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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2010 Aug 11;30(9):1703–1710. doi: 10.1161/ATVBAHA.110.209726

Endothelium-dependent Coronary Vasodilatation Requires NADPH Oxidase-derived ROS

Jun Feng 1,*, Scott M Damrauer 1,*, Monica Lee 1, Frank W Sellke 1, Christiane Ferran 1, Md Ruhul Abid 1,
PMCID: PMC2924465  NIHMSID: NIHMS221455  PMID: 20702812

Abstract

Objective

Endothelium-derived nitric oxide (NO) is important in regulating coronary vascular tone. Excess reactive oxygen species (ROS) have been shown to reduce NO bioavailability resulting in endothelial dysfunction and coronary diseases. However, the functional significance of physiological ROS levels in NO-mediated coronary vasodilatation is not known.

Methods and Results

NADPH oxidase is a major source of ROS in endothelial cells (ECs). The aim of this study was to determine the effects of reduction in NADPH oxidase-derived endogenous ROS on vascular tone in intact coronary blood vessels. Using lucigenin-based superoxide production and DCFH-DA FACS assays, we found that mouse heart ECs (MHEC) from NADPH oxidase-knockdown (p47phox−/−) animals have reduced NADPH oxidase activity (by >40%) and ROS levels (by >30%) compared to WT MHEC. Surprisingly, reduction in ROS did not improve coronary vasomotion, rather endothelium-dependent VEGF-mediated coronary vasodilatation was reduced by >50% in p47phox−/− animals. Western blots and L- citrulline assays showed significant reduction in Akt and eNOS phosphorylation, and NO synthesis, respectively, in p47phox−/− coronary vessels and MHEC. Adenoviral expression of constitutively active eNOS restored VEGF-mediated coronary vasodilatation in p47phox−/−.

Conclusions

These data suggest that endothelium-dependent VEGF regulation of coronary vascular tone requires NADPH oxidase-derived ROS to activate PI3K-Akt-eNOS axis.

Keywords: NADPH oxidase, Endothelium, Redox, Signal transduction, Coronary vascular tone

Reactive oxygen species (ROS) include molecules that have unpaired electrons, such as superoxide (O2·−), hydroxyl anion (HO·), and nitric oxide (NO·), or that have oxidizing ability but do not possess free electrons, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite (ONOO−). ROS have long been implicated in the pathogenesis of cardiovascular diseases including hypertension, atherosclerosis, diabetic vasculopathy, and heart failure 1–3. Recent findings, however, suggest that ROS play critical roles in signal transduction in vascular cells, including endothelial cells (ECs) 4–13. We and others have identified NADPH oxidase as a major source of superoxide in ECs and thus one of the important determinants of the redox state of the endothelium 14–17. The NADPH oxidase enzyme complex consists of two membrane-bound components, gp91phox (also known as Nox2) and p22phox, and several cytosolic regulatory subunits, including p47phox, p67phox, and the small GTPase Rac (Rac1 or Rac2). Upon enzyme activation, the cytoplasmic subunits translocate to the cell membrane and the resulting complex transfers electrons from NAD(P)H to molecular oxygen to form O2·−. More recently, NADPH oxidase-derived ROS have also been implicated in EC proliferation, migration, and angiogenesis 14, 15, 17.

