Mouse Model Of Pulmonary Arterial Hypertension Research Articles

Anti-inflammatory nutrition with high protein attenuates cardiac and skeletal muscle alterations in a pulmonary arterial hypertension model

Pulmonary arterial hypertension (PAH) is characterized by remodelling of the pulmonary arteries and right ventricle (RV), which leads to functional decline of cardiac and skeletal muscle. This study investigated the effects of a multi-targeted nutritional intervention with extra protein, leucine, fish oil and oligosaccharides on cardiac and skeletal muscle in PAH. PAH was induced in female C57BL/6 mice by weekly injections of monocrotaline (MCT) for 8 weeks. Control diet (sham and MCT group) and isocaloric nutritional intervention (MCT + NI) were administered. Compared to sham, MCT mice increased heart weight by 7%, RV thickness by 13% and fibrosis by 60% (all p < 0.05) and these were attenuated in MCT + NI mice. Microarray and qRT-PCR analysis of RV confirmed effects on fibrotic pathways. Skeletal muscle fiber atrophy was induced (P < 0.05) by 22% in MCT compared to sham mice, but prevented in MCT + NI group. Our findings show that a multi-targeted nutritional intervention attenuated detrimental alterations to both cardiac and skeletal muscle in a mouse model of PAH, which provides directions for future therapeutic strategies targeting functional decline of both tissues.

MiR-205-5p suppresses pulmonary vascular smooth muscle cell proliferation by targeting MICAL2-mediated Erk1/2 signaling

Pulmonary arterial hypertension (PAH) is a devastating and fatal vascular disease for which currently there is no satisfying therapy available. Excessive cell proliferation of pulmonary arterial smooth muscle cells (PASMCs) contributes significantly to PAH pathogenesis. In this study, we found that miR-205-5p was lowly expressed in hypoxia-induced PAH mouse model and hypoxia-treated PASMCs. Restoration of miR-205-5p suppressed PASMCs proliferation. In contrast, molecule interacting with CasL 2 (MICAL2) was highly expressed in hypoxia-induced PAH mouse model and hypoxia-treated PASMCs. Overexpression of MICAL2 promoted cell proliferation. Furthermore, miR-205-5p inhibited MICAL2 expression levels by targeting the MICAL2 3′ untranslated region. In addition, MICAL2 activated ERK1/2 signaling in PASMCs and ERK1/2 inhibitor blocked MICAL2-mediated-promotion effect on PASMCs proliferation. These results demonstrated that miR-205-5p suppressed PASMCs proliferation by targeting MICAL2, which activated ERK1/2 signaling. Therefore, miR-205-5p/MICAL2/Erk1/2 may serve as an ideal therapeutic target to PAH treatment.

Type 2 inositol 1,4,5-trisphosphate receptor inhibits the progression of pulmonary arterial hypertension via calcium signaling and apoptosis.

Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca2+ signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca2+ signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IP3R2), which is an intracellular Ca2+ release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IP3R2-/- mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IP3R2-/- murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca2+ entry (SOCE) was significantly enhanced in IP3R2-/- PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca2+ imaging. Furthermore, the enhancement of SOCE in IP3R2-/- PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IP3R2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IP3R in the development of PAH and may contribute to the development of targeted therapies.

Hematopoietic stem cell transplantation alters susceptibility to pulmonary hypertension in Bmpr2-deficient mice.

