Pulmonary Artery Endothelial Cells Research Articles

Abstract 11559: Acute and Chronic Effect of Heme on Vascular Function and Pulmonary Hypertension

Introduction: Patients with hemolytic anemia are at high risk of developing lung disease. We recently reported that free heme in circulation causes acute lung endothelial barrier dysfunction via activation of MKK3/p38 signaling. However, we did not evaluate the effects of chronic heme infusion in the lungs. Hypothesis: Chronic free heme infusion will cause sustained activation of the MKK3/p38 pathway leading to inflammation and vascular remodeling in the lungs. Methods: Mitogen-activated protein kinase kinase-3 knockout (MKK3-KO) mice were used for this study. Acute group mice received heme 250μM for 24-hours by i.v injection as a single dose, and the chronic group mice received 2-weeks of continuous heme i.v injection using an osmotic minipump. Human pulmonary artery endothelial cells (HPAEC) were studied in endothelial cell dysfunction experiments. Results: Heme treatment (50μM) disrupts HPAEC endothelial barrier, tube formation and decreases HPAEC migration capacity suggesting direct heme involvement in the endothelial dysfunction. Chronic heme exposure of WT mice (250uM, 2wks) increased right ventricular (RV) systolic pressure (SP) (31.89±0.88mmHg), RV-hypertrophy (0.38±0.02), and pulmonary vascular remodeling (18.47±1.15 μm) compared to the MKK3-KO group (RVSP-29.33±0.69mmHg, Fulton index-0.33±0.01, and wall thickness-13.69±1.13μm, p<0.05). Loss of tight junction proteins and activation of MKK3/p38/HSP27 axis was noticed in heme-treated WT. MKK3-KO significantly attenuated downstream targets of p38 such as Akt, STAT3, and ERK1/2. Furthermore, the MKK3-KO attenuated endothelial barrier dysfunction by maintaining the tight-junction protein, zona occludens-1 and reducing lung inflammatory cell infiltration. Cytokine multiplex analysis of plasma exhibited an inflammatory cytokine storm with a surge of 21-cytokines in WT mice, but the MKK3-KO mice were substantially protected. This includes IFN-g (1502 vs. 113 Fold Control (FC)), IL-1b (6.8 vs. 2.2 FC), IL-6 (221 vs. 6.9 FC), etc. Conclusion: Our findings suggest that free-heme triggers MKK3/p38 cascade activation resulting in the cytokines storm, endothelial barrier dysfunction, and proliferative signaling activation, all contributing to PH manifestation.

Abstract 10253: Sin3a Gene Delivery Restores Bmpr2 Expression Through Foxk2 in Pulmonary Arterial Endothelial Cells and Inhibits Sugen/Hypoxia-Induced Pah

Background: Pulmonary arterial hypertension (PAH) is a fatal lung disease of multifactorial etiology, with no curative treatment. Several studies have previously suggested that the loss of BMPR2 expression is associated with a severe hemodynamic profile and poor outcomes in PAH patients. Furthermore, preclinical studies showed that the loss of BMPR2 function is sufficient to induce a PAH-like phenotype. Recently, our group showed that SIN3a (Switch-Independent 3) plays a critical role in the epigenetic regulation of BMPR2 expression in human pulmonary arterial smooth muscle cells (hPASMC). However, our preliminary data suggested that SIN3a regulates BMPR2 expression through a different pathway independently of DNA-methylation in human pulmonary arterial endothelial cells (hPAEC). Methods/Results: We found a significant downregulation of SIN3a expression in the PAEC isolated from PAH patients. In hPAEC, our results showed that SIN3a inhibits cell proliferation, migration and upregulates BMPR2 independently of the methylation status by upregulating the FOXK2 transcription factor. Using a loss-of-function approach using a specific shRNA against FOXK2, we found that the loss of FOXK2 expression using a specific shRNA decreased the expression of BMPR2 in hPAECs. Interestingly, we found that SIN3a overexpression in FOXK2-depleted hPAEC restored the expression of BMPR2. Our results also showed that SIN3a regulates hPAEC proliferation in a FOXK2-dependant manner. Mechanistically, we found that SIN3a interacts physically with FOXK2 to promote its nuclear localization. As a result, we also found enrichment in FOXK2 abundance within the BMPR2 promoter by ChIP-qPCR in PAEC. In vivo , our results showed that restoring SIN3a expression by lentivirus-mediated gene transfer significantly decreased SuHx-induced PAH in mice, as illustrated by decreased vascular remodeling, RV hypertrophy (Fulton index, hypertrophic markers, cardiomyocyte size), and RV systolic pressure. Conclusions: Altogether, our study revealed that SIN3a plays a critical role in the regulation of BMPR2 expression through FOXK2. Additionally, our study identifies lung-targeted SIN3a gene therapy as a new promising therapeutic strategy for treating PAH patients.