ROS-mediated vascular dysfunction is, in part, caused by reduction in NO· bioavailability due to redox (O2·−)-catalyzed formation of ONOO−18–22. NO plays important roles in several vascular functions including regulation of vasomotor tone and maintenance of vascular health 23, 24. Endothelial nitric oxide synthase (eNOS) can be stimulated to produce NO by hemodynamic forces, hormones, cytokines, and growth factors, including vascular endothelial growth factor (VEGF). Once NO diffuses from the endothelium into the adjacent vascular smooth muscle (VSMC) layer, NO mediates vasorelaxation as well as regulates the balance between VSMC proliferation and apoptosis; this later function governs important aspects of vessel caliber and remodeling 24. NO-induced vasodilatation occurs via activation of the soluble guanylyl cyclase (sGC), thereby elevating cyclic guanosine monophosphate (cGMP) levels 24. Indeed, mice deficient in eNOS demonstrated impaired endothelium-dependent relaxation in conduit arteries 25. NO also exerts its effects on cardiovascular system by S-nitrosylation of protein thiols in a cGMP-independent manner 26–28. As a major source of endothelial superoxide, increases in NADPH oxidase activity contribute to the impairment of endothelium-dependent vasodilatation by reducing NO· levels. The resultant endothelial dysfunction is believed to be involved in the pathophysiology of vascular diseases 29. Thus, the balance between ROS and NO is critical for optimal endothelial and vascular function.

VEGF plays important roles in vascular protection 30, 31, hemostasis 32, 33, microvascular permeability 34–36, wound repair 37, angiogenesis of ischemic tissue 38, 39, and also regulates vasomotor tone by regulating eNOS activity 40–44. In ECs, VEGF induces NO production through PI3K-Akt-mediated phosphorylation of eNOS at S1179 40, 41, 45. We and others have previously reported that VEGF induces NADPH oxidase activity and that NADPH oxidase-derived ROS are essential for VEGF-mediated endothelial cell functions in vitro 14, 15, 46, 47. Recently, we demonstrated that VEGF-mediated activation of PI3K-Akt, but not ERK1/2, depends on NADPH oxidase-derived ROS in human coronary artery endothelial cells (HCAEC) in vitro 48. These findings present an interesting paradox: whereas ROS are required for VEGF-mediated activation of PI3K-Akt signaling that lies upstream of eNOS, ROS can also reduce bioavailability of NO. Thus, the functional outcome of physiological levels of endogenous ROS is not precisely known, i.e. do ROS exert negative effects on NO bioavailability or do they enhance NO levels by activating eNOS through VEGF-PI3K-Akt? The goal of the present study is to examine the functional consequence of a decrease in physiological NADPH oxidase activity on VEGF-mediated coronary vasodilatation. Here, we report an interesting finding that reduction in NADPH oxidase-derived ROS inhibits VEGF-mediated endothelium-dependent relaxation of coronary vessels in NADPH oxidase knockdown (p47phox−/−) mice. We also demonstrate that the defect in vasomotion is due to reduced activation of PI3K-Akt-eNOS signaling in these vessels.

Methods

Mouse Heart Endothelial Cell (MHEC) Isolation and Culture

Microvascular endothelial cells (MHEC) were isolated from hearts of 3 week-old animals as described 49. For each experiment, primary cultures of both genotypes were started simultaneously from two animal hearts each.

Assay for NADPH Oxidase Activity and Intracellular ROS Measurement

NADPH oxidase activity and intracellular levels of ROS were determined in MHEC as previously described 15, 48, 50, 51.

Ex vivo Coronary Microvessel Relaxation Studies

After cardiac harvest from 6–8 week-old mice, coronary arterioles (80–120 µm in diameter and 2 mm in length) from WT C57BL/6 (n = 6) and p47phox−/− (n = 8) mice were dissected from the surrounding tissue. Microvessel studies were performed using in vitro organ bath video microscopy as described previously 52–54. Where indicated, isolated coronary vessels were pre-treated with membrane-permeant ROS scavengers 4-Hydroxy-2,2,6,6-tetramethylpieradine-1-oxyl (TEMPOL, 1 µM) and Mn(III)tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP, 1 µM) (EMD Bioscience). See detailed Methods in Supplemental Data on-line.

eNOS Activity Assay

Mouse hearts were harvested and homogenized in 20 ml ice-cold Homogenization Buffer (25 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 7.4) per gram of tissue. eNOS activity was determined using the Nitric Oxide Synthase (NOS) Assay kit (CalBiochem) that measures conversion of [3H]-L-arginine to [3H]-L-citrulline according to the manufacturers directions. See Supplemental Data for detailed methods.

