1 multiple sclerosis and type I diabetes . Other studies have induction of Fold 0.000 0 identified a role for RGS1 in the control of lymphocyte BM ZIP homeostasis10,14, but to date no studies have investigated the 0246 8 10 15 20 25 function of RGS1 in macrophages in vascular inflammation. Ly6G-7/4himonocytes Time (h) Here we report a previously unknown role for RGS1 in Figure 1 | Rgs1 is upregulated by inflammatory stimuli in activated monocyte–macrophage trafficking in the development of vascular monocytes. (a) Confirmation of Rgs1 mRNA changes in thoracic aortas of inflammation. We identified Rgs1 as a novel candidate gene in ApoE À / À mice and wild-type controls on a high-fat diet (n ¼ 5 per group) atherosclerosis and AAA, and tested the hypothesis that RGS1 is a by qRT–PCR. (b) Rgs1 expression is correlated with the expression of key modulator of chemokine receptor activity, with critical roles the macrophage marker Cd68 in thoracic aortas of ApoE À / À mice and in regulating the vascular inflammatory response by affecting wild-type controls on a high-fat diet. Each symbol represents an individual macrophage function. We investigated the effects of Rgs1 deletion mouse (n ¼ 5 per group) by qRT–PCR. (c) qRT–PCR analysis of Rgs1 mRNA on macrophage recruitment and retention in the artery wall, and in primary cells isolated from ApoE À / À mice (n ¼ 5–6; BC, B cells; elucidate a new requirement for RGS1 in leukocyte accumulation BM, bone marrow cells; EC, endothelial cells; MF, macrophages; in atherosclerosis and AAA rupture. S, splenocytes). (d) qRT-PCR analysis of Rgs1 expression in human tissue and cells. (Mo; Blood monocytes, plaque macrophages from carotid endarterectomies (n ¼ 3), SV; Saphenous vein and IMA; internal mammary Results artery from CABGs (n ¼ 8), OA; omental artery and AAA; abdominal aortic Vascular inflammation increases Rgs1 expression. To identify aneurysm from AAA repair patients (n ¼ 8–11)). (e) SV and IMA are from genes that are specifically regulated with atherosclerosis pro- CABGs. qRT–PCR analysis of Rgs1 mRNA in Ly6G-7/4hi BM monocytes and gression, we used a whole mouse genome array to profile the peritoneal monocytes isolated from zymosan-induced peritonitis (ZIP) in À / À gene expression in the thoracic aortas from ApoE mice, ApoE À / À mice (n 6–7 per group). (f) qRT–PCR analysis of Rgs1 mRNA in À / À ¼ comparing older atherosclerotic ApoE mice with young bone marrow-derived macrophages from ApoE À / À mice stimulated with littermate animals before plaque development (Supplementary IFN-g and lipopolysaccharide (M1) and unstimulated (M0) over 24 h Table 1). Among a number of genes already known to play major presented relative to mRNA in unstimulated cells, set as 1. *Po0.05, roles in the development of atherosclerosis such as Ccl2 (MCP-1), **Po0.01 calculated using the Student’s t-test (Data in a are expressed as Rgs1 was identified as one of the novel candidate genes with mean±s.d. and data in c–f as mean±s.e.m.). higher expression in aortas from older ApoE À / À mice than in aortas from younger ApoE À / À mice. We confirmed that Rgs1 mRNA was upregulated in aortas from atherosclerotic ApoE À / À positive macrophages compared with B cells where Rgs1 is known mice compared with younger ApoE À / À mice (8 weeks, male to have a non-redundant role (Fig. 1c). In contrast, we did not mice) or wild-type C57BL/6 mice of the same age (16 weeks, male detect Rgs1 mRNA in either vascular smooth muscle cells mice) by quantitative reverse transcriptase-PCR (qRT–PCR) (VSMCs) or endothelial cells, which are also known to be (Fig. 1a). The high expression of Rgs1 was associated with the involved in atherosclerotic plaque progression (Fig. 1c). high expression of the macrophage marker Cd68 in individual To extend these findings in the mouse atherosclerosis model to animals, suggesting that macrophages may be the source of RGS1 human disease, we first evaluated Rgs1 expression in macrophages in atherosclerotic plaques (Fig. 1b). To test this hypothesis, we isolated from human carotid artery plaques obtained at quantified Rgs1 expression in different primary cells isolated from endarterectomy. Rgs1 mRNA levels were detectable in macro- ApoE À / À mice and found high Rgs1 mRNA levels in CD68 phages derived from atherosclerotic plaque (Fig. 1d), compared 2 NATURE COMMUNICATIONS | 6:6614 | DOI: 10.1038/ncomms7614 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7614 ARTICLE with blood monocytes. Because of the associations between aortic the peritoneum of Rgs1 À / À ApoE À / À mice, there was an atherosclerosis, vascular inflammation and the pathogenesis of increase in CCR5 on the circulating monocytes compared with AAA15, we next sought to determine whether Rgs1 is related to monocytes in ApoE À / À mice. In contrast, at 16 h, there was no human aneurysms. We analysed the tissue samples of AAA from difference in the cell surface level of CCR5 between Rgs1 À / À patients undergoing surgical AAA repair, in comparison with ApoE À / À and ApoE À / À monocytes (Fig. 2g). However, CCR2 non-diseased control samples of omental artery from the same surface expression was not detectable on circulating monocytes patients, and to internal mammary artery and saphenous vein from either Rgs1 À / À ApoE À / À and ApoE À / À mice after 4 h of samples from patients undergoing coronary artery bypass graft zymosan. surgery (CABG). We also measured Rgs1 in CD14-positive blood monocytes from the same AAA patients. Rgs1 was highly RGS1 modulates macrophage trafficking into the aortic wall. expressed in human AAA tissue compared with non- Since macrophage recruitment is a critical step in atherogenesis, aneurysmal vascular tissues and blood monocytes (Fig.
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