Development Of PAH Research Articles

Upregulation of G protein‐couple receptor 68 (GPCR68) in pulmonary artery smooth muscle cells from animals and humans with pulmonary hypertension

IntroductionPulmonary arterial hypertension (PAH) is a progressive and fatal disease with no cure. It is characterized by elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in part due to pulmonary vascular remodeling and sustained pulmonary vasoconstriction. The increased pulmonary arterial smooth muscle cell (PASMC) proliferation contributes to excessive pulmonary vascular remodeling while a rise in cytosolic free Ca2+concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. G‐protein coupled receptors (GPCR), which are attractive pharmacological targets, are important regulators of pulmonary vascular tone and PASMC phenotype. Stimulation of GPCR leads to Ca2+ influx and increased [Ca2+]cytdue to store‐operated Ca2+ entry (SOCE) via inositol 1,4,5‐trisphosphate (IP3) production and receptor‐operated Ca2+entry (ROCE) via diacylglycerol (DAG) production. GPCR68 (or OGR1) is a proton‐sensing GPCR that responds to extracellular acidity and regulates a variety of cellular functions. As a Gq/11‐coupled receptor, GPR68 activation is known to lead to Ca2+ release from the intracellular stores in IP3‐dependent manner but the role of GPCR68 in the development of PAH is still unknown.MethodsLung, heart, brain, liver, and kidney tissues were isolated from control rats. Pulmonary hypertension model was induced by a single dose of monocrotaline (MCT) in rats. PASMC isolated from control or MCT rats and healthy subjects or patients with idiopathic pulmonary arterial hypertension (IPAH) were cultured in 5% fetal bovine serum (FBS) smooth muscle growth media and incubated in a humidified 5% CO2 atmosphere at 37°C. Total RNA was isolated from tissue or cells using TRIZOL method. mRNA expression of GPCR68 was quantified by RT‐PCR and qRT‐PCR. The data was analyzed using ImageJ and statistical analysis was performed using paired or unpaired Student’s t‐test.ResultsRT‐PCR analysis revealed that GPCR68 is expressed in brain, lung, heart, liver, and kidney in normal rat tissues (n=5). GPCR68 mRNA level was significantly higher in lung tissue and PASMC isolated from MCT‐treated rats compared with controls. Moreover, our data shows that GPCR68 is expressed in both human PASMC and pulmonary arterial endothelial cells. qRT‐PCR showed that GPCR68 was upregulated in PASMC isolated from IPAH patients compared with cells isolated from healthy subjects.ConclusionOur finding suggest that GPR68 is involved in the development and progression of PAH and may play an important role in Ca2+ signaling and enhanced [Ca2+]cyt in PASMC from animals and humans with PAH.Support or Funding InformationThis work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL125208, HL135807, and HL126609).

LncRNA CASC2 inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation and migration by regulating the miR-222/ING5 axis

BackgroundPulmonary arterial hypertension (PAH) is often characterized by cell proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). LncRNA cancer susceptibility candidate 2 (CASC2) has been revealed to be involved in PASMC injury in hypoxia-induced pulmonary hypertension. However, the exact molecular mechanisms whereby CASC2 regulates PASMC proliferation and migration are still incompletely understood.MethodsThe expression levels of CASC2, miR-222 and inhibitor of growth 5 (ING5) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. Wound healing assay was used to analyze cell migration ability. The relationship between miR-222 and CASC2 or ING5 was confirmed using bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation assay.ResultsCASC2 was down-regulated in hypoxia-induced PASMCs in a dose- and time-dependent manner. Functional experiments showed that CASC2 overexpression could reverse hypoxia-induced proliferation and migration of PASMCs. Bioinformatics analysis indicated that CASC2 acted as a competing endogenous RNA of miR-222, thereby regulating the expression of ING5, the downstream target of miR-222, in PASMCs. In addition, rescue assay suggested that the inhibition mediated by CASC2 of hypoxia-induced PASMC proliferation and migration could be attenuated by miR-222 inhibition or ING5 overexpression.ConclusionCASC2 attenuated hypoxia-induced PASMC proliferation and migration by regulating the miR-222/ING5 axis to prevent vascular remodeling and the development of PAH, providing a novel insight and therapeutic strategy for hypoxia-induced PAH.