Increasing evidence suggests that patients with pulmonary arterial hypertension (PAH) demonstrate abnormalities in the bone marrow (BM) and hematopoietic progenitor cells. In addition, PAH is associated with myeloproliferative diseases. We have previously demonstrated that low-dose lipopolysaccharide (LPS) is a potent stimulus for the development of PAH in the context of a genetic PAH mouse model of BMPR2 dysfunction. We hypothesized that the hematopoietic progenitor cells might be driving disease in this model. To test this hypothesis, we performed adoptive transfer of BM between wild-type (Ctrl) and heterozygous Bmpr2 null (Mut) mice. Sixteen weeks after BM reconstitution, mice were exposed to low-dose chronic LPS (0.5 mg/kg three times a week for six weeks). Mice underwent right heart catheterization and tissues were removed for histology. After chronic LPS dosing, Ctrl mice in receipt of Mut BM developed PAH, whereas Mut mice receiving Ctrl BM were protected from PAH. BM histology demonstrated an increase in megakaryocytes and there was an increase in circulating platelets in Ctrl mice receiving Mut BM. These findings demonstrate that the hematopoietic stem cell compartment is involved in the susceptibility to PAH in the Mut mouse. The results raise the possibility that hematopoietic stem cell transplantation might be a potential treatment strategy in genetic forms of PAH.

Metabolic Reprogramming in the Heart and Lung in a Murine Model of Pulmonary Arterial Hypertension.

A significant glycolytic shift in the cells of the pulmonary vasculature and right ventricle during pulmonary arterial hypertension (PAH) has been recently described. Due to the late complications and devastating course of any variant of this disease, there is a great need for animal models that reproduce potential metabolic reprograming of PAH. Our objective is to study, in situ, the metabolic reprogramming in the lung and the right ventricle of a mouse model of PAH by metabolomic profiling and molecular imaging. PAH was induced by chronic hypoxia exposure plus treatment with SU5416, a vascular endothelial growth factor receptor inhibitor. Lung and right ventricle samples were analyzed by magnetic resonance spectroscopy. In vivo energy metabolism was studied by positron emission tomography. Our results show that metabolomic profiling of lung samples clearly identifies significant alterations in glycolytic pathways. We also confirmed an upregulation of glutamine metabolism and alterations in lipid metabolism. Furthermore, we identified alterations in glycine and choline metabolism in lung tissues. Metabolic reprograming was also confirmed in right ventricle samples. Lactate and alanine, endpoints of glycolytic oxidation, were found to have increased concentrations in mice with PAH. Glutamine and taurine concentrations were correlated to specific ventricle hypertrophy features. We demonstrated that most of the metabolic features that characterize human PAH were detected in a hypoxia plus SU5416 mouse model and it may become a valuable tool to test new targeting treatments of this severe disease.

Endothelial TRPV4 Channel Function is Impaired in Pulmonary Arterial Hypertension

