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2008 Annual Meeting Abstracts

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Hydrogen Sulfide is a Cytoprotectant in a Human Umbilical Vein Endothelial Cell (HUVEC) Model of Ischemia-Reperfusion Injury
Sunil P. Singh, BA, Daniel C. Rafii, BA, Jason A. Spector, MD.
Weill Medical College of Cornell University, New York, NY, USA.

PURPOSE: Despite the ubiquity of microvascular free tissue transfer, a major limitation and source of morbidity in these procedures is related to reperfusion injury of the transferred tissue. Longer periods of ischemia may result in significant reperfusion injury ultimately leading to partial flap loss or flap failure. Hydrogen sulfide, though traditionally considered a toxic species, has recently been recognized as an important endogenous signaling molecule with potential as a cytoprotectant. We propose a unique approach to significantly improving flap tolerance to ischemia by inducing a “suspended animation” like state within the flap tissue by exposure to NaHS, which can bind to cytochrome c oxidase and reversibly interrupt the process of oxidative phosphorylation. Since endothelial cells are known to be among the most sensitive components flap tissue to ischemia/reperfusion injury, we chose to establish the validity of our hypothesis using these cells.
METHODS: Human Umbilical Vein Endothelial Cells (HUVECs) were treated with varying concentrations of sodium hydrogen sulfide (10µM, 50µM, 100µM, 500µM and 1.5mM) and then subjected to 24 hours of hypoxic (1% O2) conditions followed by 6 hours of normoxia (21%), simulating reperfusion injury. A quantitative analysis of HUVEC apoptosis was performed with a Fluorescent TUNEL Assay (FTA) as an average of TUNEL positive nuclei per high powered field across five fields per slide (Fig 1A, 1B). Statistical significance was determined by ANOVA single factor with Tukey-Kramer post-hoc pair testing. All experiments were performed in duplicate.
RESULTS: FTA demonstrated an increasing cytoprotective effect of NaHS at concentrations up to 500uM compared to untreated controls. The number of apoptotic cells per high powered field decreased 50% at 10µM (p<0.05), 75% at 50µM (p<0.001) and 92% at 100µM (p<0.001) and 500µM (p<0.001) compared to untreated controls (Fig 2). Under normoxic conditions, there was no evidence of hydrogen sulfide toxicity as evidenced by a lack of apoptosis across the entire range of NaHS tested. Under hypoxic conditions, 1.5mM NaHS demonstrated a less protective effect than did any of the other concentrations with a decrease in apoptotic cell count that did not reach statistical significance, perhaps a result of sulfide toxicity.
CONCLUSION: We have demonstrated dose-dependent cytoprotective effect against ischemia/reperfusion injury with micromolar range exposure of HUVEC cells to hydrogen sulfide. These data suggest that hydrogen sulfide exposure mitigates ischemia-reperfusion injury without direct cellular toxicity in our target range, giving this molecule significant therapeutic potential as a cytoprotectant in free tissue transfer and other conditions associated with ischemic conditions.



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