Membrane serotonin transporter as a Biomarker of Pulmonary arterial hypertension in children with Congenital Heart Defect

18 young children (aged from 1 month old to 2 years old) with congenital heart disease were observed. During observation they were divided into 2 groups. Group I-8 children with CHD, not complicated by PAH. Group II 11 children with CHD, complicated by PAH. The content of serotonin and its transporter in platelets, the amount of serotonin in serum were determined. Serotonin content was determined by enzyme-linked immunosorbent assay. The study was performed using the Serotonin ELISA diagnostic kit, IBL Hamburg on the Evolis automated robotic station (BioRad). To determine the Sodium-Dependent serotonin transporter (SLC6A4) there was used the method of quantitative sandwich ELISA of Cusabio production. The main initiator of the development of PAH is development of endothelial dysfunction of the pulmonary vessels. Since the cause of this dysfunction is associated with functional disorders of the serotonergic system, our task was to determine the content of SERT and serotonin in platelets in the studied groups. We suggest that the serotonin transporter (SERT), which is responsible for the proliferation of the pulmonary artery smooth-muscle cells (PASMC), can be used as a biomarker for predicting and diagnosing PAH in children with CHD. The need for early biomarkers in the diagnosis of PAH in children is claimed by scientists from many countries of the world [1, 2]. The results of our studies showed a significant increase in the concentration of SERT in platelets in group II, compared with group I more than several times. The serotonin content in platelets in children of group II tended to decrease as compared with group I. Perhaps, an excessive increase in transporters in platelets is sufficient in itself for the induction of PASMC hyperplasia and the subsequent development of PAH. It is necessary to continue research in this direction, which will help to reveal and understand the molecular mechanisms by which SERT regulates the proliferation of PASMC and the role of serotonin and its transporter in the development of PAH in children.

Status of pulmonary artery pressures on echocardiography among high-risk newborns

Background: There is a paucity of data about the outcome of newborns who have one or more risk factors for persistent pulmonary hypertension of newborn (PPHN) and have some degrees of pulmonary hypertension not significant enough to cause hypoxemia and desaturation to be labeled as PPHN. Objective: Status of pulmonary artery pressures in high risk newborns. Materials and Methods: This was a prospective observational study, in which newborns up to 1 month of age having risk factors for PPHN were included and pulmonary hypertension was identified on echocardiography in neonates more than 48 h of life. Second screening was done at 6 weeks of life independent of subjects having PAH or not at the first screening. Results: Out of 400 cases, 26 patients were lost to follow-up, 2 cases left against medical advice, and 18 patients expired after first screening. Finally, a total of 354 cases came for follow-up at 6 weeks and repeat echocardiography was done in all cases. PAH was found in 54 cases on first screening, 12 neonates had reversal of shunt at the level of patent ductus arteriosus or patent foramen ovale, and only 2 cases had persistence PAH on second screening at 6 weeks. Perinatal asphyxia (43.9%), respiratory distress (31%), and meconium-stained amniotic fluid (30%) have shown a significant association with the development of PAH in these neonates. Development of PAH worsens the prognosis in newborns with asphyxia and sepsis. Conclusion: Newborns with risk factors have some degree of PAH, which can have an adverse impact on outcome.

Effect of Tobacco Smoke Exposure on Functional Capacity, Respiratory Muscle Strength, and Physical Activity Level in Patients with Pulmonary Arterial Hypertension: A Preliminary Study

Purpose: It is known that tobacco smoke exposure is a risk factor for the development of pulmonary arterial hypertension PAH . In addition, tobacco smoke exposure can cause numerous health problems. This study aimed to compare functional capacity, respiratory muscle strength, and physical activity level between the patients with PAH who have or do not have tobacco smoke exposure. Methods: Seventeen patients with PAH who has tobacco smoke exposure and 20 nonsmoker patients were included in this cross-sectional study. Tobacco smoke exposure was defined as current smokers, ex-smokers, and passive smokers. The study outcomes included the maximal inspiratory and expiratory pressure measurement, 6-minute walk test, and physical activity. Results: There was no significant difference for age, gender, body mass index, pulmonary arterial pressure, and functional class between the two groups p>0.05 . Inspiratory muscle strength p=0.035 , functional exercise capacity p=0.040 and physical activity level p=0.034 were lower in patients with tobacco smoke expose compared to the nonsmokers. Conclusion: The results of this study showed that functional capacity, respiratory muscle strength, and physical activity level were significantly lower in patients with tobacco smoke expose than the nonsmoker patients with PAH. These findings suggest that tobacco smoking is not only associated with PAH risk but also numerous health problems, specifically important ones for this population