Endothelial dysfunction contributes to the pathogenesis of pulmonary arterial hypertension (PAH) by reducing arterial vasodilation. Endothelium‐dependent vasodilation is an important regulator of pulmonary vascular resistance (PVR) and overall pulmonary arterial pressure (PAP). We recently identified endothelial TRPV4 (transient receptor potential vanilloid 4) channels as important regulators of vasodilation in pulmonary arteries (PAs), and showed that unitary Ca2+ influx events through TRPV4 channels, termed TRPV4 sparklets, activate endothelial nitric oxide synthase (eNOS) to cause vasodilation. Reduced NO bioavailability is a hallmark of endothelial dysfunction in PAH. However, the mechanisms underlying reduced NO bioavailability in PAH are not well understood. We hypothesized that impairment of TRPV4 channel function attenuates NO release in PAH. PAH was induced in mice using three‐week chronic hypoxia (CH) and Sugen 5416 + CH models. Mouse right ventricular pressures (RVP) were measured as an indicator of PAP. RVP was significantly higher in TRPV4−/− mice when compared to the control mice after three weeks of CH. High‐speed Ca2+ imaging was used to analyze TRPV4 sparklets in the native endothelium from resistance‐sized, fourth‐order PAs. Vascular diameter studies were performed on isolated, pressurized (15 mmHg), small PAs. Ca2+ imaging studies were also performed in PAs obtained from control and PAH patients. In CH and Sugen 5416 + CH models, TRPV4 sparklet activity and TRPV4 channel‐dependent vasodilation were significantly lower compared to normoxic control mice. However, TRPV4 mRNA expression and immunofluorescence were not altered in both mouse models of PAH, suggesting an impairment in the regulation of channel function rather than channel expression. TRPV4 sparklet activity was also consistently lower in PAs from PAH patients, when compared to control patients. Elevated levels of superoxide radicals have been shown to contribute to the development of PAH. Superoxides can react with NO to form peroxynitrite (PN). A PN scavenger, uric acid, restored TRPV4 channel activity in CH mice. We, therefore, hypothesized that PN‐induced impairment of TRPV4‐dependent Ca2+ signaling inhibits endothelial function in PAH. Indeed, PN attenuated TRPV4 sparklet activity in a reversible manner. Our results support the concept that PN‐induced regulation of TRPV4‐dependent Ca2+ signaling may contribute to the development of PAH. Moreover, altered TRPV4 channel activity may contribute to the reduced NO bioavailability in PAH.Support or Funding Information1R56HL138496, 5R00HL121484, 17PRE33660762This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the Right Ventricle as a Treatment Strategy for Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by remodeling of the pulmonary artery within the lung that leads to increased pulmonary vascular resistance (PVR). This increase in PVR can result in progressive remodeling of the right ventricle (RV) and eventual right heart failure. Current therapy aims to decrease PVR via vasodilation of the PA, but this strategy does not work for some patients, and is not a cure for the disease. This study aims to uncover novel therapeutic targets by assessing the RV for expression of G protein‐coupled receptors (GPCRs) during PAH. GPCRs are the target of more than 30% of approved drugs and regulate multiple cellular processes by activation on the cell membrane. We utilized a rat model of PAH in which Sprague Dawley rats were placed in hypoxia (10% O2) for two weeks, or normoxia as a control (n=3–4/treatment). Placement in hypoxia resulted in RV remodeling evident by an increase in the RV weight/left ventricle (LV) + septum weight (hypoxia‐treatment: 0.458 ± 0.036 vs. control rats: 0.210 ± 0.020; mean ± standard deviation, Student's t‐test p&lt;0.0001). We then utilized RNA sequencing (RNA‐seq) of the RV and the left ventricle (LV) tissue coupled with differential expression analysis to identify GPCRs expressed with significantly (false discovery rate &lt;0.1) altered expression in the RV of hypoxia‐treated rats compared to controls. In the RV, 37 GPCRs were altered in expression in hypoxia‐treated rats vs. control rats compared to 7 GPCRs altered in the LV. To examine the cell‐specific expression of these targets, cardiac myocytes and fibroblasts were isolated from a mouse model of PAH (10% O2 for 3 weeks with weekly injection of SU5416). qPCR of the target GPCRs revealed that most of the differentially expressed GPCRs were altered in the cardiac myocytes but not in the fibroblasts. These results indicate that a subset of genes is differentially expressed in the RV compared to the LV both in normoxia and hypoxia, particularly in the cardiac myocytes. This raises the possibility that treatments for PAH could be directed at GPCRs selectively expressed in RV remodeling, thus providing a novel means of treating an unmet need for patients with RV hypertrophy and failure.Support or Funding InformationSupported by NIH Grant: T32HL007444‐34 and DoD Grant: W81XWH‐14‐1‐0372This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NF-κB mediated miR-130a modulation in lung microvascular cell remodeling: Implication in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease which is associated with pulmonary arterial endothelial cells (PAEC) dysfunction and pulmonary arterial smooth muscle cells proliferation. Moreover, inflammation is contributing a critical role in EC dysfunction and remains elusive. Nuclear factor-kappa B (NF-κB) is a master transcriptional regulator in various cardiovascular pathologies; but, NF-κB's role in EC dysfunction in unknown. Our previous study using cardiac and lung specific IκBα mutant mice (3M and IKBM) showed that PAH induced right ventricular hypertrophy (RVH) was prevented in monocrotaline (MCT) treated 3M and IKBM mice, compared to the wild-type mice. Recently, microRNAs (miRNAs) have emerged as a new class of post-transcriptional regulators in vascular remodeling; but, NF-κB regulated miRNA modulation in EC dysfunction is unknown. Using miRNA array analysis, we identified miR-130a which is upregulated in MCT-induced PAH mouse model, as a possible candidate to study. We showed that overexpressing miR-130a in lung microvascular endothelial cells (MVEC) promoted activation of α-smooth muscle actin, a critical component in endothelial-to-mesenchymal transition in EC remodeling. In this study, we demonstrated that bone morphogenetic protein receptor 2 (BMPR2) was a target for miR-130a and miR-130a was regulated by NF-κB which controlled apoptosis and vascular genes in MVEC. The findings reveal that NF-κB-mediated miR-130a modulation is critical in lung vascular remodeling.

