Human Pulmonary Artery Endothelial Cells Research Articles

Upregulation of steroidogenic acute regulatory protein by hypoxia stimulates aldosterone synthesis in pulmonary artery endothelial cells to promote pulmonary vascular fibrosis.

The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension, suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in pulmonary arterial hypertension. Patients with pulmonary arterial hypertension, rats with Sugen/hypoxia-pulmonary arterial hypertension, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia-StAR signaling in HPAECs. Hypoxia (2.0% FiO2) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography-mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein-1 site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR-small interfering RNA or treated with the activator protein-1 inhibitor SR-11302 [3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid], hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in vivo. Our findings identify autonomous aldosterone synthesis in HPAECs attributable to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular remodeling and fibrosis.

Anti-endothelial cell antibodies in connective tissue diseases associated with pulmonary arterial hypertension.

To investigate the prevalence of anti-endothelial cell antibodies (AECA) in connective tissue diseases (CTD) associated with pulmonary arterial hypertension (PAH) and to corroborate the pathologic function of AECA in PAH-associated CTDs. AECA were detected by cellular enzyme-linked immunosorbent assay (ELISA) in sera of 19 PAH-associated CTD patients, 22 CTD patients without PAH involvement, and 20 age- and sex-matched healthy individuals as controls. Using IgG purified from the sera of AECA-positive, AECA-negative, and healthy subjects, the effects of AECA on the expression of ICAM-1 and the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) in cultured endothelial cells were also evaluated. A total of 12 of the 19 (63.2%) CTD patients with PAH, 9 of the 22 (40.9%) CTD patients without PAH, and 1 of the 20 (5%) healthy controls were positive for AECA, which were calculated as ELISA ratio (ER) values. ER values in PAH-associated CTD patients were significantly higher than those with CTD without PAH (3.68±2.05 versus 1.67±1.07, P<0.001). IgG purified from AECA-positive sera induced a significantly increased level of ICAM-1 expression after 48 h incubation (795.2±32.5 pg/mL) compared with AECA-negative or healthy control IgG (231.5±27.1 and 192.8±33.4 pg/mL, respectively; P<0.001). In addition, RANTES production by cultured human pulmonary arterial endothelial cells (HPAECs) increased in both a time- and concentration-dependent manner in response to incubation with purified AECA-positive IgG. AECA could be involved in CTD and might participate in the pathogenesis of PAH-associated CTD.

Endothelial apoptosis in pulmonary hypertension is controlled by a microRNA/programmed cell death 4/caspase-3 axis.

Pulmonary endothelial cell apoptosis is a transient, yet defining pathogenic event integral to the onset of many pulmonary vascular diseases such as pulmonary hypertension (PH). However, there is a paucity of information concerning the molecular pathway(s) that control pulmonary arterial endothelial cell apoptosis. Here, we introduce a molecular axis that when functionally active seems to induce pulmonary arterial endothelial cell apoptosis in vitro and PH in vivo. In response to apoptotic stimuli, human pulmonary arterial endothelial cells exhibited robust induction of a programmed cell death 4 (PDCD4)/caspase-3/apoptotic pathway that was reversible by direct PDCD4 silencing. Indirectly, this pathway was also repressed by delivery of a microRNA-21 mimic. In vivo, genetic deletion of microRNA-21 in mice (miR-21(-/-) mice) resulted in functional activation of the PDCD4/caspase-3 axis in the pulmonary tissues, leading to the onset of progressive PH. Conversely, microRNA-21-overexpressing mice (CAG-microRNA-21 mice) exhibited reduced PDCD4 expression in pulmonary tissues and were partially resistant to PH in response to chronic hypoxia plus SU 5416 injury. Furthermore, direct PDCD4 knockout in mice (PDCD4(-/-) mice) potently blocked pulmonary caspase-3 activation and the development of chronic hypoxia plus SU 5416 PH, confirming its importance in disease onset. Broadly, these findings support the existence of a microRNA-21-responsive PDCD4/caspase-3 pathway in the pulmonary tissues that when active serves to promote endothelial apoptosis in vitro and PH in vivo.