P6013Evidence for ANTXR2 as a therapeutic target on systemic-to-pulmonary shunt induced pulmonary arterial hypertension

Abstract Introduction Pulmonary arterial hypertension secondary to congenital heart disease (CHD-PAH) with systemic-to-pulmonary shunt is characterized by proliferative vascular remodeling. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) are the primary cellular bases of vascular remodeling. Anthrax toxin receptor 2 (ANTXR2) exhibits anti-proliferative properties. The effects of ANTXR2 on vascular remodeling and systemic-to-pulmonary shunt induced PAH remain unexplored. Purpose The purpose of this study was to determine the possible roles of ANTXR2 in the pathogenesis of systemic-to-pulmonary shunt induced PAH and explore its possible mechanisms. Methods Lung tissue sections from CHD-PAH patients, systemic-to-pulmonary shunt induced PAH rat model, ANTXR2−/− rats, and PASMCs were used. Immunohistochemistry, real time polymerase chain reaction, Western blot, proliferation, apoptosis, and next generation sequencing (NGS) were performed in this study. Results ANTXR2 expression was reduced in severe CHD-PAH patient lung tissue and pulmonary arterioles, as well as in lung tissues from rats with systemic-to-pulmonary shunt induced PAH. Over-expression of ANTXR2 in cultured PASMCs inhibited cell proliferation and promoted apoptosis, while knockdown of ANTXR2 promoted cell proliferation and inhibited apoptosis. Male ANTXR2−/− rats showed more severe percent medial thickness and muscularization of pulmonary arterioles than wild type (WT) rats in basal conditions, and exhibited heavier PAH following exposure to systemic-to-pulmonary shunt. To further determine the underling mechanism, NGS was performed in ANTXR2−/− rat lungs and that of WT littermates. A total of 1319 genes were found to be dysregulated, and biological processes influenced by these differentially expressed genes include negative regulation of blood vessel diameter,vasoconstriction, regulation of blood vessel diameter, regulation of blood vessel size, vascular process in circulatory system, etc. Conclusion Our work identifies a novel role for ANTXR2 in systemic-to-pulmonary shunt induced PAH based on the findings that ANTXR2 deficiency could exacerbate systemic-to- pulmonary shunt induced vascular remodeling in the development of PAH. ANTXR2 may be a potential target for CHD-PAH treatment. Acknowledgement/Funding Beijing Natural Science Foundation (7172078 and 7172182), the National Major Research Plan Training Program of China (91849111)

P1938In vitro characterization of a novel PI 3-kinase inhibitor for growth factor-induced effects in pulmonary artery smooth muscle cells

Abstract Rationale Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling in pulmonary arterioles. Increased activation of receptor tyrosine kinase (RTK)-mediated signaling pathways lead to increased proliferation and chemotaxis of pulmonary arterial smooth muscle cells (PASMCs). In previous studies, we identified phosphatidylinositol-3-kinase (PI3K) as a key enzyme for these processes. Since systemic PI3K inhibition is potentially associated with undesired side effects, we here for the first time tested the efficacy of the novel PI3K inhibitor CL27c to impede growth factor-mediated cellular effects in human PASMCs. CL27c is topically applicable and as a prodrug it is intracellularly activated and thus offers optimal efficacy in target cells while at the same time minimizing undesired effects in non-target cells. Methods Using Western blotting and immunohistochemistry, activation of the central PI3K effector AKT in human and murine lung tissue was investigated. Quantification of cell proliferation was performed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. PASMCs were incubated with CL27c [0.1; 0.3; 1; 3; 10μM] and stimulated either with PDGF-BB [30ng/ml] or with a growth factor combination (GFC) [PDGF-BB 30ng/ml, FGF 2ng/ml, EGF 0.5ng/ml, insulin 0.5μg/ml, FCS 5%]. Chemotaxis was quantified using modified Boyden chambers. The signal transduction cascade was investigated by Western blotting. Results Analysis of AKT activation revealed hyperphosphorylation in both the vascular wall and complete lung homogenates from PAH patients and hypoxia-treated mice. Examination of the mitogenic signal transduction revealed a markedly reduced phosphorylation of AKT and GSK3β in CL27c pretreated PASMCs. In addition, central cell cycle events, such as expression of cyclin D1, were reduced. This was in line with a CL27c concentration-dependent significant reduction of PDGF- and GFC-induced proliferation (control: 2.53±0.26 vs. CL27c: 0.73±0.14 and control: 5.27±0.61 vs. CL27c: 2.80±0.71 both at 10μM, p<0.001). Furthermore, PDGF- and GFC-induced chemotaxis were as well significantly reduced (100% ± 19 vs. CL27c: 24.43% ± 8.58 and 100% ± 5.76 vs CL27c: 25.23% ± 10.57 at 10μM p<0.001). Conclusion These results indicate that CL27c reduces both proliferation and chemotaxis of PASMCs. As these processes play a key role in the development and progression of PAH, this approach is currently being investigated in preclinical models for its therapeutic potential. This “prodrug” approach could avoid non-specific effects and systemic side effects of Pan-PI3K inhibition, especially since the substance is suitable for topical application (inhalation). Thus, CL27c represents a promising treatment option.