Abstract 12577: Pulmonary Endothelial Sulfatase-1 is Regulated by NEDD9 and Increased in Pulmonary Arterial Hypertension

Introduction: Oxidation of the scaffolding protein NEDD9 by aldosterone (ALDO) increases NEDD9 bioactivity in human pulmonary artery endothelial cells (HPAECs), leading to pulmonary arterial hypertension (PAH). In tumor cells, oxidizing conditions alter Sulfatase-1 (SULF-1) activity. In turn, SULF-1 catalyzes 6-O-sulfate removal from heparan sulfate proteoglycans that releases cytokines implicated in the pathogenesis of PAH. Hypothesis: NEDD9 regulates SULF-1 expression in HPAECs, which is associated with pulmonary arterial remodeling in PAH. Methods: HPAECs were treated with ALDO (10 -7 mol/L) for 24 hr and then untransfected (Un-Tx) or transfected with siRNA against NEDD9, and cell lysates were used to isolate mRNA for RNA-Seq. Separately, SULF1 expression was measured by immunoblot in HPAECs transfected with si-NEDD9 or scrambled control siRNA. Paraffin-embedded lung specimens from donor controls (N=5), patients with PAH (N=5), or the monocrotaline (MCT) (inflammatory) and SU-5416/hypoxia (angioproliferative) experimental rat models of PAH were analyzed by immunofluorescence to quantify SULF1, NEDD9, and endothelial PECAM-1. Results: In ALDO-treated HPAECs, si-NEDD9 decreased SULF1 mRNA quantity vs. Un-Tx (logFC -6.39, N=3, P<0.001, FDR<0.001). Transfection with si-NEDD9 also decreased SULF1 protein compared to si-Scr (14 ± 2.0 vs. 2.7 ± 1.0 arb. units, N=3, P<0.01) in vitro . In remodeled pulmonary arterioles from MCT-PAH and SU-5416/hypoxia-PAH, SULF1 was increased by 5.6-fold (P<0.01) and 6.6-fold (P<0.01), respectively, and correlated strongly with NEDD9 protein (r=0.91, N=9, P<0.001). The translational relevance of these data was assessed next: compared to donor controls, pulmonary endothelial SULF1 was increased in remodeled pulmonary arterioles from PAH patients in situ (0.03 ± 0.01 vs. 0.14 ± 0.03 arb. units, P<0.05). Conclusions: SULF1 is regulated by the scaffolding protein NEDD9 in HPAECs in vitro , and remodeled pulmonary arterioles from two experimental PAH models and PAH patients express increased NEDD9-SULF1. Identifying the molecular mechanism(s) regulating NEDD9-dependent regulation of SULF1 may have important implications for therapeutic target discovery to prevent vascular remodeling in PAH.

Abstract 6931: Epigenetic Modification of the Von Willebrand Factor Promotor Drives in situ Thrombosis in Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thromboembolic obstructions of the pulmonary circulation; disease progression may involve in situ thrombosis. CTEPH patients have elevated Von Willebrand Factor (VWF) plasma levels and we hypothesized that endothelial secretion of VWF in CTEPH recruits platelets to the pulmonary vascular wall, thereby driving in situ thrombosis. Methods: CTEPH specific primary pulmonary artery endothelial cells (PAEC) were isolated from pulmonary endarterectomy material and control PAECs from patients undergoing lobectomy. Endothelial-platelet interaction was studied by an human in vitro model of thrombosis. We used an antibody and knockdown mediated approach to inhibit VWF and validate its functional relevance. Cell culture findings were validated in human tissue with immunofluorescence in situ hybridization, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Results: CTEPH-PAEC showed increased platelet adhesion compared to control. This was mediated by a 5-fold enhanced VWF gene expression, resulting in higher release of VWF protein upon endothelial activation. The increase in VWF mRNA was confirmed in human pulmonary artery tissue and was associated with elevated Nuclear Factor κB 2 (NFκB2) protein expression. Epigenetic histone modifications of reduced histone tri-methylation (H3K27Me3) and increased histone acetylation (H3K27Ac) of the VWF promotor in CTEPH-PAEC, support NFκB2 binding that drives VWF transcription. Knockdown of NFκB2 normalized VWF RNA expression levels and reversed the pro-thrombotic phenotype observed in CTEPH-PAEC. Conclusions: We show experimental evidence of VWF-mediated in situ thrombosis in CTEPH. Epigenetic modifications of the VWF promotor in CTEPH-PAEC, enhances NFκB2 binding and subsequently VWF transcription and platelet adhesion. We demonstrate a direct molecular link between inflammatory pathways and thrombosis in the pulmonary vascular wall that emphasizes the central role of in situ thrombosis in CTEPH.

