NADPH oxidase modulates myocardial Akt, ERK1/2 activation, and angiogenesis after hypoxia-reoxygenation

Abstract

Recent studies have demonstrated that reactive oxygen species (ROS) mediate myocardial ischemia-reperfusion (I/R) and angiogenesis via the mitogen-activated protein kinases and the serine-threonine kinase Akt/protein kinase B pathways. NADPH oxidases are major sources of ROS in endothelial cells and cardiomyocytes. In the present study, we investigated the role of NADPH oxidase-derived ROS in hypoxia-reoxygenation (H/R)-induced Akt and ERK1/2 activation and angiogenesis using porcine coronary artery endothelial cells (PCAECs) and a mouse myocardial I/R model. Our data demonstrate that exposure of PCAECs to hypoxia for 2 h followed by 1 h of reoxygenation significantly increased ROS formation. Pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI, 10 microM) and apocynin (Apo, 200 and 600 microM), significantly attenuated H/R-induced ROS formation. Furthermore, exposure of PCAECs to H/R caused a significant increase in Akt and ERK1/2 activation. Exposure of PCAEC spheroids and mouse aortic rings to H/R significantly increased endothelial spheroid sprouting and vessel outgrowth, whereas pharmacological inhibition of NADPH oxidase or genetic deletion of the NADPH oxidase subunit, p47(phox) (p47(phox-/-)), significantly suppressed these changes. With the use of a mouse I/R model, our data further show that the increases in myocardial Akt and ERK1/2 activation and vascular endothelial growth factor (VEGF) expression were markedly blunted in the p47(phox-/-) mouse subjected to myocardial I/R compared with the wild-type mouse. Our findings underscore the important role of NADPH oxidase and its subunit p47(phox) in modulating Akt and ERK1/2 activation, angiogenic growth factor expression, and angiogenesis in myocardium undergoing I/R.

Fig. 1

A: time course of hypoxia-reoxygenation (H/R)-stimulated generation of reactive oxygen species (ROS) in porcine coronary artery endothelial cells (PCAECs). Representative images from 3 separate studies of dichlorofluorescein fluorescence in chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-loaded PCAECs taken at baseline and after exposure to 2 h of hypoxia followed by 15, 30, or 60 min of reoxygenation. B: pretreatment of PCAECs with either diphenyleneiodonium (DPI; 10 μM) or apocynin (Apo; 600 μM) caused an inhibition of H/R-induced ROS generation. C: pretreatment of PCAECs with either the NADPH oxidase inhibitors DPI (10 μM) or Apo (200 and 600 μM), followed by exposure to hypoxia for 2 h and reperfusion for 60 min, resulted in significant inhibition of H/R-induced superoxide formation as measured by lucigenin-dependent chemiluminescence (n = 3−4 cell lines, data are means ± SD. *P < 0.05 compared with H/R; #P < 0.05 compared with baseline). RLU, relative light units; Con, control.

Fig. 2

A: Western blot analysis showing time course of H/R-stimulated Akt phosphorylation in PCAECs. Phosphorylation of Akt was increased and peaked at 15 and 30 min; by 120 min phospho (p)-Akt had declined toward normal. Total Akt levels were unaltered over 120 min of reoxygenation. B: effect of NADPH oxidase inhibitors DPI (10 μM) or Apo (200 and 600 μM) on H/R-stimulated Akt phosphorylation. C: densitometric data from Western blot analyses of PCAECs showing that pretreatment with DPI and Apo significantly suppressed H/R-induced Akt phosphorylation (n = 3−4 cell lines, data are means ± SD, *P < 0.05 compared with H/R; #P < 0.05 compared with baseline). OD, optical density.

Fig. 3

A: Western blot analysis showing the time course of H/R-stimulated ERK1/2 phosphorylation in PCAECs. Increased phosphorylation was apparent at 5 min, peaked at 15 min, and remained high over the 120 min of reoxygenation. B: effect of NADPH oxidase inhibitors DPI (10 μM) or Apo (200 and 600 μM) on H/R-stimulated ERK1/2 phosphorylation. Both DPI (10 μM) and Apo (600 μM) but not 200 μM Apo suppressed H/R-stimulated ERK1/2 phosphorylation. C: densitometric data from Western blot analyses of PCAECs showing that pretreatment with DPI (10 μM) and Apo (600 μM), but not Apo (200 μM), significantly suppressed H/R-induced ERK1/2 phosphorylation (n = 4 cell lines, data are means ± SD, *P < 0.05 compared with H/R; #P < 0.05 compared with baseline).

