Katie Y. Hood

Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells: Implications in Pulmonary Arterial Hypertension.

Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16&agr;-hydroxyestrone (16&agr;OHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16&agr;OHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-&agr;, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16&agr;OHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16&agr;OHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16&agr;OHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2 whereby 16&agr;OHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.

Serotonin signaling through the 5-HT1B receptor and NADPH oxidase 1 in pulmonary arterial hypertension

Objective— Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase–derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. Approach and Results— HPASMCs from controls and PAH patients, and PASMCs from Nox1−/− mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT1B receptor signaling and Nox1, confirmed in PASMCs from Nox1−/− mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Conclusions— Serotonin can induce cellular Src-related kinase–regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT1B receptors contribute to experimental pulmonary hypertension by inducing lung ROS production. Our results suggest that 5-HT1B receptor–dependent cellular Src-related kinase-Nox1-pathways contribute to vascular remodeling in PAH.

Vascular dysfunction and fibrosis in stroke-prone spontaneously hypertensive rats: The aldosterone-mineralocorticoid receptor-Nox1 axis

Aims: We questioned whether aldosterone and oxidative stress play a role in vascular damage in severe hypertension and investigated the role of Nox1 in this process. Materials and methods: We studied mesenteric arteries, aortas and vascular smooth muscle cells (VSMC) from WKY and SHRSP rats. Vascular effects of eplerenone or canrenoic acid (CA) (mineralocorticoid receptor (MR) blockers), ML171 (Nox1 inhibitor) and EHT1864 (Rac1/2 inhibitor) were assessed. Nox1‐knockout mice were also studied. Vessels and VSMCs were probed for Noxs, reactive oxygen species (ROS) and pro‐fibrotic/inflammatory signaling. Key findings: Blood pressure and plasma levels of aldosterone and galectin‐3 were increased in SHRSP versus WKY. Acetylcholine‐induced vasorelaxation was decreased (61% vs 115%) and phenylephrine‐induced contraction increased in SHRSP versus WKY (Emax 132.8% vs 96.9%, p < 0.05). Eplerenone, ML171 and EHT1864 attenuated hypercontractility in SHRSP. Vascular expression of collagen, fibronectin, TGF&bgr;, MCP‐1, RANTES, MMP2, MMP9 and p66Shc was increased in SHRSP versus WKY. These changes were associated with increased ROS generation, 3‐nitrotyrosine expression and Nox1 upregulation. Activation of vascular p66Shc and increased expression of Nox1 and collagen I were prevented by CA in SHRSP. Nox1 expression was increased in aldosterone‐stimulated WKY VSMCs, an effect that was amplified in SHRSP VSMCs (5.2vs9.9 fold‐increase). ML171 prevented aldosterone‐induced VSMC Nox1‐ROS production. Aldosterone increased vascular expression of fibronectin and PAI‐1 in wild‐type mice but not in Nox1‐knockout mice. Significance: Our findings suggest that aldosterone, which is increased in SHRSP, induces vascular damage through MR‐Nox1‐p66Shc‐mediated processes that modulate pro‐fibrotic and pro‐inflammatory signaling pathways.

181 Nox compartmentalization and protein oxidation in vascular smooth muscle cells – implications in vascular dysfunction in hypertension