Elevated microRNA-135a is associated with pulmonary arterial hypertension in experimental mouse model.

Multiple causes are associated with the complex mechanism of pathogenesis of pulmonary arterial hypertension (PAH), but the molecular pathway in the pathogenesis of PAH is still insufficiently understood. In this study, we investigated epigenetic changes that cause PAH induced by exposure to combined Th2 antigen (Ovalbumin, OVA) and urban particulate matter (PM) in mice. To address that, we focused on the epigenetic mechanism, linked to microRNA (miR)-135a. We found that miR-135a levels were significantly increased, and levels of bone morphogenetic protein receptor type II (BMPR2) which is the target of miR-135a, were significantly decreased in this experimental PAH mouse model. Therefore to evaluate the role of miR-135a, we injected AntagomiR-135a into this mouse model. AntagomiR-135a injected mice showed decreased right ventricular systolic pressures (RVSPs), right ventricular hypertrophy (RVH), and the percentage of severely thickened pulmonary arteries compared to control scrambled miRNA injected mice. Both mRNA and protein expression of BMPR2 were recovered in the AntagomiR-135a injected mice compared to control mice. Our study understands if miR-135a could serve as a biomarker helping to manage PAH. The blocking of miR-135a could lead to new therapeutic modalities to alleviate exacerbation of PAH caused by exposure to Th2 antigen and urban air pollution.

Functional magnetic resonance imaging for in vivo quantification of pulmonary hypertension in the Sugen 5416/hypoxia mouse.

What is the central question of this study? Non-invasive, quantitative methods to assess right cardiac function in mice with pulmonary hypertension have not been demonstrated. What is the main finding and its importance? This study shows the potential of magnetic resonance imaging to estimate right ventricular ejection fraction and measure spatial, dynamic changes in cardiac structure in the Sugen 5416/hypoxia mouse model of pulmonary hypertension. Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary artery pressures and right heart failure. Mouse models of PAH are instrumental in understanding the disease pathophysiology. However, few methods are available to evaluate right cardiac function in small animals. In this study, magnetic resonance imaging was used to measure in vivo cardiac dimensions in the Sugen 5416/hypoxia mouse model. Pulmonary hypertension (PH) was induced in C57BL/6 mice by 3weeks of exposure to 10% oxygen and vascular endothelial growth factor receptor inhibition (20mgkg-1 SU5416). Control mice were housed in room air and received vehicle (DMSO). Right ventricular pressures were recorded with a pressure-conductance transducer. Short-axis contiguous 1-mm-thick slices were acquired through the heart and great vessels using a fast low-angle shot (FLASH)-cine sequence. Thirteen images were collected throughout each cardiac cycle. Right ventricular systolic pressure was elevated in PH mice (23.6 ± 6 versus 41.0 ± 11mmHg, control versus PH, respectively; P<0.001, n=5-11). Right ventricular wall thickness was greater in PH than in control mice at end diastole (0.30 ± 0.05 versus 0.48 ± 0.06mm, control versus PH, respectively; P<0.01, n=6), but measurements were not different at end systole (control versus PH, 0.59 ± 0.11 versus 0.70 ± 0.11mm, respectively). Right ventricular ejection fraction was decreased in PH mice (72 ± 3 versus 58 ± 5%, control versus PH, respectively; P<0.04, n=6). These data demonstrate that magnetic resonance imaging is a precise method to monitor right ventricular remodelling and cardiac output longitudinally in mouse models of PH.