MicroRNA‐301b reduces PPARγ expression in transgenic sickle mice and in hemin‐treated human pulmonary artery endothelial cells (1089.22)

Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis resulting in the release of hemin into the blood. Our preliminary microarray data indicate that PPARγ expression is significantly reduced in hemin‐treated human pulmonary artery endothelial cells (HPAECs). To examine post‐transcriptional mechanisms of PPARγ regulation in pulmonary hypertension in SCD (SCD‐PH), Townes knockin SCD mice aged 12‐weeks were gavaged daily with vehicle or rosiglitazone (RSG, 10 mg/kg/day) for 10 d. PPARγ was decreased and ET‐1 expression was increased in sickle mice (SS) compared to littermate control mice (AA). Using an in vitro model that mimics acute extracellular hemin release, HPAECs were treated with DMSO or hemin (5 μM) for 72 h ± RSG (10 μM) for the final 24 h. Similarly, hemin reduced PPARγ and increased ET‐1 expression in HPAECs. MicroRNA (miR‐301b), which negatively regulates PPARγ, was increased in SCD mice and in hemin‐treated HPAECs. In contrast, PPARγ activation attenuates increases in miR‐301b levels in SS mice and in hemin‐induced HPAECs. These findings suggest that hemolysis in SCD increases miR‐301b to reduce PPARγ and increase ET‐1 levels. These results suggest novel pathways for SCD‐PH pathogenesis and therapy.Grant Funding Source: Supported by AHA‐SDG 13SDG14150004,CEB F16788‐00, Atlanta VA 1I01BX001910, NIH HL074518, DK102167

Combinatorial treatment with epigenetic modifiers inhibits thrombin‐induced eNOS/RhoA signaling and restores AJ integrity (1176.7)

Objective: Treatment of acute lung injury (ALI) is to target the pathways that lead to profound dysregulation of the restrictive endothelial barrier, and thereby restore lung microvascular endothelial barrier function and inhibit protein‐rich tissue fluid accumulation. Here we test the hypothesis that epigenetic modifiers TSA (Trichostatin‐A, HDAC inhibitor) and Aza (5‐aza‐2‐deoxycytidine, DNA methyl transferase inhibitor) together inhibit the thrombin induced eNOS/RhoA signaling and restore adherens junctions (AJs) integrity and diminished endothelial hyperpermeability.Results: The signaling, AJ integrity and endothelial permeability were assessed by RT‐PCR, immunofluoresence, Western analysis, and transendothelial resistance (TER). Our data show that combination of TSA+Aza is more effective in inhibiting thrombin‐induced lung hyperpermeability. We used an in vitro culture system of TER to determine the effect of TSA+Aza on thrombin‐induced change in resistance over the human pulmonary arterial endothelial cells. TSA+Aza treatment ameliorated the thrombin‐induced endothelial barrier disruption. The Western analysis data show that the treatment of TSA+Aza decreases the levels of thrombin induced RhoA and eNos signaling in lung endothelial cells.Conclusions: Our data indicate, for the first time, that TSA+Aza exert a protective role in preventing lung microvascular hyperpermeability and endothelial barrier disruption in thrombin‐induced lung injury, and thus have great potential as novel therapeutic agents

ADAM17 cleaves sphingosine 1 phosphate receptor 1 and regulates downstream signaling in endothelial cells (851.1)

