Effects of carbon monoxide (CO) on vascular endothelial cells under hypoxia and in the presence of cell-free hemoglobin and sickle red blood cells

Wednesday, October 26, 2011 — Poster Session III
10:00 a.m. – Noon	Natcher Conference Center	FDA/CBER	TECH-9

Authors

• C Reiter
• JL Miller
• AI Alayash

Abstract

Objective: Cell-free Hb and elevated endothelin-1 (ET-1) contribute to sickle-cell-induced vascular occlusion. We hypothesized that CO reduces ET-1 expression and mitochondrial stress in hypoxic endothelial cells. Methods: Bovine aortic endothelial cells (BAECs) were treated with CO-releasing molecule-2 (CORM-2) under normoxia and hypoxia. BAECs were grown on transwell-filters under hypoxic conditions and treated with Hb ligated to CO (Hb-CO) to measure HIF-1α expression as an index of tissue oxygenation. BAECs were analyzed for mitochondrial oxygen consumption using XF24 technology (Seahorse Biotechnology) in response to CORM-2, Hb, Hb-CO, normal RBCs, and sickle RBCs. Results: CORM-2 (100μM) reduced HIF-1α and ET-1 expression in normoxic and hypoxic BAECs. On transwell-filters, Hb reduced HIF-1α expression under hypoxic conditions, but Hb-CO did not change HIF-1α expression. CORM-2 (50μM) had no effect on mitochondrial oxygen consumption, but higher doses of CORM-2 (100-500μM) dose-dependently increased oxygen consumption and reduced mitochondrial respiratory capacity. Intact RBCs or sickle cells had no effect on mitochondrial oxygen consumption, but Hb and Hb-CO (0.05mM) increased oxygen consumption without altering respiratory capacity. Conclusions: CO reduced HIF-1α and ET-1 expression and, at low doses, had no effect on endothelial mitochondria oxygen consumption. Hb-CO technology may promote vasodilation during vascular occlusive attacks in sickle cell disease.

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