STARS knockout attenuates hypoxia-induced pulmonary arterial hypertension by suppressing pulmonary arterial smooth muscle cell proliferation.

STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein, which expressed early in cardiac development and involved in pathological remodeling. Abundant evidence indicated that STARS could regulate cell proliferation, but it's exact function remains unclear. In this study, we aimed to investigate the role of STARS in the proliferation of pulmonary arterial smooth muscle cells (PASMC) and the potential effect on the progression of pulmonary arterial hypertension (PAH). In this study, we established a PAH mouse model through chronic hypoxia exposure as reflected by the increased RVSP and RVHI. Western blot and RT-qPCR detected the increased STARS protein and mRNA levels in PAH mice. Next, we cultured the primary PASMC from PAH mice. After STARS overexpression in PASMC, STARS, SRF and Egr-1 were up-regulated significantly. The MTT assay revealed an increase in cell proliferation. Flow cytometry showed a marked inhibition of cell apoptosis. However, STARS silence in PASMC exerted opposite effects with STARS overexpression. SRF siRNA transfection blocked the effects of STARS overexpression in PASMC. In order to further confirm the role of STARS in PAH mice in vivo, we exposed STARS knockout mice to hypoxia and found lower RVSP and RVHI in knockout mice as compared with controls. Our results not only suggest that STARS plays a crucial role in the development of PAH by increasing the proliferation of PASMC through activation of the SRF/Egr-1 pathway, but also provides a new mechanism for hypoxia-induced PAH. In addition, STARS may represent a potential treatment target.

BMPR2 gene delivery reduces mutation-related PAH and counteracts TGF-β-mediated pulmonary cell signalling.

Idiopathic, familial and secondary pulmonary arterial hypertension (PAH) are associated with reduced bone morphogenetic protein receptor type 2 (BMPR2) expression, and in some contexts, TGF-β upregulation. Our aims were to assess BMPR2 gene therapy in a PAH mouse model and to assess the impact on TGF-β signalling. Using a targeted in vivo gene delivery approach, we assessed the impact of BMPR2 gene delivery in a transgenic mouse model in which PAH was first induced by doxycycline driven expression of a dominant negative BMPR2 mutant (R899X). We also assessed the impact of BMPR2 gene delivery on TGF-β-induced changes in cell signalling in human pulmonary vascular endothelial and smooth muscle cells. In the mouse model, changes in TGF-β levels were not detected, but BMPR2 gene delivery reversed the increase in right ventricle systolic pressure (RVSP) and Fulton Index (FI), associated with a trend to increased pulmonary endothelial nitric oxide synthase (eNOS) gene expression. In vitro, BMPR2 gene transfer reduced TGF-β effects on Smad2, Smad1/5/8 and Erk1/2 phosphorylation in human pulmonary arterial smooth muscle cells (HPASMC). BMPR2 was also found to upregulate nitric oxide (NO) production in lung derived human microvascular endothelial cells (HMVEC-L). This study provides further evidence that BMPR2 modulation may have therapeutic potential. See Editorial, page 406.

Mammalian target of rapamycin overexpression antagonizes chronic hypoxia-triggered pulmonary arterial hypertension via the autophagic pathway.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disorder with high morbidity and mortality, and is characterized by excessive growth of endothelial cells. Recently, the mammalian target of rapamycin (mTOR) has attracted increasing attention due to its potential as a therapeutic target against certain diseases associated with proliferative and metabolic abnormalities. However, the effect on mTOR on PAH has not yet been elucidated. In the present study, a marked downregulation of mTOR was observed in PAH patients. Following construction of a mouse model of PAH by chronic exposure to hypoxia, adenovirus-mediated upregulation of mTOR significantly attenuated right ventricular systolic pressure, right ventricular hypertrophy and wall thickness of pulmonary arterioles, indicating a protective effect of mTOR on PAH. Further analysis confirmed that mTOR overexpression inhibited autophagy triggered by hypoxia through blocking light chain 3 II expression and increasing p62 levels. In vitro, hypoxia enhanced the proliferation of human pulmonary artery endothelial cells (PAECs), which was markedly abrogated by mTOR overexpression. Of note, upregulation of mTOR inhibited the hypoxia-induced autophagy pathway, which contributed to cell proliferation, while silencing of autophagy by RNA interference with ATG5 significantly inhibited cell proliferation. In conclusion, the results of the present study suggested a potential protective effect of mTOR on the progression of PAH by suppressing PAEC proliferation through blocking the autophagic pathway. Therefore, the present study suggested that mTOR is a promising therapeutic agent against PAH.