Sphingosine‐1‐ phosphate receptor 1 (S1PR1) is a high affinity seven‐membrane G protein coupled receptor for bioactive lipid sphingosine‐1‐ phosphate (S1P). S1PR1 plays a critical role in regulating several endothelial cell functions such as proliferation, migration and importantly enhances endothelial barrier function. However, mechanism regulating S1PR1 cell surface retention remains unclear. Members of the ADAM (a disntintegrin and metalloproteases) family of proteases are known for cleaving the ectodomain of a variety of cell surface receptors thereby regulating their activities. In our present study, we addressed the possibility that ADAM family of proteases target S1PR1 and thereby downregulate it’s signaling in endothelial cells. We found that treatment of human pulmonary arterial endothelial (HPAE) cells with phorbol‐12‐myristate 13‐acetate (PMA), an activator of ADAM proteases, induces cleavage of S1PR1 within 2 hrs, while treatment with a broad range MMP inhibitor (TAPI‐0), stabilized S1PR1 expression. Additionally, PMA‐cleaved S1PR1 failed to localize on the cell surface rendering it refractive to exogenous S1P ligation and in enhancing the endothelial barrier. Also, Transendothelial resistance after ADAM17 deletion clearly shows enhanced barrier function. Over expression or depletion of ADAM17, but not ADAM10, in HPAE cells altered S1PR1 cell surface expression and endothelial barrier function by S1P identifying a novel role of ADAM17 as the protease responsible for targeting S1PR1.

0027: NGF contributes to medial and intimal remodelling of pulmonary arteries in pulmonary hypertension: receptors and signalling pathways involved in smooth muscle and endothelial cell proliferation

We have previously identified critical roles for the nerve growth factor NGF in pathophysiology of pulmonary hypertension (PH). We have here evaluated whether NGF may contribute to medial and intimal remodelling of pulmonary arteries in vivo in PH rat models. We have also investigated the NGF receptors and signalling pathways involved in vitro in NGF-induced proliferation of human pulmonary arterial smooth muscle (hPASMC) and endothelial cells (hPAEC). Experimental PH in the rat was induced either by a single injection of monocrotaline (MCT, 60mg/kg), or after 28 days of chronic hypoxia (CH, 0.5atm). Anti-NGF blocking antibodies (10μg/kg ip) were administered as a preventive or a curative treatment. Pulmonary arterial pressure was assessed after 28 days. Medial and intimal remodelling of pulmonary arteries were then evaluated on lung sections after hematoxylin/eosin or Van Gieson stainings. hPASMC or hPAEC proliferation was assessed by the BrdU technique, and involvement of the NGF receptors TrkA and p75 NTR was studied by both pharmalogical and SiRNA approaches. In vivo , NGF inhibition with anti-NGF blocking antibodies both prevented and reversed medial and intimal remodelling of pulmonary arteries in MCT and CH rats. In vitro , NGF-induced hPASMC proliferation occurred at low concentrations and only involved TrkA/PI3-kinase dependent signalling pathways. NGF-induced hPAEC proliferation occurred at higher concentrations and involved both TrkA and p75 NTR receptors activating PI3-kinase independent/ NF?B-dependent signalling pathways. We show here that NGF contributes to medial and intimal remodelling of pulmonary arteries in vivo in PH rat models. This may occur through stimulation of both hPASMC and hPAEC proliferation, but through distinct receptors and signalling pathways in these two different cell types, thus suggesting that blocking both NGF receptors may be of therapeutical interest in PH.

Exogenous Hydrogen Sulfide (H2S) Protects Alveolar Growth in Experimental O2-Induced Neonatal Lung Injury

BackgroundBronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD.MethodsWe took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo.Results In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation.ConclusionsH2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.

Aberrant Chloride Intracellular Channel 4 Expression Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension

Chloride intracellular channel 4 (CLIC4) is highly expressed in the endothelium of remodeled pulmonary vessels and plexiform lesions of patients with pulmonary arterial hypertension. CLIC4 regulates vasculogenesis through endothelial tube formation. Aberrant CLIC4 expression may contribute to the vascular pathology of pulmonary arterial hypertension. CLIC4 protein expression was increased in plasma and blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension and in the pulmonary vascular endothelium of 3 rat models of pulmonary hypertension. CLIC4 gene deletion markedly attenuated the development of chronic hypoxia-induced pulmonary hypertension in mice. Adenoviral overexpression of CLIC4 in cultured human pulmonary artery endothelial cells compromised pulmonary endothelial barrier function and enhanced their survival and angiogenic capacity, whereas CLIC4 shRNA had an inhibitory effect. Similarly, inhibition of CLIC4 expression in blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension attenuated the abnormal angiogenic behavior that characterizes these cells. The mechanism of CLIC4 effects involves p65-mediated activation of nuclear factor-κB, followed by stabilization of hypoxia-inducible factor-1α and increased downstream production of vascular endothelial growth factor and endothelin-1. Increased CLIC4 expression is an early manifestation and mediator of endothelial dysfunction in pulmonary hypertension.