NADPH oxidases (Noxs) are a major source of reactive oxygen species (ROS) in vascular cells. ROS are important signalling molecules with diverse actions. Mechanisms underlying differential ROS effects may relate, in part, to subcellular localization and Nox isoform specificity. We investigated the compartmentalization of Noxs and ROS in vascular smooth muscle cells (VSMC) and questioned whether these phenomena are altered in hypertension. VSMCs isolated from mesenteric arteries of Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. Subcellular compartmentalization of Noxs was evaluated by immunoblotting after organelle fractionation. ROS levels were measured by chemiluminescence (O2-) and amplex red (H2O2) in the absence or presence of of ML171 (Nox1 inhibitor), GKT136901 (Nox1/4 inhibitor), mito-tempol (mitochondrial-targeted antioxidant) and 4-PBA (ER stress inhibitor). Protein oxidation was assessed using the fluorescent probe DCP-Rho1 for protein sulfenylation and the oxyblot assay for protein carbonylation. Oxidation of protein tyrosine phosphatases (PTP) was evaluated by immunoblotting and Peroxiredoxin (Prx) oxidation was assessed by one-dimensional isoelectric focusing. Vascular reactivity was assessed by myography ± DTT (reducing agent) and peroxiredoxin inhibitor (Conoidin A). Expression of Nox1, Nox2 and Nox4 was greater in total cell homogenates from SHRSP versus WKY. Nox isoforms were detected in plasma membrane, ER and nucleus in both strains, but not in the mitochondria. Basal ROS generation was increased in SHRSP cells. In WKY only Nox1 inhibition decreased Ang II-induced ROS generation. Inhibition of Nox1 and Nox4 decreased basal and Ang II-induced ROS in SHRSP. Additionally, mito-tempol and 4-PBA reduced basal ROS generation in SHRSP. Analysis of protein oxidation revealed increased protein carbonylation and PTP oxidation in SHRSP. Furthermore, oxidation of the antioxidant enzymes Prxs was increased in SHRSP. Prx2, localised in the cytosol, and mitochondrial Prx3 were more oxidised in SHRSP cells than WKY cells. Noradrenaline-induced vascular contraction was reduced by DTT and Conoidin A. Our data demonstrate that Noxs are expressed in an organelle-specific manner, with Nox1,2,4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHRSP VSMCs Nox expression, ROS generation and protein oxidation are increased. Inhibition of oxidation attenuated vascular reactivity. These findings suggest an important role for Nox1/4 in oxidative stress and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

OS 02-01 ROLE OF ALDOSTERONE AND NADPH OXIDASE 1 IN HYPERTENSION-ASSOCIATED VASCULAR AGING.

Objective: In hypertension, the vasculature undergoes alterations that resemble those seen in aging. The involvement of aldosterone and Noxs in the mechanisms underlying age-associated vascular damage is unclear. We postulated that aging-like changes in the vasculature is amplified in hypertension due to increased aldosterone-induced Nox-redox signalling. Design and method: We assessed vascular aging in arteries from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Vascular smooth muscle cells (VSMC) were also used. Blood pressure was measured by tail-cuff. Vascular function was analysed by wire myography and structure by pressure myography. Gene expression was assessed by qPCR and protein levels by immunoblotting. Plasma Gal-3, aldosterone and peroxynitrite were measured by ELISA. Results: BP was increased only in SHRSP rats. Vascular hypercontractility in aged WKY was similar to that in SHRSP rats and normalised by inhibition of Nox1. Plasma aldosterone levels were elevated in SHRSP (p < 0.05; vs. WKY) and eplerenone normalised hypercontractility only in SHRSP. Increased vascular stiffness and fibrosis were observed in arteries from SHRSP compared to adult WKY rats. mRNA expression of Nox2, NoxA1 and NoxO1 was increased (p < 0.05; vs. WKY), while Nox4, p22phox and p47 phox gene levels were unchanged, in vessels from aged WKY and SHRSP rats. Nox1 mRNA expression and increased peroxynitrite were only increased in SHRSP rats (p < 0.05; vs. WKY). Aldosterone-induced Nox1 mRNA expression was exaggerated in VSMCs from SHRSP rats. p66SHC, an important pro-aging molecule, was activated in SHRSP and aged WKY arteries. In VSMCs, p66shc activation by aldosterone was blocked by ML171 (Nox1 inhibitor) and blunted in the vasculature from Nox1 KO mice. Conclusions: In conclusion, aging-like vascular phenotypes are associated with increased levels of Nox1 related genes and can be normalised by Nox1 inhibitors. Increased activation of Nox1 by aldosterone may contribute to an ‘aging’ vascular phenotype in hypertension.

OS 15-08 Ang(1-7) INFLUENCES ET-1 SIGNALING THROUGH MAS: ETBR INTERACTIONS: IMPLICATIONS IN PULMONARY HYPERTENSION.