Direct and indirect protection of right ventricular function by estrogen in an experimental model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) results in right ventricular (RV) dysfunction and failure. Paradoxically, women are more frequently diagnosed with PAH but have better RV systolic function and survival rates than men. The mechanisms by which sex differences alter PAH outcomes remain unknown. Here, we sought to study the role of estrogen in RV functional remodeling in response to PAH. The SU5416-hypoxia (SuHx) mouse model of PAH was used. To study the role of estrogen, female mice were ovariectomized and then treated with estrogen or placebo. SuHx significantly increased RV afterload and resulted in RV hypertrophy. Estrogen treatment attenuated the increase in RV afterload compared with the untreated group (effective arterial elastance: 2.3 ± 0.1 mmHg/μl vs. 3.2 ± 0.3 mmHg/μl), and this was linked to preserved pulmonary arterial compliance (compliance: 0.013 ± 0.001 mm(2)/mmHg vs. 0.010 ± 0.001 mm(2)/mmHg; P < 0.05) and decreased distal muscularization. Despite lower RV afterload in the estrogen-treated SuHx group, RV contractility increased to a similar level as the placebo-treated SuHx group, suggesting an inotropic effect of estrogen on RV myocardium. Consequently, when compared with the placebo-treated SuHx group, estrogen improved RV ejection fraction and cardiac output (ejection fraction: 57 ± 2% vs. 44 ± 2% and cardiac output: 9.7 ± 0.4 ml/min vs. 7.6 ± 0.6 ml/min; P < 0.05). Our study demonstrates for the first time that estrogen protects RV function in the SuHx model of PAH in mice directly by stimulating RV contractility and indirectly by protecting against pulmonary vascular remodeling. These results underscore the therapeutic potential of estrogen in PAH.

MiR‐17~92 promotes hypoxia‐induced pulmonary hypertension by regulating proliferation and contraction of pulmonary arterial smooth muscle cells (LB779)

Introduction: Recent studies suggest that microRNAs (miRNAs) play important roles in development and pathogenesis of human diseases. We sought to identify novel miRNAs that may be involved in the pathogenesis of pulmonary arterial hypertension (PAH) and to investigate underlying mechanisms.Methods and Results: We performed miRNA qPCR array using pulmonary arterial smooth muscle cells (PASMC) isolated from lungs of normal donor and PAH patients. We found that all of the six members of the miR‐17~92 cluster were down‐regulated in PAH PASMCs. In hypoxia‐induced PAH mouse model and cultured human PASMCs, we found that short term hypoxia induced miR‐17~92 whereas prolonged hypoxia suppressed miR‐17~92. We generated a strain of SMC‐specific miR‐17~92 knockout (sm‐17~92‐/‐) mouse by crossbreeding miR‐17~92 fl/fl mice with sm22α‐Cre mice and subsequent backcrossing. We exposed 8‐10 week old sm‐17~92‐/‐ mice along with their wild type littermates to hypoxia (10% O2) or normoxia for five weeks. We showed that knockout of miR‐17~92 in SMC attenuated hypoxia‐induced increase in measured their right ventricular systolic pressure (RVSP), RV hypertrophy (RV/(LV+S) ratio), and pulmonary arterial wall thickness, suggesting that SMC‐specific miR‐17~92 is essential for the development of PAH. To investigate the underlying mechanism, we over‐expressed the miR‐17~92 cluster in human PASMC by a lenti‐virus encoding this miRNA cluster. Overexpression of mi‐17~92 induced expression levels of SMA, SM22α, Calponin, and PCNA and BrdU incorporation. Human PASMC from idiopathic PAH (IPAH) patients expressed decreased levels of SMA, SM22α, and Calponin, whereas over‐expression of miR‐17~92 restored the expression of SMA, SM22α, and Calponin in PASMCs from IPAH patient.Summary: We provide evidence that miR‐17~92 is dysregulated in hypertensive PASMC and suppression miR‐17~92 in SMC is sufficient to inhibit hypoxia‐induced PAH in vivo. We also show that miR‐17~92 induces PASMC proliferation and expression of contractile proteins. These results suggest that miR‐17~92 may promote PAH by regulating vascular remodeling and that miR‐17~92 may represent a novel therapeutic target for the treatment of PAH. Support: NIH HL110829 and Gilead Sciences Research Scholars Program.Grant Funding Source: Support by NIH HL110829 and Gilead Sciences Research Scholars Program.

