Abstract 2058: Tetrahydrobiopterin-dependent eNOS Coupling Attenuates Vein Graft Atherosclerosis Through Inhibition of MCP-1 Mediated Monocyte Recruitment To Sites Of Vascular Injury
A key feature of endothelial damage following vascular injury is the production of reactive oxygen species (ROS) and the initiation of an inflammatory response through redox sensitive pathways. In the vasculature endothelial nitric oxide synthase (eNOS) may be critical to these pathways. Tetrahydrobiopterin (BH4), an essential eNOS cofactor which regulates NOS synthesis of either nitric oxide or ROS, has reduced bioavailability in vascular disease states favouring ROS production. We hypothesized that maintaining sufficient endothelial BH4 would attenuate ROS mediated inflammation and associated vascular remodeling. To investigate this we used a transgenic mouse with endothelial over-expression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GCH). GCH/ApoE-KO or ApoE-KO littermates underwent venous bypass grafting (n=28). Vein grafts (VG) were harvested 28 days post surgery. BH4 levels in aortas were 8-fold higher in GCH/ApoE-KO mice (P<0.01). In keeping with this, endothelial O2- production was significantly attenuated in GCH/ApoE-KO mice measured both by dihydroethidium fluorescence (77%, P<0.001) and lucigenin enhanced luminescence (54%, P<0.001). VG lesion area was reduced by 61% (P<0.001) in GCH/ApoE-KO mice compared with ApoE-KO controls. There was a marked reduction in VG macrophage content in GCH/ApoE-KO mice assessed by MAC-3 (51%, P<0.05) and MAC-1 (50%, P<0.05) immunohistochemistry as well as macrophage marker CD68 mRNA levels (P<0.05). To investigate whether the reduction in macrophages in GCH/ApoE-KO VGs was mediated by alterations in chemokine expression, we measured MCP-1, a recognized mediator of atherosclerosis. MCP-1 mRNA (P<0.05) and tissue (90%, P<0.05) levels were significantly reduced in aortas of GCH/ApoE-KO mice compared to ApoE-KO. Finally, MCP-1 receptor CCR2 mediated cell migration was significantly reduced in GCH/ApoE-KO aortas compared with ApoE-KO. In summary, increased endothelial BH4 levels reduce vein graft lesion area and macrophage content through a reduction in NOS-dependent O2- signaling. These findings highlight the importance of endothelial redox signaling in the response to vascular injury and identify MCP-1 as a critical mediator of vein graft atherosclerosis.