Elucidating a potential role for CCR5 and its ligands MIP-1α, MIP-1β and RANTES in saphenous vein graft disease

Katie Jones1, Rhoda Kuc1, Janet Maguire1, Sidath Katugampola2, Anthony Davenport1

1University of Cambridge, Cambridge, UK, 2Pfizer Global Research and Development, Sandwich, Kent, UK

Coronary artery bypass graft surgery commonly utilises the long saphenous vein, however graft failure is frequent owing to saphenous vein graft disease, both early thrombotic events and later intimal hyperplasia proceeding to atheroma. The mechanisms behind intimal hyperplasia and atherosclerotic disease are similar to native atherogenesis, but progression of graft atheroma is more rapid (Motwani and Topol, 1998). Vein graft intimal hyperplasia can be investigated in vitro using human saphenous vein organ culture (Porter, 2002).

The chemokine receptor CCR5 and its high affinity ligands MIP-1α, MIP-1β and RANTES have been implicated in atherosclerosis through studies in cultured vascular smooth muscle cells (Schecter et al., 2000), Apoe-/-CCR5-/- mice (Quinones et al., 2007) and human tissues (Jones et al., 2008). CC-chemokine inhibition reduced vein graft atheroma in Apoe-/- mice (Ali et al., 2005), suggesting that CCR5 and its ligands could have a potential role in the formation of vein graft disease. Our aim was to determine whether CCR5 and its ligands are present in graft saphenous vein, using organ culture to investigate a functional role for CCR5 in vein intimal hyperplasia.

Tissue was from coronary artery bypass graft surgery (normal saphenous vein) or heart transplant (saphenous vein graft). Dual-labelling fluorescent immunohistochemistry (IHC) was performed on sections (n = 3) of graft saphenous vein using antibodies against human CCR5, MIP-1α, MIP-1β and RANTES. Antibodies against von Willebrand Factor (endothelial cell marker), smooth muscle α-actin, and CD68 (macrophage marker) were co-incubated and immunoreactivity detected by confocal microscopy. Saphenous vein segments were cultured with CCR5-specific antagonists maraviroc (Dorr et al., 2005) and PF-232796 (1 μM) or vehicle control for 14 days. Neointimal area was expressed as a percentage of total intimal and medial area (neointimal index).

IHC localised CCR5-like immunoreactivity (LI) and immunoreactivity for CCR5 ligands to the neointima and media of vein graft. The use of cell markers suggested some co-localisation of CCR5 and its ligands with endothelial cells, smooth muscle cells and macrophages. Preliminary results from vein culture show development of intimal hyperplasia with a trend towards inhibition by CCR5 antagonists (neointimal index: vehicle 12.5±9.1%; maraviroc 5.4±1.6%; PF-232796 3.1±1.5%; n=3) suggesting this model is appropriate to investigate a role for CCR5 in vein intimal hyperplasia.

The detection of CCR5 and ligand immunoreactivity in graft saphenous vein suggests potential involvement of this system in lesion development. Vein organ culture is a promising model to investigate a functional role for CCR5 in intimal hyperplasia.

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