Western Blot Analyses

Mouse whole hearts and coronary vessels with surrounding cardiac tissues were harvested separately for total protein. Western blots were carried out as previously described 55, 56. Coronary vessels from two hearts were pooled for each sample. Anti-phospho (473) Akt (p-Akt), anti-phospho (1179) eNOS (p-eNOS), Anti-phospho ERK1/2 (p-ERK1/2), anti-Akt and anti eNOS antibodies were from Cell Signaling. MHEC were grown on gelatin-coated plates to 80%-90% confluency, serum starved for 24h and then treated with VEGF (100 ng/ml) for the times indicated. Cell lysates were prepared for Western blot analysis as described 48.

Adenoviruses

Replication-deficient adenoviruses encoding the cDNAs of β-galactosidase (Ad-lacZ), GFP (Ad-GFP) and S1179D eNOS (Ad-S1179D eNOS-GFP) were described previously 41, 57, 58.

Adenoviral Expression of Constitutively Active S1179D eNOS in Coronary Vessels

6–8 week old p47phox−/− mice underwent in situ coronary artery perfusion with replication-deficient adenovirus encoding control genes (Ad-lacZ; Ad-GFP) or Adv-S1179D eNOS 59, 60. Mice were anesthetized, exsanguinated, and perfused with saline via infusion into the apex of the left ventricle. 5 × 109 pfu of recombinant adenovirus in 0.9% saline was then perfused into the aortic root in a retrograde fashion at a rate of 40 µL/min over 20 min. The heart was explanted and cultured in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin, and 25 µg/ml gentamicin for 16 hrs at 37°C, 5% CO2. See Supplemental Data for further details.

Immunohistochemistry

See Supplemental Data on-line.

Statistical Analyses

All values are presented as mean ± SEM where appropriate. A value of P<0.05 between experimental groups was considered to represent a significant difference (*). Nonlinear regression modeling utilizing the extra sum-of-squares F test to compare slopes (Prism 5. Graph Pad Software) was used to analyze microvessel reactivity data.

Results

p47phox−/− MHEC Have Reduced NADPH Oxidase Activity and ROS Levels

We first wanted to determine the levels of NADPH oxidase activity and total ROS in endothelial cells (ECs) of genetically modified p47phox subunit-knockout (p47phox−/−) animals. Using a low concentration-based lucigenin assay and DCF-DA-based FACS analysis, we determined 41±4.7% reduction in NADPH oxidase activity (Figure 1A) and 32.2±6.4% reduction in total ROS levels (Figure 1B) in p47phox−/− MHEC compared to WT MHEC. These findings suggest that endothelial NADPH oxidase activity and ROS levels are significantly reduced in the absence of p47phox.

Figure 1.

Figure 1

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Endothelial cells from p47phox−/− mice have reduced NADPH oxidase activity and ROS levels. A, Protein extracts from MHEC were subject to lucigenin assay to determine NADPH oxidase activity. p47phox−/− MHEC show marked reduction in NADPH oxidase activity compared to WT (C57BL/6). *p <0.05. B, p47phox−/− and WT MHEC were subject to FACS for intracellular ROS production using DCFH-DA. DCF fluorescence of control cells (WT) was arbitrarily set at 100%. *p <0.05. All experiments were performed in triplicate and the data shown are means ± SEM.