TUG1 Regulates Pulmonary Arterial Smooth Muscle Cell Proliferation in Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease, characterized by a persistent elevation of pulmonary arterial pressure and pulmonary vascular remodelling. Recent studies implicated that long noncoding RNAs (lncRNAs) play important roles in the development of various diseases. However, the underlying mechanisms of lncRNAs in PAH remain unclear. Here we show evidence for the modulation of human pulmonary smooth muscle cell (HPASMC) proliferation and vascular remodelling by lncRNA taurine upregulated gene1 (TUG1). MethodsTUG1 expression and localization was detected by real-time polymerase chain reaction (PCR) and fluorescence in situ hybridization. Proliferation and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), western blot, bromodeoxyuridine incorporation, flow cytometry, scratch-wound assay, 4′,6-diamidino-2-phenylindole (DAPI), and caspase-3 activity. Luciferase activity and microscale thermophoresis were used to identify biomolecular interactions. The right ventricular systolic pressure and right ventricular hypertrophy were measured to evaluate cardiopulmonary function. ResultsTUG1 was upregulated in the pulmonary arteries of mice after a hypoxic assault and showed a significant increase in patients with PAH. TUG1 knockdown significantly prevented the development of PAH in vivo. Moreover, TUG1 promoted the proliferative responses of HPASMCs, including cell viability, 5-bromodeoxyuridine incorporation, the expression of proliferating cell nuclear antigen, and cell-cycle progression. All these functions of TUG1 were likely to be associated with miR-328. ConclusionsThe present study indicates that TUG1, a novel potential target for the treatment of PAH, is necessary for HPASMC proliferation and pulmonary vascular remodelling.

Unique clinical and autoantibody profile of a large Asian Indian cohort of scleroderma-do South Asians have a more aggressive disease?

