CCL18 as a Mediator of the Pro‐Fibrotic Actions of M2 Macrophages in the Vessel Wall during Hypertension

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, chemokine (C‐C motif) ligand 18 (CCL18), which signals via its cognate chemokine receptor, CCR8. Pro‐fibrotic actions of CCL18 have been reported in the lung (Atamas et al ., 2003), yet whether these effects are also observed in the heart and vasculature remains to be seen. In addition, the localisation and expression of CCR8 in the vascular wall has not been investigated. Aims To (i) determine if angiotensin II augments CCL18 production from human primary M2 macrophages,(ii) identify vascular targets of CCL18 and (iii) investigate the ability of CCL18 to promote fibrosis. Methods Human primary macrophages were treated with the M2‐polarising stimulus, interleukin‐4 (IL‐4; 0.05–50 ng/ml, 6–72 h) and CCL18 expression was measured (qRT‐PCR, ELISA). Following incubation with a sub‐maximal concentration of IL‐4 (0.5 ng/ml, 24 h), angiotensin II (100 pM) was added for a further 48 h in the presence or absence of the angiotensin II type 1 receptor (AT 1 R) antagonist, candesartan (1 μM), and effects on CCL18 expression were measured. Blood pressures of saline or angiotensin II (0.7 mg/kg/d, 28 d; s.c. )‐treated male C57BL/6J mice were 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. Type 1 collagen was measured via western blotting in CCL18 (3–300 ng/ml, 72 h)‐treated human cardiac fibroblasts. Results IL‐4 caused concentration‐ and time‐dependent increases in macrophage CCL18 mRNA (up to 1700‐fold, P<0.05, n=4–7) and protein (up to 500‐fold, P<0.05, n=5–7) expression. In M2‐polarised macrophages (0.5 ng/ml IL‐4), angiotensin II increased CCL18 protein levels by a further 40% (P<0.05, n=5), an effect which was partially attenuated by candesartan. In aortas from hypertensive mice, CCL8 mRNA expression was elevated by 3‐fold (p<0.05, n=4–8), and the chemokine was found to co‐localise with the M2 macrophage marker CD206. Importantly there was a 50% increase in the number of CCL8 + M2 macrophages in the aorta of hypertensive versus normotensive mice (P<0.05, n=6). CCR8 was also expressed in the endothelium, adventitia and perivascular fat of hypertensive vessels. Finally, treatment of human cardiac fibroblasts with CCL18 increased pro‐ and mature type 1 collagen expression by up to 4‐fold and 1.5‐fold respectively (n=4). 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 cardiac fibroblasts. Thus, CCL18 and its receptor CCR8, may serve as potential targets for the treatment of vascular and cardiac fibrosis during hypertension. Support or Funding Information NHMRC Project Grant APP1041326