VEGF-induced Vasodilatation is Inhibited in Coronary Vessels of p47phox−/− Mice

Endothelium-dependent vasodilatation is dependent on NO bioavailability. Since MHEC from p47phox−/− mice have significantly reduced NADPH oxidase activity and ROS, we wanted to examine endothelium-dependent coronary vasodilatation in these animals. Surprisingly, VEGF induced coronary vasodilatation was reduced by 50±6.4% in p47phox−/− mice compared to WT (Figure 2A). This apparently counterintuitive finding led us to examine whether NO production in endothelium and NO-response in vascular smooth muscle cells are intact in p47phox−/− mice. To that end, coronary arterioles from WT and p47phox−/− mice were subject to relaxation responses to ADP, a nucleotide that releases NO from the endothelium in a PI3K-Akt-independent manner 61, and to SNP, an NO donor that acts directly on vascular smooth muscle. ADP- (Figure 2B) and SNP-induced (Figure 2C) vasodilatation of the coronary arterioles from p47phox−/− mice were comparable to that of WT animals, suggesting that the ability of the endothelium to produce NO in response to non-VEGF stimuli and that the response of smooth muscle cells to NO in these vessels were unaffected. Furthermore, endothelium-dependence of VEGF- and ADP-mediated but not SNP-mediated vasodilatation was confirmed by endothelial denudation in WT coronary vessels as described in Methods (data not shown). Taken together, these findings suggest that VEGF-stimulated endothelium-dependent vasodilatation is specifically affected in the coronary blood vessels in p47phox−/− mice.

Figure 2.

Figure 2

Figure 2

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VEGF-induced, but not ADP- or SNP-mediated, vasodilatation is inhibited in coronary vessels of p47phox−/− mice. A, endothelium-dependent dilatation of coronary arterioles from p47phox−/− (n = 8) and WT (n = 6) mice in response to VEGF. B, endothelium-dependent ADP-mediated vasodilatation of coronary arterioles from p47phox−/− (n = 8) and WT (n = 6) mice. C, endothelium-independent dilatation of coronary arterioles from p47phox−/− (n = 8) and WT (n = 6) mice in response to NO-donor, sodium nitroprusside (SNP).

PI3K-Akt-eNOS Signaling and NO Synthesis are Reduced in Coronary Vessels of p47phox−/− Mice

NO synthesis in endothelium is known to be induced by PI3K-Akt-mediated activation of eNOS 62–64. Recently, we have demonstrated that VEGF-mediated activation of PI3K-Akt is dependent on NADPH oxidase-derived ROS in human ECs in vitro 48. Combined with our current findings, this led us to examine whether the observed defect in coronary vessel relaxation in p47phox−/− mice resulted from reduced basal activation of redox-sensitive PI3K-Akt-eNOS. To test this, we measured activation levels of PI3K-Akt-eNOS signaling and NO levels in p47phox−/− coronary vessels using Western blots and citrulline assay, respectively. Figure 3A demonstrates that phosphorylation levels of Akt and eNOS in p47phox−/− coronary vessels are reduced by 61±4.8% and 34.5±6.3%, respectively, compared to that of WT mice. NO synthesis in coronary vessels of p47phox−/− mice was also reduced by 30.8±4.2% compared to that of WT (Figure 3B). These findings suggest that genetic deletion of NADPH oxidase subunit, p47phox, results in reduced activation of PI3K-Akt-eNOS signaling and NO synthesis in the coronary vasculature. However, there was no difference in the expression levels of VEGF receptors (VEGFR1 and VEGFR2) (Supplemental Figure IA) or phosphorylation levels of ERK1/2 (Figure 3A) in coronary vessels of WT and p47phox−/− mice, suggesting that NADPH oxidase activity selectively modulates the PI3K-Akt-eNOS pathway.

Figure 3.

Figure 3

Figure 3

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PI3K-Akt-eNOS (but not ERK1/2) activation and NO synthesis are reduced in coronary vessels of p47phox−/− mice. A, Western blots of protein extracts from p47phox−/− and WT coronary vessels. Blots shown are representative of three independent experiments. The lower panel shows quantitative densitometric analysis of three Western blots using NIH J image (mean ±SEM.). *p<0.05. B, NO production is decreased in p47 phox−/− heart and coronary vessels as measured by 3H-L-arginine-dependent citrulline assay. All experiments were performed in triplicate and the data shown are means ± SEM. *p<0.05.