BDNF secretion by human pulmonary artery endothelial cells in response to hypoxia

Within human pulmonary artery, neurotrophin growth factors [NTs; e.g. brain-derived neurotrophic factor (BDNF)] and their high-affinity receptors (tropomyosin-related kinase; Trk) and low-affinity receptors p75 neurotrophin receptor (p75NTR) have been reported, but their functional role is incompletely understood. We tested the hypothesis that BDNF is produced by human pulmonary artery endothelial cells (PAECs). In the context of hypoxia as a risk factor for pulmonary hypertension, we examined the effect of hypoxia on BDNF secretion and consequent autocrine effects on pulmonary endothelium. Initial ELISA analysis of circulating BDNF in 30 healthy human volunteers showed that 72h exposure to high altitude (~11,000ft, alveolar PO2=100mmHg) results in higher BDNF compared to samples taken at sea level. Separately, in human PAECs exposed for 24h to normoxia vs. hypoxia (1–3% O2), ELISA of extracellular media showed increased BDNF levels. Furthermore, quantitative PCR of PAECs showed 3-fold enhancement of BDNF gene transcription with hypoxia. In PAECs, BDNF induced NO production (measured using an NO-sensitive fluorescent dye DAF2-DA) that was significantly higher under hypoxic conditions, an effect also noted with the TrkB agonist 7,8-DHF. Importantly, hypoxia-induced NO was blunted by neutralization of secreted BDNF using the chimeric TrkB-Fc. Both hypoxia and BDNF increased iNOS (but not eNOS) mRNA expression. In accordance, BDNF enhancement of NO in hypoxia was not blunted by 50nM l-NAME (eNOS inhibition) but substantially lower with 100μM l-NAME (eNOS and iNOS inhibition). Hypoxia and BDNF also induced expression of hypoxia inducible factor 1 alpha (HIF-1α), a subunit of the transcription factor HIF-1, and pharmacological inhibition of HIF-1 diminished hypoxia effects on BDNF expression and secretion, and NO production. These results indicate that human PAECs express and secrete BDNF in response to hypoxia via a HIF-1-regulated pathway.

Atrial natriuretic peptide inhibits lipopolysaccharide-induced acute lung injury

ObjectivesWe recently reported that administration of atrial natriuretic peptide during the perioperative period has prophylactic effects with respect to not only cardiovascular but also respiratory complications following pulmonary resection. However, its mechanisms are not well understood. The objective of the present study was to investigate the mechanism of the prophylactic effects of atrial natriuretic peptide in an acute lung injury model. MethodsFor the evaluation of the early phase of pulmonary inflammation, in vitro and in vivo studies using lipopolysaccharide were used. In the in vitro study, the effects of atrial natriuretic peptide on the induction of E-selectin by lipopolysaccharide in human pulmonary artery endothelial cells were evaluated. In the in vivo study, the effects of atrial natriuretic peptide on lipopolysaccharide-induced inflammatory cell infiltration and cytokine levels including tumor necrosis factor-alpha and interleukin-6 in the bronchoalveolar lavage fluid in the lungs of C57/B6 mice were examined. The number of myeloperoxidase-positive staining cells in the tissue sections of the lung of lipopolysaccharide-administered C57/B6 mice was also evaluated. ResultsAtrial natriuretic peptide significantly attenuated the up-regulation of E-selectin expression induced by lipopolysaccharide in human pulmonary artery endothelial cells. There were significantly lower cell counts and levels of tumor necrosis factor-alpha and interleukin-6 in the bronchoalveolar lavage fluid of atrial natriuretic peptide-treated mice compared to control mice after lipopolysaccharide injection. In addition, there were significantly fewer myeloperoxidase-positive cells in atrial natriuretic peptide-treated mice than in control mice after lipopolysaccharide injection. ConclusionsAtrial natriuretic peptide had a protective effect in the lipopolysaccharide-induced acute lung injury model. Atrial natriuretic peptide may be of value in therapeutic strategies aimed at the treatment of acute lung injury such as pneumonia or acute respiratory distress syndrome.

Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

G protein–dependent basal and evoked endothelial cell vWF secretion

Abstractvon Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13−/−;Gα12−/− mice that could be normalized by infusion of human vWF. Blood from Gα12−/− mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12−/−, whereas thrombin-induced vWF secretion was defective in both EC-Gαq−/−;Gα11−/− and Gα12−/− mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

HIV-1/Cocaine Induced Oxidative Stress Disrupts Tight Junction Protein-1 in Human Pulmonary Microvascular Endothelial Cells: Role of Ras/ERK1/2 Pathway

Intravenous drug use (IVDU) is the major risk factor in the development of HIV-related pulmonary arterial hypertension (HRPAH); however, the pathogenesis of HRPAH in association with IVDU has yet to be characterized. Endothelial injury is considered to be an initiating factor for pulmonary vascular remodeling in animal models of PAH. Our previous study shows that simultaneous exposure to HIV-Trans-activator of transcription (Tat) and cocaine exacerbates both disruption of tight junction proteins and permeability of human pulmonary artery endothelial cells compared with either treatment alone. We here now demonstrate that this HIV-Tat and cocaine mediated endothelial dysfunction accompanies with increase in hydrogen peroxide and superoxide radicals generation and involves redox sensitive signaling pathway. Pretreatment with antioxidant cocktail attenuated the cocaine and Tat mediated disassembly of Zonula Occludens (ZO)-1 and enhancement of endothelial monolayer permeability. Furthermore, inhibition of NADPH oxidase by apocynin or siRNA-mediated knockdown of gp-91phox abolished the Tat/cocaine-induced reactive oxygen species (ROS) production, suggesting the NADPH oxidase mediated generation of oxidative radicals. In addition, ROS dependent activation of Ras and ERK1/2 Kinase was observed to be mediating the TJP-1 disassembly, and endothelial dysfunction in response to cocaine and Tat exposure. In conclusion, our findings demonstrate that Tat/cocaine -mediated production of ROS activate Ras/Raf/ERK1/2 pathway that contributes to disruption of tight junction protein leading to pulmonary endothelial dysfunction associated with pulmonary vascular remodeling.

CYP2J2 and EETs Protect against Oxidative Stress and Apoptosis in Vivo and in Vitro Following Lung Ischemia/Reperfusion

Background: Cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acids to epoxyeicosatrienoic acids (EETs). EETs exert various biological effects, including anti-inflammatory, anti-apoptotic, pro-proliferation, pro-angiogenesis, anti-oxidation, and anti-fibrosis effects. However, little is known about the role of CYP2J2 and EETs in lung ischemia/reperfusion injury. In this study, we examined the effects of exogenous EETs or CYP2J2 overexpression on lung ischemia/reperfusion injury in vivo and in vitro. Methods and Results: CYP2J2 gene was stably transfected into rat lungs via pcDNA3.1-CYP2J2 plasmid delivery, resulting in increased EETs levels in the serum and lung. A rat model of lung ischemia/reperfusion injury was developed by clamping the left lung hilum for 1 hour, followed by reperfusion for 2 hours. We found that CYP2J2 overexpression markedly decreased the levels of oxidative stress and cell apoptosis in lung tissues induced by ischemia/reperfusion. Moreover, we observed that exogenous EETs, or CYP2J2 overexpression, enhanced cell viability, decreased intracellular reactive oxygen species (ROS) generation, inhibited mitochondrial dysfunction, and attenuated several apoptotic signaling events in a human pulmonary artery endothelial cells (HPAECs)-based anoxia/reoxygenation model. These apoptotic events included activation of NADPH oxidase, collapse of mitochondrial transmembrane potential, and activation of pro-apoptotic proteins and caspase-3. These effects were mediated, at least partially, by the PI3K/Akt signaling pathway. Conclusion: These results reveal that CYP2J2 overexpression and exogenous EETs can protect against oxidative stress and apoptosis following lung ischemia/reperfusion in vivo and in vitro, suggesting that increasing the level of EETs may be a novel promising strategy to prevent and treat lung ischemia/reperfusion injury.