Abstract 13033: Cavin/Caveolin Interaction Modulates BMP/Smad Signaling in Pulmonary Artery Endothelial Cells

Background: Pulmonary hypertension (PH) is a progressive disease associated with poor prognosis. Caveolin-1 (Cav1) and Cavin-1 are components of caveolae, and both interact with each other and influence the composition and stabilization of caveolae. Cav1 is identified as a related gene of pulmonary arterial hypertension (PAH). Gene mutation of bone morphogenetic protein type II receptor (BMPRII) is the most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Methods and Results: As in previous reports, Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy. Caveolae disappeared in pulmonary microvascular endothelial cells of Cav1 knockout mice. We then examined the role of Caveolin-Cavin and the association with BMP/Smad signaling in human pulmonary artery endothelial cells (hPAECs). Cav1 knockdown in hPAECs reduced BMPRII at the plasma membrane and Smad 1/5/9 phosphorylation. Cav1 knockdown also significantly increased hypoxia-induced apoptosis in hPAECs. Cav1 was associated with BMPRII at the membrane of hPAECs. In addition, Cavin-1 inhibited the interaction of BMPRII with Cav1 and reduced BMPRII localization on the membrane of hPAECs. Cavin-1 knockdown reversed BMPRII localization at the plasma membrane and Smad 1/5/9 phosphorylation induced by Cav1 knockdown. Both Cavin-1 and BMPRII were associated with the Cav1 scaffolding domain. These results suggest that Cavin-1 interacts with Cav1 and inhibits the association of Cav1 with BMPRII, resulting in modulating Smad signaling pathway. Conclusions: Cavin-1 and BMPRII are competitively associated with Cav1 at the plasma membrane and modulating BMP/Smad signal transduction in PAECs. Our findings reveal a previously undescribed mechanism via Cavin-Caveolin interaction in BMP/Smad signaling-modulated PH development.

Abstract 12319: 16alpha-Hydroxyestrone Downregulates SOX17 During the Development of Pulmonary Arterial Hypertension

Introduction: Sexual dimorphism in pulmonary arterial hypertension (PAH) is attributed, in part, to estrogen signaling. 16α-hydroxyestrone (16αOHE) is considered a major contributor to PAH pathogenesis. Recent genetic studies have also suggested that deficiency of SOX17, an endothelial cell (EC)-specific transcription factor, contributes to PAH risk. While functional studies of SOX17 are absent, we hypothesized that 16αOHE contributes to PAH, in part, via, SOX17 downregulation. Methods/Results: Sox17 expression was reduced in 3 animal PAH models and in human pulmonary artery ECs (HPAECs) isolated from patients with PAH (vs controls). Inducible Tie2-specific Sox17 knockout ( Sox17 EC-/- ) mice exhibited increased right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and PA wall thickness (PAWT) after chronic hypoxia (CH) (Fig 1A). Inducible Tie2- Sox17 transgenic overexpressing ( Sox17 Tg ) mice attenuated CH-induced PH (Fig 1B). While not evident across murine sex, Sox17 expression was increased in baseline lungs from male compared to female rats. Supporting in silico evidence of estrogen response elements (ERE) on the SOX17 promoter, HPAECs exposed to 16αOHE reduced SOX17 expression and promoter luciferase activity via ERα, which was partly negated by serial ERE mutagenesis (Fig 1C ) . Lungs from ERα loss-of-function mutant rats (vs control) confirmed in vivo reductions of Sox17 expression. Sox17 Tg mice attenuated 16αOHE-mediated PH after CH (Fig 1D). To translate these data, we identified a functional coding SNP in the ESR1 gene (encoding ERα), rs746432, previously shown to reduce transcriptional activity of ERα. The SNP was associated with reduced pulmonary vascular resistance in patients with PAH (n=702, Fig 1E) in adjusted analyses. Conclusion: Validating genetic studies, SOX17 deficiency augments preclinical PAH. 16αOHE mediates PH development via downregulation of SOX17, linking SOX17 genetics with the observed sexual dimorphism.

Abstract 12410: The Critical Role of Mitochondrial Dysfunction in the Impaired Angiogenesis Associated With the Pulmonary Hypertensive Phenotype