VEGF-induced Activation of PI3K-Akt-eNOS, but not ERK1/2, is Inhibited in p47phox−/− MHEC

Next, we wanted to examine whether VEGF-induced activation of PI3K-Akt-eNOS signaling required NADPH oxidase activity in murine endothelium. To that end, we performed Western blots for the phosphorylation of Akt, eNOS and ERK1/2 using cell lysates from WT and p47phox−/− MHEC. VEGF-induced phosphorylation of Akt and eNOS, but not ERK1/2, was inhibited in MHEC from p47phox−/− mice, suggesting that selective activation of PI3K-Akt-eNOS by VEGF depends on NADPH oxidase activity in murine endothelial cells (Supplemental Figure 1B). However, there was no difference in the expression levels of VEGF receptors (VEGFR1 and VEGFR2) between WT and p47phox−/− MHEC (Supplemental Figure IA). Taken together, these results suggest that VEGF-PI3K-Akt-eNOS axis (but not ERK1/2) in MHEC is dependent on NADPH oxidase activity.

Expression of Activated eNOS Compensates for p47phox−/− Effects on Coronary Vasodilatation

In order to examine whether reduced activation of PI3K-Akt-eNOS is responsible for the inhibition of VEGF-induced vasodilatation, we introduced replication-deficient control adenovirus (Ad-lacZ or Ad-GFP) or adenovirus expressing constitutively active eNOS (Ad-S1179D eNOS-GFP) 41 into the coronary vessels of p47phox−/− mice (Figure 4) as described in Methods. Gene transfer to the endothelium was confirmed by β-galactosidase staining (data not shown) and co- immunofluorescence staining for GFP and anti-CD31/PECAM (Figure 4A). Overexpression of eNOS in the endothelium of the Ad-S1179D eNOS-transduced coronary vessels was further confirmed by Western blots (Figure 4B) and immunohistochemistry (Figure 4C). Ad-S1179D eNOS-transduced p47phox−/− coronary vessels demonstrated 1.84±0.22-fold increase in eNOS activity compared to Ad-GFP-transduced vessels, as measured by citrulline assay (Figure 4D).

Figure 4.

Figure 4

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Adenoviral expression of constitutively active S1179D-eNOS in the coronary vessels of p47phox−/− mice. A, Expression of constitutively active GFP-tagged S1179D-eNOS by gene transfer in the coronary vessels of p47phox−/− mice. Cross-section of coronary arteries demonstrating negative control (Ad-lacZ; upper panels) and GFP fluorescence (Ad-S1179D-eNOS-GFP; lower panels) in the endothelium. Co-localization of endothelium is shown using anti-CD31 antibody immunofluorescence (red). Green, GFP; blue, DAPI (nuclei); Arrow, blood vessel. B, Expression of eNOS protein as detected by Western blot analysis in p47phox−/− coronary vessels transduced with Ad-GFP or Ad-S1179D-eNOS. Protein extracts from two pooled hearts were used for each experiment. The membrane was stripped and reprobed using anti-Akt antibody as loading control. C, Expression of eNOS protein in the coronary vessels transduced with control (Ad-GFP) or Ad-S1179D-eNOS was detected by immunohistochemistry using anti-eNOS antibody. D, Nitric oxide production was 1.8-fold higher in Ad-S1179D eNOS-transduced p47phox−/− coronary vessels compared to Ad-GFP. All experiments were performed in triplicate and the data shown are means ± SEM. *P<0.05.

Finally, we examined whether expression of activated eNOS in the endothelium of intact coronary blood vessels could compensate for the reduced vasodilatory activity in NADPH oxidase knockdown mice. Figure 5A demonstrates that expression of activated eNOS (rAd.S1179D nos3) restored VEGF response in the coronary vessels of p47phox−/− mice to the WT levels. Together, these findings suggest that activation of PI3K-Akt-eNOS signaling is inhibited in p47phox−/− coronary vessels, which results in defective vasodilatation in response to VEGF.

Figure 5.

