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OR1-4

CCL18 as a potential mediator of the pro-fibrotic actions of M2 macrophages in the vessel wall during hypertension

[Speaker] Barbara K Kemp-Harper:1
[Co-author] Caitlin V Lewis:1, May Zhu:1, Maggie Lieu:1,2, Seyuri Moodley:1, Yan Wang:1, Robert E Widdop:1, Chrishan S Samuel:1, Grant R Drummond:2
1:Department of Pharmacology, Monash University, Australia, 2:Department of Physiology, Anatomy & Microbiology, La Trobe University, Australia

Background: M2 macrophages contribute to vascular fibrosis and stiffening in hypertension (Moore et al., 2015). A potential mediator of these actions is the macrophage-derived pro-fibrotic chemokine, CCL18. However, the role of CCL18 and its cognate receptor, CCR8 in hypertension and vascular fibrosis has not been investigated. This study aimed to determine if angiotensin II augments CCL18 production from human primary M2 macrophages, identify vascular targets of CCL18 and investigate the ability of CCL18 to promote fibrosis.
Methods: Human primary macrophages were treated with a submaximal concentration of the M2-polarising stimulus, interleukin-4 (IL-4; 0.5 ng/ml, 24h), followed by addition of angiotensin II (100 pM, 48h) and CCL18 expression measured (ELISA). Type 1 collagen was detected via western blotting in CCL18 (3-300 ng/ml, 72h)-treated human aortic adventitial fibroblasts. The blood pressure (BP) of saline or angiotensin II (0.7 mg/kg/d, 28 d; s.c.)-treated male C57BL/6J mice was measured by tail cuff. Localisation and expression of CCR8 and the murine functional analogue of CCL18, CCL8, were assessed in the thoracic aorta by immunohistochemistry and qRT-PCR, respectively.
Results: In M2-polarised macrophages, angiotensin II increased CCL18 protein levels by a further 40% (P<0.05, n=5), and CCL18 treatment enhanced collagen I protein expression by 4-fold in human aortic adventitial fibroblasts (n=4). Angiotensin II infusion significantly elevated systolic BP (151±5 mmHg) as compared to saline treated mice (111±5 mmHg). In aortas from hypertensive mice, mRNA expression of CCL8 was elevated 3-fold (P<0.05, n=4-8), and CCL8 expression in the aortic wall was co-localised with the M2 macrophage marker CD206. Quantification of CCL8+CD206+ cells revealed a 50% increase in CCL8+ M2 macrophages in the aorta of hypertensive mice (P<0.05, n=6). CCR8 expression was also evident in the endothelium, adventitia and perivascular fat.
Conclusions: Angiotensin II increases CCL18 generation from M2 macrophages, which may target CCR8 expressing endothelial, adventitial and/or perivascular fat cells to promote fibrosis and vascular stiffening. CCL18 also promotes collagen synthesis from human adventitial aortic fibroblasts. Thus, CCL18 and its receptor CCR8, may serve as potential targets for the treatment of vascular fibrosis during hypertension.

Moore JP, et al., (2015). Am J Physiol Heart Circ Physiol, 309(5): H906-H917

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