Abstract 119: Ccl18 as a Potential Mediator of the Pro-Fibrotic Actions of M2 Macrophages in the Vessel Wall During Hypertension

M2 macrophages contribute to vascular fibrosis and stiffening in hypertension and may mediate these actions via release of the pro-fibrotic chemokine, CCL18, which they release to a greater extent than TGF-β1. The role of CCL18 and its cognate receptor, CCR8 in hypertension and vascular fibrosis has not been investigated. This study aimed to identify vascular targets of CCL18 and explore its ability to promote fibrosis. Blood pressure (BP) of saline or angiotensin II (0.7 mg/kg/day, 28 days; 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. Human aortic adventitial fibroblasts (AoAFs) and endothelial cells were treated with the pro-fibrotic agent TGF-β1 (10 ng/ml) or CCL18 (3-300 ng/ml) for 3-72h or 7d, respectively. In human AoAFs, expression of pro-collagen I, mature collagen I and α-SMA and were measured (qRT-PCR, Western blotting). Endothelial-mesenchymal transition was measured via VE-cadherin and α-SMA protein expression. Angiotensin II infusion significantly elevated systolic BP (151±5 mmHg, p<0.05) as compared to saline treated mice (111±5 mmHg, n=8). 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. CCR8 expression was evident in the endothelium, adventitia and perivascular fat. In human AoAFs, TGF-β1 caused a 2-fold increase in protein expression of collagen I (24h, p<0.01) and α-SMA (24-72h, p<0.05). CCL18 (300 ng/ml) did not change α-SMA expression, but increased the protein expression of pro-collagen I by 2-fold (24h; p<0.01), and elevated mature collagen I by 3.6-fold (72h; p<0.05). In human aortic endothelial cells, CCL18 (10 ng/ml) induced a trend towards reduced VE-cadherin, while increasing α-SMA expression (1.5-fold, p<0.05). CCL18 stimulates collagen synthesis from human AoAFs and may promote endothelial-to-mesenchymal cell transition. Expression of its receptor in the vascular wall suggests that the CCL18-CCR8 axis may serve as a potential target for the treatment of vascular fibrosis during hypertension.