Fig. 4

Effect of H/R on Akt, ERK1/2 phosphorylation, and ROS formation in wild-type (WT) and p47^phox−/− mouse heart microvascular endothelial cells (MHMECs). A: exposure of WT MHMECs to hypoxia for 2 h followed by reoxygenation for 5, 15, 30, 60, and 120 min resulted in a gradual increase in Akt phosphorylation. p47^phox−/− MHMECs failed to show an increase in Akt phosphorylation in response to H/R (n = 3 cell lines). B: time course of H/R-stimulated ERK1/2 phosphorylation in WT and p47^phox−/− MHMECs. Exposure of WT MHMECs to H/R resulted in a significant increase in phosphorylated ERK1/2. Cells isolated from p47^phox−/− mice failed to show an increase in ERK1/2 phosphorylation (n = 3 cell lines). C: H/R- induced intracellular ROS formation was markedly attenuated in p47^phox−/− MHMECs compared with WT MHMECs (n = 4 −5 cell lines). D: H/R-induced superoxide production was significantly suppressed in p47^phox−/− MHMECs (black bars) as compared with WT MHMECs (white bars) (n = 5 cell lines, data are means ± SD, #P < 0.05 compared with baseline). E and F: time course and patterns of I/R-induced ERK1/2 (E) and Akt (F) phosphorylation in WT (△) and p47^phox−/− (▲) mice [n = 4−5 mice, data are means ± SD, *P < 0.05 compared with ischemia-reperfusion (I/R); #P < 0.05 compared with control]. KO, knockout.

Fig. 5

Representative images showing the sprouting of capillary-like structures from collagen-embedded PCAEC spheroids over time following exposure to H/R. A: 24-h control. B: H/R, 24 h. C: H/R, 48 h. D: H/R, 72 h. Untreated PCAEC spheroids had a low level of spontaneous sprouting. Twenty-four hours of exposure to H/R led to growth of sprouts from the spheroids. Exposure to H/R for 48−72 h resulted in a complex network of tubules sprouting from the spheroids.

Fig. 6

A: representative images of the effects of inhibitors of NADPH oxidase, phosphatidylinositol 3-kinase (PI3K), and ERK1/2 on H/R-stimulated sprouting from PCAEC spheroids at 24 h. B: quantitative analysis of sprout length from PCAEC spheroids exposed to H/R. Treatment with DPI (10 μM), Apo (200 μm and 600 μM), Wortmannin (Wort; 500nM), or PD-98059 (25 μM) suppressed the H/R-stimulated increase in sprout length (n = 4 cell lines, data are means ± SD; *P < 0.05 compared with H/R; #P < 0.05 compared with baseline). C: images of WT and p47^phox−/− MHMEC spheroids exposed to H/R for 24 h. D: quantitative analysis of sprout length from WT (white bars) and p47^phox−/− (black bars) MHMEC spheroids exposed to H/R. H/R was less effective in stimulating spheroid spourting in p47^phox−/− MHMECs compared with WT MHMECs (n = 3 cell lines, data are means ± SD, *P < 0.05 compared with H/R; #P < 0.05 compared with non-H/R).

Fig. 7

A: images of the effects of NADPH oxidase inhibitors on H/R-stimulated vessel outgrowth from WT and p47^phox−/− mouse aortic rings at days 5−7. WT aortic rings exposed to H/R demonstrated increases in vessel outgrowth at 5−7 days, whereas vessel outgrowth from p47^phox−/− mouse aortic rings was minimal at days 5−7. B: quantitative analysis of the area of vessel outgrowth (in arbitrary units) from WT (white bars) and p47^phox−/− (black bars) mouse aortic rings. Exposure to H/R significantly increased the outgrowth area in WT mouse aortic rings. The outgrowth area was also increased in the p47^phox−/− mouse aortic rings exposed to H/R compared with baseline, but this increase was less than that in H/R-stimulated WT aortic rings (n = 4−5, data are means ± SD, *P < 0.05 compared with H/R; #P < 0.05 compared with baseline).

Fig. 8

A: Western blot analysis showing time course and patterns of H/R-induced VEGF expression in MHMECs isolated from WT or p47^phox−/− mice. Exposure of WT MHMECs (△) to H/R increased VEGF expression at 4 and 8 h, whereas exposure of p47^phox−/− MHMECs (▲) to H/R failed to induce VEGF expression (n = 3 cell lines, data are means ± SD, *P < 0.05 compared with H/R; #P < 0.05 compared with baseline). B: Western blot analysis reveals that exposure of I/R increases myocardial VEGF expression in WT (white bars) but not in p47^phox−/− mice (black bars, n = 4−5 mice, data are means ± SD, #P < 0.05 compared with control).