Introduction: Mitochondrial dysfunction (MD) is known to be tightly associated with the pathobiology of pulmonary arterial hypertension (PAH). Recently, we discovered that rats reproducing a human mutation in NFU1 (NFU1 G206C ) have a severely altered mitochondrial function and spontaneously develop PAH characterized by increased right ventricle (RV) systolic pressure, RV hypertrophy, and severe obliterative disease of small pulmonary arteries. Hypothesis: In this study, we investigated whether MD induced by NFU1 mutation is sufficient to impair pulmonary angiogenesis contributing to PAH pathogenesis. Methods: Angiogenesis was assessed by micro-computed tomography of the pulmonary vasculature, microangiography of small pulmonary arteries (PAs), and histological evaluation of pulmonary capillary density. The angiogenic capacity was also investigated in vitro using cell migration and tube formation assays and analysis of EC tip cell population by flow cytometry. Results: MD significantly reduced the complexity of the pulmonary vascular network and prevented its further advancement (PA density (mean intensity/pxl) in WT vs. NFU1 G206C at 6 and 10 wks of age is 0.07±0.004 vs. 0.03±0.002 and 0.1±0.004 vs. 0.04±0.003; p<0.0001; N=5). NFU1 G206C rats had a reduced number of PA branches and junctions and impaired PA capillary density. PA endothelial cells isolated from NFU1 G206C rats demonstrated a strongly attenuated cell migration (~30% of WT) and tube formation capacity and a ~2.2 fold decrease in the number of CD-304 positive EC tip cells. Supplementation of NFU1 G206C rats with lipoic acid (LA, 1.70 mM in drinking water) aimed to attenuate the LA-synthase insufficiency secondary to NFU1 mutation, restored mitochondrial function, preserved the complexity of PA vascular network at WT level, and averted the PAH phenotype. Conclusions: We conclude that MD plays a causative role in the impaired pulmonary angiogenesis. Indeed, the lungs of NFU1 G206C rats fully reproduced the “dead-tree” picture of pulmonary vasculature described in PAH patients. At the same time, normalized mitochondrial function was sufficient to preserve pulmonary angiogenesis and prevent the PAH, suggesting the protective role of MD ameliorative approaches.

CYP2J2 and EETs protect against pulmonary\xa0arterial hypertension with lung ischemia\u2013reperfusion injury in vivo and in vitro

BackgroundCytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs), which exert anti-inflammatory, anti-apoptotic, pro-proliferative, and antioxidant effects on the cardiovascular system. However, the role of CYP2J2 and EETs in pulmonary arterial hypertension (PAH) with lung ischemia–reperfusion injury (LIRI) remains unclear. In the present study, we investigated the effects of CYP2J2 overexpression and exogenous EETs on PAH with LIRI in vitro and in vivo.MethodsCYP2J2 gene was transfected into rat lung tissue by recombinant adeno-associated virus (rAAV) to increase the levels of EETs in serum and lung tissue. A rat model of PAH with LIRI was constructed by intraperitoneal injection of monocrotaline (50 mg/kg) for 4 weeks, followed by clamping of the left pulmonary hilum for 1 h and reperfusion for 2 h. In addition, we established a cellular model of human pulmonary artery endothelial cells (HPAECs) with TNF-α combined with anoxia/reoxygenation (anoxia for 8 h and reoxygenation for 16 h) to determine the effect and mechanism of exogenous EETs.ResultsCYP2J2 overexpression significantly reduced the inflammatory response, oxidative stress and apoptosis associated with lung injury in PAH with LIRI. In addition, exogenous EETs suppressed inflammatory response and reduced intracellular reactive oxygen species (ROS) production and inhibited apoptosis in a tumor necrosis factor alpha (TNF-α) combined hypoxia-reoxygenation model of HPAECs. Our further studies revealed that the anti-inflammatory effects of CYP2J2 overexpression and EETs might be mediated by the activation of PPARγ; the anti-apoptotic effects might be mediated by the PI3K/AKT pathway.ConclusionsCYP2J2 overexpression and EETs protect against PAH with LIRI via anti-inflammation, anti-oxidative stress and anti-apoptosis, suggesting that increased levels of EETs may be a promising strategy for the prevention and treatment of PAH with LIRI.Graphical

MiR-1226-3p Promotes eNOS Expression of Pulmonary Arterial Endothelial Cells to Mitigate Hypertension in Rats via Targeting Profilin-1.

In pulmonary arterial hypertension (PAH), microRNAs (miRNAs) are related with dysfunction of pulmonary arterial endothelial cells. miR-1226-3p was found to be downregulated in the serum of PAH patients, while few studies have illustrated the regulation mechanism of miR-1226-3p on PAH. In this study, we aimed to systematically investigate the role of miR-1226-3p in PAH. Sprague-Dawley (SD) rats were treated with monocrotaline (MCT) to establish the PAH models. The right ventricular systolic pressure (RVSP), ratio of the right ventricle to the left ventricle with septum (RV/(LV+S) ratio), and nitric oxide (NO) content were used to reflect the symptom of the rats. The rat models were used to observe the regulation mechanism of miR-1226-3p on PAH, and dual-luciferase reporter assay was used to verify the binding effect of miR-1226-3p to Pfn1. Besides, the qRT-PCR and western blot were used to measure the expression levels of miR-1226-3p and some keys proteins such as eNOS and Pfn1, respectively. The results showed that the PAH models were established successfully. The RVSP levels and the RV/(LV+S) ratio of the PAH rats were higher than those indexes in normal rats, while the NO content showed the opposite trends. Besides, the decreased miR-1226-3p and eNOS were, respectively, found in the PAH rats and rPAECs, and overexpressed miR-1226-3p could reverse the disadvantages of the PAH rats including increased RVSP, high RV/(LV+S) ratio, and decreased NO content. Furthermore, miR-1226-3p could directly target the 3′-UTR of Profilin-1 (Pfn1). Overexpressed Pfn1 led to decreased eNOS, while miR-1226-3p could partly inhibit the expression of Pfn1 and increase the expression level of eNOS in rPAECs. In summary, this study suggests miR-1226-3p as a protector to increase eNOS, improve NO content in rPAECs of the PAH rats via targeting Pfn, and finally protect the rats from the injury induced by PAH.

Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension.

Background: The hypoxia-induced pro-proliferative and anti-apoptotic characteristics of pulmonary arterial endothelial cells (PAECs) play critical roles in pulmonary vascular remodeling and contribute to hypoxic pulmonary arterial hypertension (PAH) pathogenesis. However, the mechanism underlying this hypoxic disease has not been fully elucidated.Methods: Bioinformatics was adopted to screen out the key hypoxia-related genes in PAH. Gain- and loss-function assays were then performed to test the identified hypoxic pathways in vitro. Human PAECs were cultured under hypoxic (3% O2) or normoxic (21% O2) conditions. Hypoxia-induced changes in apoptosis and proliferation were determined by flow cytometry and Ki-67 immunofluorescence staining, respectively. Survival of the hypoxic cells was estimated by cell counting kit-8 assay. Expression alterations of the target hypoxia-related genes, cell cycle regulators, and apoptosis factors were investigated by Western blot.Results: According to the Gene Expression Omnibus dataset (GSE84538), differentiated embryo chondrocyte expressed gene 1-peroxisome proliferative-activated receptor-γ (Dec1-PPARγ) axis was defined as a key hypoxia-related signaling in PAH. A negative correlation was observed between Dec1 and PPARγ expression in patients with hypoxic PAH. In vitro observations revealed an increased proliferation and a decreased apoptosis in PAECs under hypoxia. Furthermore, hypoxic PAECs exhibited remarkable upregulation of Dec1 and downregulation of PPARγ. Dec1 was confirmed to be crucial for the imbalance of proliferation and apoptosis in hypoxic PAECs. Furthermore, the pro-surviving effect of hypoxic Dec1 was mediated through PPARγ inhibition.Conclusion: For the first time, Dec1-PPARγ axis was identified as a key determinant hypoxia-modifying signaling that is necessary for the imbalance between proliferation and apoptosis of PAECs. These novel endothelial signal transduction events may offer new diagnostic and therapeutic options for patients with hypoxic PAH.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

Involvement of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) in endothelial dysfunction associated with pulmonary hypertension

AimsIncreased proliferation, inflammation, and endothelial microparticle (EMP) generation in the pulmonary vasculature lead to endothelial dysfunction in pulmonary hypertension (PH). Interestingly, MK2, a downstream of p38MAPK, is a central regulator of inflammation, proliferation, and EMP generation in cardiovascular diseases. However, the role of MK2 in pulmonary endothelial dysfunction remains unexplored. Main methodsThe Human Pulmonary Artery Endothelial cells (HPAECs) were exposed to hypoxia (1% O2) for 72 h, and MK2 inhibition was achieved by siRNA treatment. Western blotting, qualitative RT-PCR, immunocytochemistry, flow cytometry and enzyme-linked immunoassays were conducted to study pathological alterations and molecular mechanisms. Neoangiogenesis was studied using cell migration and tubule formation assays. For in vivo study, Male Sprague Dawley rats and MK2 knock-out mice with littermate control were treated with monocrotaline (MCT) 60 mg/kg and 600 mg/kg, respectively (s.c. once in rat and weekly in mice) to induce PH. MMI-0100 (40 μg/kg, i.p. daily for 35 days), was administered in rats to inhibit MK2. Key findingsMK2 inhibition significantly decreased inflammation, cell proliferation, apoptosis resistance, and improved mitochondrial functions in hypoxic HPAECs. Hypoxia promoted cell migration, VEGF expression, and angiogenesis in HPAECs, which were also reversed by MK2 siRNA. MK2 inhibition decreased EMP generation and increased the expression of p-eNOS in hypoxic HPAECs, a marker of endothelial function. Furthermore, MK2 deficiency and inhibition both reduced the EMP generation in mice and rats, respectively. SignificanceThese findings proved that MK2 is involved in endothelial dysfunction, and its inhibition may be beneficial for endothelial function in PH.

