Abstract 236: Endothelial Nitric Oxide Production Inhibits Leukocyte Endothelial Cell- But Not Platelet Endothelial Cell Interactions
Background: We previously found that endothelial and neuronal nitric oxide synthase (NOS) decreases atherosclerosis, while inducible NOS accelerates plaque formation in apolipoproteinE knockout mice (apoE−/−). Here, we evaluate the contribution of eNOS to NO and superoxide production and its modulation of leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) cell interactions in atherosclerosis.
Methods and Results: Intravital microscopy studies of carotid arteries revealed that rolling, transiently and firmly adherent L/E-interactions are significantly elevated in apoE/eNOS double knockout mice (apoE−/−/eNOS−/−;n=16) compared to apoE−/− (n=23,p<0.01), while P/E-interactions did not differ. eNOS deletion resulted in 60% reduction in vascular NO production in apoE−/−, measured by electron paramagnetic resonance (EPR) with Fe-(DETC)2 (p<0.0001). Circulating, nitrosyl hemoglobin levels EPR measurement, considered a marker for NO-bioavailability in the blood, were reduced in apoE−/−/eNOS−/− (n=11) compared to apoE−/− (n=13,p<0.01). Vascular superoxide production was increased in apoE−/− (n=14, p<0.00001) and apoE−/−/eNOS−/− (n=13,p<0.05), compared to C57Bl6 mice (n=9). Pharmacological inhibition of eNOS using L-NIO (n=22) and genetic eNOS deletion (n=13) reduced vascular superoxide production (p<0.05), suggesting that eNOS is partially uncoupled in apoE−/−vessels. Vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression were high in apoE−/ −/eNOS−/ − (n=9), compared to apoE−/ − (n=20,p<0.05). Additionally, vascular macrophage infiltration measured by vascular CD14 RNA expression (n=17,p<0.00002) and immunohistochemistry for MOMA-2 in the plaques (n=9,p<0.05) were increased in apoE−/ −/ eNOS−/ −, compared to apoE−/ −.
Conclusion: We conclude that eNOS derived NO production is an important inhibitor of L/E-interactions but not P/E- interactions in atherosclerosis. Despite the expression of other NOS isoforms in the plaque, eNOS is the major contributor of vascular NO production and circulating nitrosyl hemoglobin levels in apoE−/ −. Uncoupling of eNOS occurs in apoE−/ − atherosclerosis. Increased VCAM-1 expression in apoE−/ −/eNOS−/ − provides a mechanism for the hyperadhesive state.