Inhibition of Nuclear Factor-κB in the Lungs Prevents Monocrotaline-Induced Pulmonary Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with significant morbidity and mortality in patients with various lung and heart diseases. PAH is characterized by vascular obstruction which leads to a sustained increased pulmonary vascular resistance, vascular remodeling, and right ventricular hypertrophy and failure. Limited PAH therapies indicate that novel approaches are urgently needed for the treatment of PAH. Nuclear factor-κB (NF-κB) has been shown to play an important role in different cardiac pathologies; however, the role of NF-κB remains limited in the setting of PAH. Here, we investigated whether NF-κB inhibition in the lungs using Club (Clara) cell-10 promoter driving IκBα mutant had any effect in monocrotaline (MCT)-induced PAH mouse model. Our data revealed that MCT-induced PAH and right ventricular hypertrophy were associated with NF-κB activation, inflammatory response, and altered expression of bone morphogenetic protein receptor 2, inhibitor of differentiation, and Notch-3 signaling molecules in wild-type mice; and all these alterations were prevented in IκBα mutant mice treated with MCT. Moreover, endothelial cell apoptosis and endothelial-to-mesenchymal transition occurred in the lungs of MCT-treated wild-type mice and were restored in IκBα mutant+MCT mice, indicating an association with NF-κB signaling. In lung microvascular endothelial cells, IκBα (AA) mutant plasmid restored the decreased bone morphogenetic protein receptor 2 protein level and reversed the endothelial-to-mesenchymal transition process induced by transforming growth factor-β1. We conclude that NF-κB regulates bone morphogenetic protein receptor 2-inhibitor of differentiation-Notch-3 axis genes and the subsequent endothelial cell apoptosis and endothelial-to-mesenchymal transition events in the lungs, providing new mechanistic information about MCT-induced PAH and right ventricular hypertrophy.

Progressive right ventricular functional and structural changes in a mouse model of pulmonary arterial hypertension.

Right ventricle (RV) dysfunction occurs with progression of pulmonary arterial hypertension (PAH) due to persistently elevated ventricular afterload. A critical knowledge gap is the molecular mechanisms that govern the transition from RV adaptation to RV maladaptation, which leads to failure. Here, we hypothesize that the recently established mouse model of PAH, via hypoxia and SU5416 treatment (HySu), captures that transition from adaptive to maladaptive RV remodeling including impairments in RV function and decreases in the efficiency of RV interactions with the pulmonary vasculature. To test this hypothesis, we exposed C57BL6 male mice to 0 (control), 14, 21, and 28 days of HySu and then obtained synchronized RV pressure and volume measurements in vivo. With increasing HySu exposure duration, arterial afterload increased monotonically, leading to a continuous increase in RV stroke work, RV fibrosis, and RV wall stiffening (P < 0.05). RV contractility increased at 14 days of HySu exposure and then plateaued (P < 0.05). As a result, ventricular–vascular coupling efficiency tended to increase at 14 days and then decrease. Our results suggest that RV remodeling may begin to shift from adaptive to maladaptive with increasing duration of HySu exposure, which would mimic changes in RV function with PAH progression found clinically. However, for the duration of HySu exposure used here, no drop in cardiac output was found. We conclude that the establishment of a mouse model for overt RV failure due to PAH remains an important task.