Cavin-1 modulates BMP/Smad signaling through the interaction of Caveolin-1 with BMPRII in pulmonary artery endothelial cells

Abstract Background Pulmonary hypertension (PH) is a progressive disease associated with poor outcomes. Caveolin-1 (Cav1) and Cavin-1 are components of caveolae, and Cav1 is identified as a related gene of pulmonary arterial hypertension (PAH). Gene mutations of bone morphogenetic protein type II receptor (BMPRII) is the most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Purpose Our study aims to investigate the relationship between Caveolin-Cavin system and BMP/Smad signaling pathway in pulmonary artery endothelial cells (PAECs). [Methods] Cav1 knockout mice were used to assess PH, and caveolae in PAECs were observed by electron microscope. After knocking down Cav1 and/or Cavin-1 in human PAECs (hPAECs) using siRNA, we evaluated the phosphorylation of Smad by Western blotting. Apoptosis was explored by flow cytometry. To assess the interaction between Cav1 and BMPRII, and the effect of Cavin-1 for this interaction and BMP/Smad signaling, we performed immunoprecipitation, Co-immunostaining, Proximal Ligation Assay (PLA), GST pulldown assay, and Western blotting. Results As in previous reports, Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy and PAECs isolated from Cav1 knockout mice showed caveolae disappearance. Cav1 knockdown in hPAECs reduced BMPRII at the plasma membrane and Smad 1/5/9 phosphorylation. Cav1 knockdown also significantly increased hypoxia-induced apoptosis in hPAECs. Co-immunostaining revealed that Cav1 was associated with BMPRII at the membrane of hPAECs. Cavin-1 inhibited the interaction of BMPRII with Cav1 and reduced BMPRII localization on the membrane of hPAECs. GST pulldown assay revealed that Cavin-1 and BMPRII were associated with Cav1 through the scaffolding domain in Cav1. These findings suggest that Cavin-1 and BMPRII are competitively associated with Cav1. Cavin-1 knockdown improved the interaction between Cav1 and BMPRII and inhibited both BMPRII reduction at the plasma membrane and Smad 1/5/9 dephosphorylation. Conclusions Cavin-1 affects the interaction of Cav1 with BMPRII at the plasma membrane and modulates BMP/Smad signaling in PAECs. The binding of Cavin-1 to Cav1 enhances the interaction between BMPR2 and Cav1, resulting in stabilization of BMPRII localization at the plasma membrane in PAECs and prevention of BMP/Smad signaling attenuation, which is important for PAH development. Funding Acknowledgement Type of funding sources: None.

Effects of varying shear stress profiles on the regulation of pulmonary artery endothelial cell gene expression: a focus on selected mediators of vascular tone, inflammation and angiogenesis

Abstract Background Pulmonary arterial hypertension (PAH) is a complex pathology characterized by obliterative vascular remodeling that leads to right heart failure and death. Predisposition to PAH is associated with mutations in the BMPR2 gene in approximately 70–80% of familial cases and around 30% for that of sporadic PAH. The study of the pathogenetic basis of PAH is often performed in static endothelial cultures. Such two-dimensional, isolated cell microenvironments fail to consider the heterogeneity in mechanical stress acting on endothelial cells in various regions of the pulmonary vascular tree. In the remodeled pulmonary vasculature, low and oscillatory shear stress is observed in the proximal pulmonary artery with high shear stress in distal pre-capillary pulmonary arterioles. Therefore, the impact of varied shear profiles (including both laminar and oscillatory flow) on pulmonary artery endothelial cell (and that of BMPR2-deplete) gene expression of common vasoactive (EDN1, ENOS), proinflammatory (IL6, IL8) and angiogenic mediators (ANG2, VEGFA), are poorly described. Purpose To evaluate the effects of shear stress magnitude, including unidirectional and oscillatory flow on BMPR2-knockdown human pulmonary artery endothelial cell (HPAEC) gene expression of EDN1, ENOS, IL6, IL8, ANG2 and VEGFA. Methods HPAECs were transfected with siRNA directed against BMPR2 (siB2) or with a non-targeting control (siCon). Cells were exposed to 10 hours of laminar or oscillatory flow (1Hz; 1.5 dyn/cm2, 15 dyn/cm2 or 90 dyn/cm2) using a parallel-plate fluid flow chamber system. Measurement of mRNA expression was performed using qPCR. Results Shear stress intensity and flow type (unidirectional and oscillatory) mediated diverse effects on HPAEC gene expression across the markers studied. Changes in gene expression were calculated relative to that of static siCon-transfected HPAECs and in such a manner are summarized as fold changes in the table below. Asterisks are shown where significant fold differences are reported. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. aP≤0.05, bP≤0.05, cP≤0.05, denote comparisons between groups. Of note, no significant differences in gene expression were observed between static siCon and static siB2. Conclusions For the markers studied, different magnitudes of shear stress and flow profiles (together with BMPR2 loss) exhibit varied patterns of gene expression in the pulmonary vascular endothelium. As such, this illustrates the need for wider study of in vitro endothelial-shear stress interactions in understanding mechanisms of remodeling in PAH. Funding Acknowledgement Type of funding sources: None. Table 1

A rare nonsense variant of the X-gene in a paediatric patient with severe peripheral pulmonary stenosis

