M. Polcz1, P. Komalavilas1, J. Cheung-Flynn1, C. Brophy1 1Vanderbilt University Medical Center,Department Of Surgery,Nashville, TN, USA
Introduction:
Exposure of human saphenous vein to normal saline (NS), a non-buffered, acidic solution (pH 5.0-5.6) results in endothelial injury and impaired endothelial function, potentially resulting in decreased patency of vein grafts used for bypass. Previous data have suggested that NS induced injury leads to membrane disruption, ATP release, activation of the P2X7 receptor and p38 MAPK pathway. This leads to an amplifying response resulting in inflammation and endothelial dysfunction. We hypothesized that increases in proinflammatory cytokine IL-1β, after activation of the P2X7R/p38 MAPK pathway, directly participates in endothelial dysfunction after injury.
Methods:
To determine the effects of IL-1β on endothelial-dependent relaxation, rat aorta (RA, n=12-20) were treated with IL-1β at 10, 50, and 100ng/ml in a muscle bath for 3 hours. Additionally, co-treatment with SB203580 (SB; 20µM), a p38 MAPK inhibitor, was performed.
To determine whether endothelial dysfunction associated with IL-1β is related to decreased nitric oxide (NO) bioavailability, human saphenous vein endothelial cells (HSVEC; n=4-5) were treated with IL-1β (10ng/ml) for 3, 6, and 24 hours. Western Blot analysis of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, and arginase II levels were performed. Protein-antibody complexes were visualized and quantified using the Odyssey Infrared Imaging System (LiCor Biosciences, Lincoln, NE). Protein levels were normalized to GAPDH level and phosphorylation was calculated as a ratio of the phosphorylated protein to total protein.
Results:
Treatment of RA with IL-1β led to a dose-dependent impairment of endothelial relaxation of pre-contracted tissues. This effect of IL-1β on endothelial dysfunction was prevented by cotreatment with SB, suggesting that IL-1β signals via a p38 MAPK-associated pathway. Additionally, treatment of HSVEC with IL-1β resulted in increased relative levels of arginase II at 6 and 24 hours but had no effect on eNOS levels or phosphorylation in HSVEC (Figure 1).
Conclusion:
IL-1β promotes endothelial dysfunction through increased arginase II levels resulting in decreased NO bioavailability. These data provide a plausible hypothesis by which NS-induced injury leads to inflammation and endothelial dysfunction which may be associated with eventual vein graft failure.