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking. In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

Gene Transfer Therapy by Either Type 1 or Type 2 Adeno-Associated Virus Expressing Human Prostaglandin I2 Synthase Gene is Effective for Treatment of Pulmonary Arterial Hypertension

Prostaglandin I(2) (PGI(2)) plays an important role in the clinical treatment of pulmonary arterial hypertension (PAH). However, the administration of PGI(2) involves continuous intravenous infusion using an indwelling catheter, which limits the patient's quality of life and increases the risk of infection. We therefore investigated whether human PGI(2) synthase (hPGIS) gene transfer using an adeno-associated virus (AAV) vector is still effective in a mouse model of PAH and tested for differences in the therapeutic efficacy of PAH among AAV serotypes. The PAH was induced by subjecting mice to hypoxia (10% O(2)). Type 1 AAV expressing hPGIS (AAV1-hPGIS) or type 2 AAV expressing hPGIS (AAV2-hPGIS) was injected into the thigh muscle of mice. Both vectors expressing hPGIS produced strong hPGIS protein expression in the mouse thigh skeletal muscles after 8 weeks of hypoxia. The administration of AAV1-hPGIS or AAV2-hPGIS also significantly inhibited the hypoxia-induced increase in right ventricular systolic pressure, the ratio of right ventricular weight to body weight (RV/BW), and the ratio of RV weight to left ventricular plus septal weight (RV/LV + S), and significantly attenuated the hypoxia-induced increase in medial wall thickness of peripheral pulmonary arteries. Furthermore, there were no significant differences in the degree of amelioration in RV systolic pressure, RV/BW, RV/LV + S, and percentage of wall thickness of peripheral pulmonary arteries between AAV1-hPGIS and AAV2-hPGIS administrations. In conclusion, we revealed that type 1 and type 2 AAV are equally effective for the treatment of PAH in a hypoxia-induced mouse model. Gene-transfer therapy using AAV expressing hPGIS is, therefore, a potential therapeutic breakthrough for PAH.

Impaired Natural Killer Cell Phenotype and Function in Idiopathic and Heritable Pulmonary Arterial Hypertension

Beyond their role as innate immune effectors, natural killer (NK) cells are emerging as important regulators of angiogenesis and vascular remodeling. Pulmonary arterial hypertension (PAH) is characterized by severe pulmonary vascular remodeling and has long been associated with immune dysfunction. Despite this association, a role for NK cells in disease pathology has not yet been described. Analysis of whole blood lymphocytes and isolated NK cells from PAH patients revealed an expansion of the functionally defective CD56(-)/CD16(+) NK subset that was not observed in patients with chronic thromboembolic pulmonary hypertension. NK cells from PAH patients also displayed decreased levels of the activating receptor NKp46 and the killer immunoglobulin-like receptors 2DL1/S1 and 3DL1, reduced secretion of the cytokine macrophage inflammatory protein-1β, and a significant impairment in cytolytic function associated with decreased killer immunoglobulin-like receptor 3DL1 expression. Genotyping patients (n=222) and controls (n=191) for killer immunoglobulin-like receptor gene polymorphisms did not explain these observations. Rather, we show that NK cells from PAH patients exhibit increased responsiveness to transforming growth factor-β, which specifically downregulates disease-associated killer immunoglobulin-like receptors. NK cell number and cytotoxicity were similarly decreased in the monocrotaline rat and chronic hypoxia mouse models of PAH, accompanied by reduced production of interferon-γ in NK cells from hypoxic mice. NK cells from PAH patients also produced elevated quantities of matrix metalloproteinase 9, consistent with a capacity to influence vascular remodeling. Our work is the first to identify an impairment of NK cells in PAH and suggests a novel and substantive role for innate immunity in the pathobiology of this disease.