Abstract Background Most cases of peripheral pulmonary stenosis (PPS) associated with William's syndrome are usually very mild or resolve spontaneously. However, we encountered a paediatric case of William's syndrome (WS) with severe PPS. In this case, we investigate whether the patient's genetic background was responsible for the severity of the PPS. Case presentation Shortly after birth, the male patient was suspected as WS because of his peculiar facial appearance and PPS. Fluorescence in situ hybridization identified microdeletion in the 7q11.23 region, and he had a confirmed diagnosis of WS. PPS became worsened subsequently, and a cardiac catheterization was performed at the age of 7 years. Due to severe multiple PPS, the right ventricular pressure exceeded the left ventricular pressure. At the age of 12, he had haemoptysis, which required ventilatory management. A pseudoaneurysm of the right pulmonary artery was identified and a right middle lobectomy was performed. Methods and results First, we performed multiplex ligation-dependent probe amplification and whole exome sequencing, considering the possibility of homozygous mutations in the region responsible for WS (7q11.23) or a larger deletion of this region in the patient. The deletion was confirmed to be a heterozygous deletion with a typical range for WS. Therefore, we hypothesized that the causative gene of severe PPS may exist separately from the region responsible for WS. After reviewing the result of the whole exome sequencing, we identified a rare nonsense variant of the “X”-gene as the disease-causing gene. The X-gene is involved in the suppression of vascular smooth muscle cell proliferation and the generation of pulmonary arteriolar dilator “Y”. The X-gene has been reported as a disease-causing gene in patients with pulmonary arterial hypertension. Based on these previous reports, we assumed that the X gene variant could be a cause of PPS; therefore, we performed functional analysis. We first introduced a mutant X-gene construct into human pulmonary arterial endothelial cells (hPAECs) and human pulmonary arterial smooth muscle cells and found that the mutant significantly reduced the production of Y compared to the wild-type X-gene. When transfected into hPAECs, the mutant X gene significantly increased cell proliferation compared to the wild-type X-gene. The hPAECs also showed reduced caspase 3/7 activity and reduced apoptosis compared to wild-type X-gene transfected cells. In the patient's own lung pathology specimens, irregular fibrous thickening of the intima of the intrapulmonary arteries was observed, which supported the results of the functional analysis. Conclusion Our study showed that an X-gene variant could lead to reduced Y production in the pulmonary arteries, abnormal proliferation of hPAECs, and the development of severe PPS. As Y-replenishing drugs are already in clinical use, the results may lead to the development of medical treatments for severe PPS. Funding Acknowledgement Type of funding sources: None.

Senescent endothelial cells exacerbate pulmonary hypertension through notch-mediated juxtacrine interaction with pulmonary artery smooth muscle cells

Abstract Background Despite recently developed clinical therapies, vascular remodelling in pulmonary arterial hypertension (PAH) progressively worsen. Hemodynamic unloading has been proposed to normalize the remodelled pulmonary vascular structures in the lungs. Recently, it has been reported that cellular senescence was associated with the irreversibility of pulmonary vascular structures after hemodynamic unloading. Purpose This study aims to elucidate the role of senescent endothelial cells (ECs) in the pathogenesis of PAH. Methods We generated EC-specific progeroid mice in which ECs undergo premature senescence by overexpressing the dominant-negative form of telomere repeat-binding factor 2 under the control of the VE-cadherin promoter. Following three weeks of hypoxia exposure, the PH phenotypes were assessed by RVSP, lung histology, and RT-qPCR. The interaction of human pulmonary artery ECs (hPAECs) and human pulmonary artery smooth muscle cells (hPASMCs) was indirectly and directly explored through the co-culture system. Gamma-secretase inhibitor (DAPT) was administrated to inhibit Notch signalling both in the in-vitro and in-vivo study. Results EC-specific progeroid mice showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced medial SMCs proliferation in the distal pulmonary arteries. Contact-mediated interaction with senescent hPAECs increased proliferation and migration capacities in hPASMCs, while no such effects were detected in the absence of ECs-SMCs contact. Consistently, senescent ECs highly expressed Notch ligands, thus activated Notch signalling in hPASMCs, leading to increased Notch target genes in hPASMCs. Pharmacological inhibition of Notch signalling attenuated the enhanced SMCs proliferation and migration induced by senescent hPAECs, as well as the worsened PH phenotypes in EC-specific progeroid mice. Conclusions Our data established a crucial role of senescent ECs in the PAH pathogenesis through the dysregulated SMC functions via juxtacrine signaling. Senescent ECs are attracting targets for further pathological-targeted therapy to cure PAH completely. Funding Acknowledgement Type of funding sources: None.

Angiostatic Peptide, Endostatin, Predicts Severity in Pediatric Congenital Heart Disease-Associated Pulmonary Hypertension.

