Selective Endothelin A receptor antagonism attenuates neointimal lesion development in the mouse femoral artery

Karolina Duthie, Eileen Miller, Jessica Ivy, John McShane, David Webb, Patrick Hadoke. University of Edinburgh, BHF/ University Centre for Cardiovascular Science, EH16 4TJ, United Kingdom.

Although non-selective Endothelin (ET) A/B receptor antagonists inhibit neointimal lesion formation (Reel et al., 2005), studies in transgenic mice suggest that the ETB receptor has a role in attenuating neointimal remodelling (Murakoshi et al 2002). This suggests that selective ETA-receptor antagonism may be a better therapeutic option for inhibiting neointimal proliferation. Therefore, this study addressed the hypothesis that the potent, selective ETA receptor antagonist sitaxsentan would inhibit neointimal proliferation in a mouse model of intra-luminal injury.

Methods: The ability of sitaxsentan to selectively block ETA receptors in femoral arteries isolated from adult, male C57Bl6 mice (25-35g) was assessed using isometric wire myography. Femoral arteries were suspended in a myograph chamber (containing physiological salt solution at 37oC) and cumulative concentration-response curves obtained for ET-1 (10-11-10-6M), phenylephrine (PhE; 10-9-10-4M) and acetylcholine (ACh; 10-9-10-4M) after incubation (30min) with antagonist (sitaxsentan; 10-9-10-7M) or vehicle (water; n = 6-7). Adult, male C57Bl6 mice (25-35g) underwent femoral artery wire-injury (under anaesthesia by inhalation of isoflurane) and received either ETA antagonist (sitaxsentan; 15mg/kg/day in chow) or vehicle (n = 7-8) for 5 weeks (from 1 week before arterial injury). Femoral arteries were harvested 28 days after arterial injury for analysis of lesion size and composition. Data are mean+s.e.mean, where n indicates the number of different mice, and were analysed using Student’s unpaired t-test.

Results: In isolated arteries, sitaxsentan produced a concentration-dependent rightward shift of the ET-1-mediated contraction (pD2; 7.90+0.14 Control vs 6.85+0.15 100nM sitaxsentan; P<0.0005) but had no effect on responses to ACh or PhE. Femoral artery injury produced large, concentric fibro-proliferative neointimal lesions. Administration of sitaxsentan reduced lesion (40.2+3.0% vs 22.7+4.5%; P = 0.007), but not lumen (49.9+6.3% vs 66.5+9.1%; P = 0.15), size. Sitaxsentan administration also altered lesion composition with collagen content reduced (30.4+5.3% vs 13.7+1.5%; P = 0.02) but macrophage and α-smooth muscle actin immunoreactivity unchanged.

Conclusions: These results are consistent with selective antagonism of ETA receptors in femoral artery smooth muscle inhibiting neointimal lesion development, although reduced collagen staining suggests lesion vulnerability may be increased.

Murakoshi et al (2002) Circulation 106:1991-1998.

Reel,et al., (2005) J Pharm Pharmacol, 57, 1599-1608.