Selective Recapitulation of Conserved and Nonconserved Regions of Putative NOXA1 Protein Activation Domain Confers Isoform-specific Inhibition of Nox1 Oxidase and Attenuation of Endothelial Cell Migration

Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O2·-) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O2·- generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O2·- production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.

Nitrosation-dependent caveolin 1 phosphorylation, ubiquitination, and degradation and its association with idiopathic pulmonary arterial hypertension.

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%-80%, similar to that observed in IPAH patient-derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.

Abstract 9809: Upregulation of Aldosterone by Steroidogenesis Acute Regulatory Protein (StAR) is Associated With Connective Tissue Growth Factor-Mediated Pulmonary Vascular Fibrosis in Pulmonary Arterial Hypertension In Vivo

Hyperaldosteronsim is associated with increased pulmonary vascular remodeling in patients with pulmonary arterial hypertension (PAH). In cardiomyocytes, aldosterone (ALDO) promotes collagen deposition by increasing levels of connective tissue growth factor (CTGF). The role of CTGF in ALDO-mediated vascular remodeling in PAH, however, is not known. We hypothesized that upregulation of CTGF by ALDO is a novel signaling mechanism that modulates pulmonary vascular fibrosis in PAH. To test this hypothesis, cultured human pulmonary artery endothelial cells were exposed to ALDO (10 -9 -10 -7 mol/l) for 24 hr and changes in CTGF levels were assessed by immunoblotting. Compared to vehicle control, ALDO induced a dose-dependent increase in CTGF expression (3.4 ± 0.2 vs. 6.7 ± 1.9 vs. 10.4 ± 1.6 vs. 17.6 ± 1.9 units, P=0.02). We next investigated the effect of ALDO on CTGF in PAH in vivo. Compared to controls, rats exposed to SU-5416 (20 mg/kg) and hypoxia (10% O 2 ) (Su/Hyp) for 21 d demonstrated increased pulmonary artery systolic pressure (PASP) (26 ± 2 vs. 81 ± 5 mmHg, P&lt;0.05), which was associated with a 397% (P&lt;0.02) increase in plasma ALDO levels. Next, we investigated changes in levels of steroidogenic acute regulatory protein (StAR) as a mechanism by which to account for hyperALDO in PAH. Compared to controls, immunohistochemistry revealed increased StAR in the pulmonary arterioles of Su/Hyp-PAH rats (19.2 ± 0.9 vs. 30.2 ± 0.5 units, P&lt;0.01) and in patients with PAH (14.3 ± 1.4 vs. 19.3 ± 0.9 units, P&lt;0.02). Immunohistochemistry revealed that compared to controls, Su/Hyp-PAH rats expressed increased levels of CTGF (88.1 ± 11.5 vs. 171.8 ± 5.9 units, P&lt;0.01) and collagen III (61.5 ± 18.7 vs. 126.5 ± 6.3 units, P&lt;0.01) in pulmonary arterioles. ALDO antagonism with eplerenone (0.6 mg/g chow) decreased CTGF and collagen III levels in SU/Hyp-PAH by 81% (p&lt;0.01) and 38% (P&lt;0.01), respectively, to decrease PASP (81 ± 5 vs. 62 ± 7 mmHg, P&lt;0.05). These data demonstrate that upregulation of StAR is associated with hyperALDO in PAH, which promotes vascular fibrosis through a mechanism involving CTGF. Identifying StAR and/or CTGF as novel treatment target(s) to attenuate pulmonary vascular fibrosis has potential therapeutic implications for patients with PAH.