BackgroundEndostatin, an angiogenic inhibitor, is associated with worse pulmonary arterial hypertension (PAH) outcomes in adults and poor lung growth in children. This study sought to assess whether endostatin is associated with disease severity and outcomes in pediatric PAH.Methods and ResultsSerum endostatin was measured in cross‐sectional (N=160) and longitudinal cohorts (N=64) of pediatric subjects with PAH, healthy pediatric controls and pediatric controls with congenital heart disease (CHD) (N=54, N=15), and adults with CHD associated PAH (APAH‐CHD, N=185). Outcomes, assessed by regression and Kaplan‐Meier analysis, included hemodynamics, change in endostatin over time, and transplant‐free survival. Endostatin secretion was evaluated in pulmonary artery endothelial and smooth muscle cells. Endostatin was higher in those with PAH compared with healthy controls and controls with CHD and was highest in those with APAH‐CHD. In APAH‐CHD, endostatin was associated with a shorter 6‐minute walk distance and increased mean right atrial pressure. Over time, endostatin was associated with higher pulmonary artery pressure and pulmonary vascular resistance index, right ventricular dilation, and dysfunction. Endostatin decreased with improved hemodynamics over time. Endostatin was associated with worse transplant‐free survival. Addition of endostatin to an NT‐proBNP (N‐terminal pro‐B‐type natriuretic peptide) based survival analysis improved risk stratification, reclassifying subjects with adverse outcomes. Endostatin was secreted primarily by pulmonary artery endothelial cells.ConclusionsEndostatin is associated with disease severity, disease improvement, and worse survival in APAH‐CHD. Endostatin with NT‐proBNP improves risk stratification, better predicting adverse outcomes. The association of elevated endostatin with shunt lesions suggests that endostatin could be driven by both pulmonary artery flow and pressure. Endostatin could be studied as a noninvasive prognostic marker, particularly in APAH‐CHD.

EPAS1 (Endothelial PAS Domain Protein 1) Orchestrates Transactivation of Endothelial ICAM1 (Intercellular Adhesion Molecule 1) by Small Nucleolar RNA Host Gene 5 (SNHG5) to Promote Hypoxic Pulmonary Hypertension.

[Figure: see text].

ENDOTHELIAL BMPR2 LOSS PROMOTES ALTERED IL-15 SIGNALING, CONTRIBUTING TO IMMUNE DYSFUNCTION IN PULMONARY HYPERTENSION

<h3>BACKGROUND</h3> Natural killer (NK) cell impairment is a feature of pulmonary arterial hypertension (PAH) and contributes to disease development in mouse models of NK cell deficiency (Ormiston et al, Circulation, 2012; Ratsep et al, AJP Lung, 2018). PAH is also strongly associated with mutations in BMPR2, the gene encoding the type-II bone morphogenetic protein receptor. Genetic and experimental evidence point to a loss of BMPR2 in the pulmonary endothelium as a critical contributor to disease development. However, the impact of endothelial BMPR2 loss on NK cell impairment in PAH remains unknown. <h3>METHODS AND RESULTS</h3> SiRNA-mediated silencing of BMPR2 in human pulmonary artery endothelial cells (HPAEC) significantly reduced the surface presentation and secretion of the α-type receptor for interleukin-15 (IL-15Rα), a major regulator of NK cell survival, proliferation and activity. Assessment of IL-15Rα transcriptional variants by RNA-Seq and glycosylation by immunoblotting demonstrated that this reduction was not due to changes in receptor transcription or processing. Treatment of HPAECs with the matrix metalloproteinase inhibitor Batimastat also ruled out a role for altered IL-15Rα cleavage with BMPR2 silencing. Instead, confocal microscopy identified impaired trafficking of IL-15Rα through the trans-Golgi-network (TGN) in BMPR2-silenced cells. To evaluate the effects of impaired IL-15 mediated signaling and NK cell loss on PAH development, NK cell-deficient Il15-/- rats and immune-competent Il15+/+ littermates were exposed to either the monocrotaline (MCT) or SUGEN/hypoxia models of experimental pulmonary hypertension. While both male and female Il15-/- rats developed more severe disease than their wildtype counterparts in the SUGEN/Hypoxia model, only male Il15-/- rats demonstrated this enhanced severity in the MCT model, which is the only one of the two models to reproduce the sex-based difference in disease severity that is observed in human PAH patients. Assessment of genes associated with sex hormone signaling and metabolism identified a potential increase in pulmonary Cyp1b1 expression (n=5-7, p=0.08) in male MCT-challenged Il15-/- rats relative to Il15+/+ controls. <h3>CONCLUSION</h3> We have identified the loss of IL-15 signaling as a novel BMPR2-dependent contributor to NK cell impairment and pulmonary arterial hypertension pathogenesis. Ongoing work includes assessing the contribution of sex hormones to immune-mediated disease processes in PAH.

Autophagy, TERT, and mitochondrial dysfunction in hyperoxia.

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1β, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.