Figure 5

Figure 5

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A, Activated eNOS expression compensates for the NADPH oxidase knockdown effects on coronary vessels. VEGF-induced endothelium-dependent dilatation of coronary arterioles from p47phox−/− mice transduced with Ad-GFP (n = 4) or Ad-S1179D-eNOS (nos3)-GFP (n = 4). P<0.0001. B, Proposed model for NADPH oxidase-dependent activation of eNOS and coronary vasodilatation. Reduction in coronary vasodilatation in NADPH oxidase-knockdown mice appears to be due to defective activation of the redox-sensitive VEGF signaling pathway (shown in colored box), PI3K-Akt-eNOS, in the coronary endothelium. ERK1/2 activation is independent of the redox level in endothelium.

Reduction in ROS Inhibits VEGF-induced Coronary Vasodilatation

Next, we wanted to test whether ROS have a direct role on VEGF-induced coronary vasodilatation. To that end, we treated the coronary arteries of WT mice with two different cell-permeant ROS scavengers, TEMPOL (broad-spectrum free radical scavenger) and MnTBAP (superoxide scavenger/dismutase that does not scavenge NO), and measured VEGF-induced vasodilatation. Both TEMPOL and MnTBAP significantly inhibited VEGF-induced coronary vasodilatation (Figure 6) but not SNP-mediated vasodilatation (data not shown), providing direct evidence that decreasing ROS impairs endothelium-dependent coronary vasodilatation. Whereas TEMPOL decreases ROS levels, MnTBAP specifically reduces superoxide levels by converting O2·− to H2O2 and thus increases overall H2O2 levels. Inhibitory effects of both TEMPOL and MnTBAP suggest a greater role for superoxide compared to H2O2 in VEGF-induced coronary vasodilatation. However, our data cannot exclude requirement for proper subcellular localization of H2O2 in VEGF-induced activation of signaling pathways and coronary vasodilatation. Future studies will address these important questions.

Figure 6.

Figure 6

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ROS scavengers inhibit VEGF-induced vasodilatation in coronary vessels of WT mice. Isolated coronary vessels were treated with membrane-permeant ROS scavenger 4-Hydroxy-2,2,6,6-tetramethylpieradine-1-oxyl (TEMPOL, 1 µM) and Mn(III)tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP, 1 µM) for 15 min. Endothelium-dependent dilatation of coronary arterioles from WT (n = 4) mice in response to VEGF was assayed as described in Methods. For statistical significance, nonlinear regression modeling was used to compare slopes (Prism 5, Graph Pad Software) of the microvessel reactivity data.

Discussion

The goal of the present study was to determine the role of endogenous NADPH oxidase-derived ROS, i.e. physiological levels of ROS in coronary vascular function. Specifically, we wanted to examine whether reduction in endogenous NADPH oxidase activity would improve coronary vascular function, e.g., vasodilatation, by increasing NO bioavailability. To that end, we have utilized an NADPH oxidase-knockdown mouse model (p47phox−/−) that has significantly reduced ROS levels in coronary endothelium. The results presented here using intact coronary blood vessels and isolated murine heart ECs (MHEC) suggest that endothelium-dependent coronary vasodilatation operates through NADPH oxidase-mediated activation of PI3K-Akt-eNOS signaling and NO synthesis in EC. These data also provide evidence, for the first time, that reduction in endogenous NADPH oxidase activity can result in coronary vascular dysfunction by decreasing redox-mediated activation of eNOS.