A single-centre retrospective study was conducted using electronic medical records (EMR) of inpatients and outpatients with the diagnosis of "scleroderma" or "systemic sclerosis" visiting our clinic over the preceding 5years. A total of 327 patients' charts met our selection criteria; 301 were females. The median (IQR (inter quartile range)) age at onset of first non-Raynaud's symptom was 34.67 (27-43) years and median (IQR) disease duration prior to presentation to our department was 2.5 (1-5) years. Of these, 310 (94.8%) belonged to diffuse systemic sclerosis variety, 13 (4%) had limited systemic sclerosis, and 4 (1.2%) were of sine scleroderma type. A total of 289/302 (95.7%) patients were positive for ANA; of them, 245/327 (74.9%) were Scl-70 antibody-positive and 4% were CENP antibody-positive. Interstitial lung disease (ILD) was present in 288/327 (88.1%) patients. Among patients with available baseline forced vital capacity (FVC) data, 20% had a normal lung function and 28.4% had severe restriction. Pulmonary hypertension as assessed by echocardiography was present in 8.1% of patients. A significant association of Scl-70 antibody positivity with the presence of interstitial lung disease (ILD) (p = 0.000) and pulmonary hypertension (p = 0.035) was seen. On the other hand, presence of CENP antibody showed a protective trend against muscle weakness and/or muscle enzyme elevation (p = 0.052). Presence of arthritis was protective against development of digital ulceration (p = 0.021) and PAH (0.004). Patients younger than 40years of age had significantly higher frequency of Scl-70 positivity (p = 0.038), whereas CENP antibody positivity was more likely in those aged > 40years (p = 0.002). Younger age of onset and high prevalence of Scl-70 antibody are unique South Asian features common with large Indian, Thai, and Chinese series. High prevalence of ILD is a feature common to Indian and Chinese series. Strong correlation of Scl-70 antibody with younger age and pulmonary hypertension were unique features of our cohort. • Asian Indian scleroderma patients are younger by 2 decades compared to Caucasian series. • Higher prevalence of Scl-70 antibody, its association with young age, interstitial lung disease and pulmonary hypertension are features of our cohort. • High prevalence of interstitial lung disease (88.1%) was noted ; among those with baseline spirometry data (141/327), two thirds(66%) had moderate to severe restriction. • Younger age at onset, higher prevalence of Scl-70 antibody are features common to other Indian, Thai and Chinese series.

2018 AHA Late-Breaking Basic Science Abstracts.

2018 AHA Late-Breaking Basic Science Abstracts.

LncRNA H19 promotes the proliferation of pulmonary artery smooth muscle cells through AT1R via sponging let-7b in monocrotaline-induced pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is related to inflammation, and the lncRNA H19 is associated with inflammation. However, whether PDGF-BB-H19-let-7b-AT1R axis contributes to the pathogenesis of PAH has not been thoroughly elucidated to date. This study investigated the role of H19 in PAH and its related mechanism.MethodsIn the present study, SD rats, C57/BL6 mice and H19−/− mice were injected with monocrotaline (MCT) to establish a PAH model. H19 was detected in the cytokine-stimulated pulmonary arterial smooth muscle cells (PASMCs), serum and lungs of rats/mice. H19 overexpression and knockdown experiments were also conducted. A dual luciferase reporter assay was used to explore whether let-7b is a sponge miRNA of H19, and AT1R is a novel target of let-7b. A CCK-8 assay and flow cytometry were used to analyse cell proliferation.ResultsThe results showed that H19 was highly expressed in the serum and lungs of MCT-induced rats/mice, and H19 was upregulated by PDGF-BB in vitro. H19 upregulated AT1R expression via sponging miRNA let-7b following PDGF-BB stimulation. AT1R is a novel target of let-7b. Moreover, the overexpression of H19 and AT1R could facilitate PASMCs proliferation in vitro. H19 knockout protected mice from pulmonary artery remodeling and PAH following MCT treatment.ConclusionOur study showed that H19 is highly expressed in MCT-induced rodent lungs and upregulated by PDGF-BB. The H19-let-7b-AT1R axis contributed to the pathogenesis of PAH by stimulating PASMCs proliferation. The H19 knockout had a protective role in the development of PAH. H19 may be a potential tar-get for the treatment of PAH.

Abstract 17190: Dual Hit of HIV-1 and Opioids Promote Oxidative Stress Mediated Pulmonary Endothelial Dysfunction