Objective: Ang(1–7) has been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the importance of ET-1 in PH pathophysiology and endothelial dysfunction, we questioned whether Ang(1–7) influences ET-1 signaling in endothelial cells and whether Ang(1–7) treatment influences the ET-1 system in PH. Design and Method: Human endothelial cells (hEC) were stimulated with ET-1 in absence/presence of Ang(1–7). Mas and ETBR interaction was observed by immunoprecipitation. To characterize physical interactions, we utilized novel technology, employing a library of peptides spanning the MasR sequence, to define sites of ETBR binding. To investigate pathophysiological significance of our findings, we investigated whether Ang(1–7) treatment ameliorates PH. Hypoxia was used to induce PH in mice: normoxic (NV) and hypoxic vehicle (HV), normoxic (NA) and hypoxic PH (HA) treated with Ang(1–7) 30 &mgr;g/kg/day. Results: Ang(1–7) increases ET-1 release (125%) and ETBR protein (50%). ET-1-induced increases in VCAM-1 protein (38%) and TNF&agr; production (30%) were blocked by Ang(1–7). Pro-inflammatory effects were dependent on NO. Ang(1–7) increased NO production (257%) in a Mas and ETBR-dependent manner. Mutagenesis studies identified regions conferring specificity for ETBR binding. Peptide disruptors to prevent Mas/ETBR interaction were used for in vitro validation. We previously demonstrated in hEC that Ang(1–7) stimulates eNOS phosphorylation (180%), an effect inhibited by pre-incubation with peptide disruptors. In HP mice, RVSP (18.7 NV vs. 47.6mmHg HV, p < 0.05) RVH (0.19 NV vs. 0.28 HV, p < 0.01) and ET-1 levels (0.8 NV vs 2.4pg/ml HV, p < 0.05) were increased and blocked by Ang(1–7). Hypercontractility in pulmonary arteries of HV mice was attenuated by Ang(1–7). Conclusions: These findings indicate that vasoprotective effects of Ang(1–7) may be mediated through Mas:ETBR dimerization. In vivo studies support a relationship between Ang(1–7)/Mas and ET-1 systems. In conclusion we have identified a novel link between Ang(1–7) and ET-1 through physical interactions between Mas and ETBR.

JS ISH-ECCR-2 ANG-(1-7) AND ET-1, A NEW PARTNERSHIP.

ACE2 and Ang-1-7 have been shown to protect against pulmonary hypertension (PH). Mechanisms for this remain unclear. Considering the important role of ET-1 in the pathophysiology of PH and endothelial dysfunction, we questioned whether Ang-(1-7) influences ET-1 signaling in endothelial cells and whether Ang-(1-7) treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in the absence/presence of Ang 1-7 and showed that Ang 1-7 increased preproET-1 mRNA levels, ET-1 release, and ETBR protein levels. ET-1 increases in e-selectin mRNA, VCAM-1 protein and TNFα production were blocked by Ang 1-7. Pro-inflammatory effects were dependent on NO production. Ang 1-7 increased NO production in a Mas and ETBR-dependent manner. An interaction between Mas and ETBR was observed by immunoprecipitation. To further characterise a physical interaction between Mas/ETBR, we utilised novel technology, employing a library of overlapping peptides scanning the entirety of the MasR sequence, to define the interaction sites for ETBR binding. By substitution or sequence truncation we identified two distinct regions on the MasR that confer specificity for ETBR binding. Peptides that disrupt each of these regions to prevent Mas/ETBR interaction were developed for in vitro validation. To investigate the pathophysiological significance of our findings, we investigated whether Ang-(1-7) treatment ameliorates PH and whether this is associated with changes in ET-1 status. Hypobaric hypoxia was used to induce PH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with orally active Ang 1-7 30 μg/kg/day for 14 days. In HP mice, RVSP, RVH and ET-1 levels were increased and blocked by Ang 1-7 treatment. Hyper-contractility and endothelial dysfunction in pulmonary arteries of HP mice compared to NC was attenuated by Ang 1-7. These findings indicate that vasoprotective effects of Ang-(1-7) may be mediated through dimerization of MAS:ETBR. In vivo studies support a relationship between the Ang-(1-7)/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang-(1-7) and ET-1 through physical interactions between MAS and ETBR.

Nicotinamide adenine dinucleotide phosphate oxidase–mediated redox signaling and vascular remodeling by 16α-hydroxyestrone in human pulmonary artery cells

Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16&agr;-hydroxyestrone (16&agr;OHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16&agr;OHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-&agr;, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16&agr;OHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16&agr;OHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16&agr;OHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2 whereby 16&agr;OHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase–Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery CellsNovelty and Significance

Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16&agr;-hydroxyestrone (16&agr;OHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16&agr;OHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-&agr;, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16&agr;OHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16&agr;OHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16&agr;OHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2 whereby 16&agr;OHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase–Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery CellsNovelty and Significance: Implications in Pulmonary Arterial Hypertension

Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16&agr;-hydroxyestrone (16&agr;OHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16&agr;OHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-&agr;, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16&agr;OHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16&agr;OHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16&agr;OHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2 whereby 16&agr;OHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH, particularly important in women.