Our data demonstrate that genetic knockdown of NADPH oxidase subunit, p47phox, results in significant decrease in NADPH oxidase activity and ROS levels in coronary blood vessels. We also show that coronary vessels of these animals have reduced PI3K-Akt-eNOS activity, NO production, and VEGF-induced vasodilatation. Interestingly, coronary vasodilatation by ADP, a PI3K-Akt-independent activator of eNOS 61, was unaffected in p47phox−/− mice, providing additional support for specificity of the defective VEGF activation of PI3K-Akt-eNOS axis in this animal. Furthermore, our results demonstrate that VEGF-induced activation of PI3K-Akt-eNOS, but not ERK1/2, signaling is impaired in NADPH oxidase-knockdown MHEC. These findings are in accordance with our previous report that VEGF-mediated activation of PI3K-Akt-forkhead signaling, but not ERK1/2, is dependent on NADPH oxidase activity in HCAEC in vitro 48. Taken together, we hypothesize that NADPH oxidase-derived ROS modulate activation of PI3K-Akt-eNOS in endothelium and thus regulate VEGF-induced coronary vasodilatation. Our data demonstrating that expression of activated eNOS compensates for the functional loss of upstream PI3K-Akt-eNOS activity in p47phox−/− coronary vessels support this hypothesis. Future studies aimed at modulating coronary vasodilatation using loss- or gain-of-function mutants of eNOS-activating signaling intermediates (e.g. Akt, PI3K, c-Src) including upstream Ser/Thr kinases (e.g. PKCδ, AMPK, PKA) should further our understanding about their individual contributions to ROS-mediated eNOS activity, and thus in the regulation of coronary vasomotor tone.

ROS have been previously shown to increase NO release in blood vessels and in endothelial cell culture in vitro 20, 63, 65. A recent study by Fulton and coworkers reported that superoxide production by overexpression of nox5 increased eNOS activity in aorta and cultured ECs 66. However, nox5-induced activation of eNOS was mediated through enhanced association of eNOS with hsp90 without altering eNOS phosphorylation 66. Another study reported that Rac1 regulated vasomotor response and angiogenesis by transcriptional upregulation of eNOS 67. To the best of our knowledge, the current study is the first to demonstrate that endogenous NADPH oxidase-derived ROS play an important role in endothelium-dependent vasodilatation in intact coronary blood vessels. Our data also provide evidence for a novel mechanism by which NADPH oxidase-derived ROS modulate PI3K-Akt activation and thus, regulate downstream phosphorylation and post-translational activation of eNOS in coronary endothelium. Additionally, ROS scavengers inhibited endothelium-dependent coronary vasodilatation in WT animals providing further support for a positive role for ROS in coronary vasomotion. These results are in contrast with a long-held notion that reduction of ROS will improve global vascular functions by increasing bioavailability of NO. Rather, our data support a critical positive role for endogenous NADPH oxidase-derived ROS in NO production in intact coronary vessels.

In summary, we demonstrate that endothelium-dependent coronary vasodilatation operates through NADPH oxidase-derived ROS-mediated activation of PI3K-Akt-eNOS signaling and NO synthesis in endothelium (Figure 5B). Together with our previous findings using human coronary ECs 48, the present study suggests that redox sensitivity of PI3K-Akt-eNOS signaling may be evolutionarily conserved in mammalian coronary vessels. The redox dependence of eNOS in the coronary vasculature may have important functional implications: 1) it may help maintain critical balance between superoxide and NO levels in coronary vessels, and thus ensure NO bioavailability during oxidative stress, 2) it may synchronize peroxynitrite levels with redox content of the vasculature, and finally, 3) it may represent a coordinated feedback loop that enhances eNOS activity by ROS to counterbalance reduction in NO bioavailability. Studies are undergoing to address these important questions.

Supplementary Material

Supp1

Acknowledgments

a) Acknowledgments- We thank Dr. William Sessa of Yale University School of Medicine for the generous gift of Ad-S1179D eNOS adenoviruses. We are also grateful to Dr. Sessa for critically reading the manuscript and for his valuable suggestions. b) Sources of Funding-This work was supported in part by American Heart Association Grant and Department of Medicine, BIDMC (MRA), by NIH grants 5T32HL007734-14 (SMD), HL080130 (CF), HL-46716 and HL-69024 (FWS). c) Disclosure- The authors have no conflict of interest to be disclosed.

Footnotes

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