Introduction: HIV related pulmonary hypertension is one of the serious non-infectious cardiovascular complications in HIV infected individuals, especially among intravenous drug users. We previously demonstrated that opioids in combination with viral protein(s) result in reactive oxygen species (ROS) mediated induction of autophagy in pulmonary endothelial cells leading to shift in the cells from apoptosis to apoptosis resistant proliferative status associated with angio-proliferative remodeling observed in PAH. Here, we delineate the major sources of HIV-1 and morphine induced oxidative burst and its role in development of PAH using in-vivo model. Methods: Human pulmonary microvascular endothelial cells (HPMECs) were treated with morphine and HIV-Tat in the presence or absence of ROS scavengers followed by measurement of ROS. Mitochondrial specific superoxide radicals were measured with MitoSOX dye and ratio of mitochondrial DNA to nuclear DNA was determined by real-time PCR. To assess the in-vivo effect of HIV-1 and morphine on pulmonary vascular dysfunction, male HIV-Tg/WT Fischer rats (n=7-13per group) were subcutaneously administered 10 mg/kg body wt. morphine or saline daily IP for 21 days. Right ventricle systolic pressure (RVSP) and fulton Index were measured along with immunohistochemistry analysis of lungs. Result: Increased oxidative stress in HPMECs in response to combined treatment of morphine and tat (M+T) was noted to be contributed by mitochondria, NADPH oxidase and xanthine oxidase as observed by the decrease in ROS production of M+T treated cells on pre-treatment with ROS scavenger viz., Mitotempo (mitochondria), VAS3947 (NADPH oxidase) and allopurinol (Xanthine oxidase). However, M+T mediated early apoptosis and late hyperproliferation of HPMECs was prevented in presence of NADPH oxidase inhibitor. In parallel we observed elevated mitochondrial specific ROS at 2 hr and increased mitophagy at 3h followed by increase in total mitochondrial mass at 4-6h of MT treatment. Further, we observed significant increase in RVSP and RV hypertrophy in HIV-Tg rats exposed to morphine compared to WT rats. Immunohistochemical analysis also showed significant pulmonary vascular remodelling including endothelial proliferation in lung sections from HIV+morphine rats. Conclusion: Our findings suggest NADPH oxidase specific ROS role in autophagy-dependent increased proliferation of M+T treated HPMECs. Further our in-vivo findings provide evidence of potentiation of PAH development with the dual hit HIV-1 and opioids.

Involvement of S100A4/Mts1 and associated proteins in the protective effect of fluoxetine against MCT – Induced pulmonary hypertension in rats

BackgroundPulmonary arterial hypertension (PAH) is a complex pulmonary vasculature disease characterized by remodeling of the pulmonary vessels and a persistent increase in the pulmonary vascular resistance (PVR) with a poor prognosis. Serotonin increases the expression of S100A4/Mts1, which in turn stimulates the proliferation and migration of human pulmonary artery smooth muscle cells through the interaction with RAGE (receptor for advanced glycation end products) and thus S100A4/Mts1 has been implicated in the development of PAH in vitro. Fluoxetine, a selective serotonin re-uptake inhibitor has been shown to protect against PAH. The current study was designed to test whether S100A4 and its associated proteins connected in the development of PAH in vivo as well as to investigate the involvement of those proteins in the protective effect of fluoxetine against PAH. MethodsMCT-induced PAH models were established in Wistar rats by a single intraperitoneal injection of MCT (60 mg/kg). Fluoxetine (2 and 10 mg/kg/day) was intragastrically administered once a day for 3 weeks along with controls. The detection methods followed include Hematoxylin and Eosin (H&E) staining, immunohistochemistry, western blotting and real-time reverse transcription-polymerase chain reaction (RT-PCR). ResultsMCT induced pulmonary hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy significantly increased the expressions of S100A4 and RAGE in the pulmonary arteries, lungs and right ventricle (RV). Fluoxetine dose-dependently inhibited MCT-induced pulmonary arterial hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy and reduced the S100A4 and RAGE. Further analysis revealed that fluoxetine alleviated both the increase of p53, MMP13, MMP2 and MMP9 and the decrease of pp53Ser15 and MDM2 in lungs and RV tissues of MCT-induced PAH rats. ConclusionFrom the present investigation it could be concluded that S100A4/Mts1 and its associated proteins are involved in the evolution of MCT-induced PAH in rats and fluoxetine inhibits MCT-induced PAH in rats mainly through S100A4/RAGE signaling axis and involved factors.

Macrophage-derived extracellular vesicles mediate smooth muscle hyperplasia: role of altered miRNA cargo in response to HIV infection and substance abuse.