Abstract 18761: Non-Muscle Myosin Light Chain Kinase Mediates Vascular Endothelial Growth Factor Induced Vascular Hyperpermeability via Specificity Protein 1 During Asthmatic Inflammation

INTRODUCTION: Increased vascular permeability and accumulation of alveolar fluid are cardinal features of lung inflammation and asthma. Two targets for these responses are vascular endothelial growth factor (VEGF) and the non-muscle isoform of myosin light chain kinase (nmMLCK), a key regulator of vascular barrier function via actin-myosin mediated cell contraction. Both VEGF and nmMLCK mRNA and protein expression are upregulated in asthmatic patients. Supported by preliminary in vitro studies that demonstrate increased VEGF-induced nmMLCK expression, we propose a linkage between these two asthma targets and hypothesize that nmMLCK mediates VEGF induced vascular hyperpermeability in asthmatic inflammation. Methods &amp; Results: mRNA quantified by qPCR showed increases in nmMLCK mRNA in lung biopsies from human asthmatic patients and ovalbumin (OVA, 0.4 mg/kg) challenged murine asthma model. OVA-challenged mice overexpressing nmMLCK in the endothelium (nmMLCKec/ec) exhibit increased airway pressure time index (APTI) and BAL protein levels compared to WT, whereas OVA-challenged nmMLCK-/- mice demonstrated significant attenuation of increases in APTI and BAL protein levels. Human pulmonary artery endothelial cells (HPAEC) treated with recombinant hVEGF (100ng/ml) increased nmMLCK mRNA and protein expression. In silico analysis of nmMYLK 5’ UTR 2.4 kb region and in vitro luciferase promoter assay in HPAEC revealed transcription factor binding sites of downstream effectors of VEGF signaling, with -400bp upstream of TSS being the critical region, which included the binding sites for the transcription factor, Sp1. Consistent with this, reduced Sp1 expression by siRNA attenuated VEGF-induced decrease in transendothelial electrical resistance (TER) as measured by electrical cell-substrate impedance sensing system. CONCLUSIONS: Our data demonstrate that nmMLCK induced by VEGF is mediated through Sp1 in lung EC. nmMLCK is a novel asthmatic target and represents a potential candidate for therapeutic modulation of asthmatic inflammation.

Telmisartan attenuates monocrotaline-induced pulmonary artery endothelial dysfunction through a PPAR gamma-dependent PI3K/Akt/eNOS pathway

BackgroundPulmonary artery endothelial dysfunction has been demonstrated in pulmonary arterial hypertension (PAH). Telmisartan has beneficial effects in endothelial function in PAH patients; however, the underlying mechanisms for these effects remain unknown. AimsIn this study, we observed the effects of telmisartan on monocrotaline (MCT)-induced Sprague Dawley (SD) rat model of PAH. MethodsAfter a single-dose injection of MCT (60 mg/kg), oral administration of telmisartan (10 mg/kg/d) was started from day 1 to day 28 or with saline as MCT control. The vasorelaxation and remodelling of pulmonary arteries; the expression of peroxisome proliferator-activated receptor γ (PPARγ), Akt, eNOS; levels of phosphorylation of Akt (p-Akt) and phosphorylation of eNOS (p-eNOS) were analysed in isolated rat pulmonary arteries and cultured human pulmonary artery endothelial cells (HPAECs). ResultsCompared to MCT control group, telmisartan treatment ameliorated pulmonary artery endothelial dysfunction and remodelling, prevented the elevation of right ventricular systolic pressure (RVSP) induced by MCT. Immunoblotting results indicated lower levels of PPARγ, p-Akt and p-eNOS in pulmonary arteries treated with MCT alone and levels were significantly restored by co-treatment with telmisartan. In isolated pulmonary arteries, the impaired endothelium-dependent vasorelaxation of pulmonary arteries was improved following incubation with telmisartan for 12 h, whereas this effect was blocked by the inhibition of either PPARγ or phosphoinositide 3-kinase (PI3K) signals transduction. In cultured HPAECs, treatment with telmisartan increased PPARγ expression and promoted the phosphorylation of Akt and eNOS, thereby increasing the production of NO. These effects were abolished by the inhibition of PPARγ or PI3K. ConclusionTelmisartan protected against endothelial dysfunction in MCT-induced PAH through a PPARγ-dependent PI3K/Akt/eNOS pathway. Thus, telmisartan may be a promising therapeutic strategy for patients with a high risk of PAH.