Abstract 1153: NADPH Oxidase-Derived Reactive Oxygen Species Are Required for Flow-Induced Dilation of Human Coronary Arterioles
NADPH oxidase is an important source of vascular reactive oxygen species (ROS), particularly in cardiovascular disease. In human coronary arterioles (HCAs), H2O2 mediates bradykinin (BK)- and flow-induced dilation. While mitochondria have been identified as a critical source of ROS in response to shear stress, NADPH oxidase itself is mechanosensitive to shear stress and is also activated by mitochondria-derived ROS in animal models; therefore, we hypothesized that NADPH oxidase contributes to flow-induced H2O2 generation and dilation of HCAs. Expression of NADPH oxidase subunits in human right atrial appendages was determined by immunohistochemistry (IHC). H2O2 production was assayed in isolated HCAs by semi-quantitative histofluorescence using the peroxide-sensitive dye DCFH-DA. HCAs were cannulated and pressurized, and diameter changes were measured by videomicroscopy in the presence of L-NAME (10−4 M, a nitric oxide synthase inhibitor) and indomethacin (10−5 M, a cyclooxygenase inhibitor). IHC revealed prominent expression of gp91phox, the catalytic subunit of NADPH oxidase, as well as regulatory subunits p22, p47, and p67, in HCA endothelium. In HCAs incubated with DCFH-DA, flow (shear stress ≈20 dyn/cm2) and BK (10−6 M) induced H2O2 formation in the presence of intraluminal gp91scram-tat (5x10−5 M, a scrambled control peptide, fluorescence ratio vs. baseline 2.32±0.27 and 1.96±0.29, respectively, n = 2 and 4, p<0.05 vs. baseline) but not gp91ds-tat (5x10−5 M, a specific peptide inhibitor of NADPH oxidase, 1.00±0.18 and 0.98±0.06, respectively, n = 2 and 4, p<ns). In HCAs constricted 30 –50% with endothelin-1, incremental increases in the transluminal pressure gradient produced a flow-induced dilation (65±6% at 100 cmH2O, n = 6) that was inhibited by gp91ds-tat (19±8% at 100 cmH2O, n = 3, p<0.05) but not by gp91scram-tat (58±5% at 100 cmH2O, n = 3, p=ns). However, dilation to papaverine (an endothelium-independent vasodilator) was not inhibited by either gp91ds-tat or gp91scram-tat (9±34% and 97±3%, n = 2 and 2, respectively, versus 95±2% with vehicle, n = 4, at 10−4 M). We conclude that NADPH oxidase is a functionally relevant source of H2O2 in HCAs, and that this enzyme complex participates in flow-induced vasodilation in the human heart.