Our previous studies consistently demonstrate enhanced pulmonary vascular remodeling in HIV–infected intravenous drug users, and in simian immunodeficiency virus–infected macaques or HIV-transgenic rats exposed to opioids or cocaine. Although we reported an associated increase in perivascular inflammation, the exact role of inflammatory cells in the development of pulmonary vascular remodeling remains unknown. In this study, HIV–infected and cocaine (H+C)–treated human monocyte derived macrophages released a higher number of extracellular vesicles (EVs), compared to HIV-infected or uninfected cocaine-treated macrophages, with a significant increase in the particle size range to 100–150 nm. Treatment of primary human pulmonary arterial smooth muscle cells (HPASMCs) with these EVs resulted in a significant increase in smooth muscle proliferation. We also observed a significant increase in the miRNA-130a level in the EVs derived from H+C-treated macrophages that corresponded with the decrease in the expression of phosphatase and tensin homolog and tuberous sclerosis 1 and 2 and activation of PI3K/protein kinase B signaling in HPASMCs on addition of these EVs. Transfection of HPASMCs with antagomir-130a–ameliorated the EV-induced effect. Thus, we conclude that EVs derived from H+C-treated macrophages promote pulmonary smooth muscle proliferation by delivery of its prosurvival miRNA cargo, which may play a crucial role in the development of PAH.—Sharma, H., Chinnappan, M., Agarwal, S., Dalvi, P., Gunewardena, S., O’Brien-Ladner, A., Dhillon, N. K. Macrophage-derived extracellular vesicles mediate smooth muscle hyperplasia: role of altered miRNA cargo in response to HIV infection and substance abuse.

Is pulmonary vascular disease reversible with PPAR ɣ agonists?

Severe angio-obliterative PAH remains a disease characterized by great morbidity and shortened survival. Unfortunately, the only currently available treatments for angio-obliterative changes are palliative in the form of pulmonary vasodilators evolving from the phosphodiesterase inhibitor sildenafil to endothelin receptor antagonist: Bosentan; while the only definitive treatment is lung transplantation which remains dependent on the availability of donors and the transplant policies which vary widely from a country to another. PPARs, especially the γ isoform, are largely expressed in pulmonary artery endothelial cells and smooth muscle cells. They are also found on endothelial progenitor cells. Several previous studies have highlighted the role of PPAR γ agonists in reversal of vascular remodeling especially in coronary, carotid and peripheral vascular disease atherosclerotic plaques. Experimental studies have also revealed that PPAR γ activation affects many different pathways; thus, the effect of PPAR γ is multifaceted, affecting almost every pathobiological pathway involved in the development of PAH simultaneously. We thereby hypothesize that PPAR γ agonists may play a key role in reversing severe pulmonary angio-obliterative changes and promote microvascular regeneration which may substitute the need for heart-lung transplantation in such patients.

EZH2 Inhibition Ameliorates Transverse Aortic Constriction-Induced Pulmonary Arterial Hypertension in Mice.

Background EPZ005687 is a selective inhibiter of methyltransferase EZH2. In this article, we investigated the protective role and mechanism of EPZ005687 in transverse aortic constriction-induced pulmonary arterial hypertension in mice. Methods We assigned 15 (6–8 weeks old) male balb/c mice to 3 groups randomly: Sham control + DMSO group, TAC + DMSO group, and TAC + EPZ005687 group (10 mg kg−1, once a week for 4 weeks). On day 28 following TAC operation, the right ventricular systolic blood pressure (RVSBP) was measured, and lung tissues were collected for laboratory examinations (DHE, Western blot, real-time PCR, and ChIP). Results Murine PAH model was successfully created by TAC operation as evidenced by increased RVSBP and hypertrophic right ventricle. Compared with the sham control, TAC-induced PAH markedly upregulated the expression of EZH2 and ROS deposition in lungs in PAH mice. The inhibiter of methyltransferase EZH2, EPZ005687 significantly inhibits the development of TAC-induced PAH in an EZH2-SOD1-ROS dependent manner. Conclusion Our data identified that EZH2 serves a fundamental role in TAC-induced PAH, and administration of EPZ005687 might represent a novel therapeutic target for the treatment of TAC-induced PAH.

Coupling Factor 6 Is Upregulated in Monocrotaline-induced Pulmonary Arterial Hypertension in Rats

BackgroundPulmonary arterial hypertension (PAH) is a rare and fatal disease. The prostacyclin (PGI2) pathway is a well-known therapeutic target for PAH. As a novel vasoconstrictive peptide, coupling factor 6 (CF6) has been recognized as the only endogenous inhibitor of PGI2. However, the relationship between CF6 and PAH is still unknown. In this study, we investigated the involvement of CF6 in PAH in rats. MethodsA total of 80 Sprague-Dawley rats were randomly divided into 2 groups: a control group (with saline intraperitoneally) and a monocrotaline (MCT)-treated group (with MCT 60mg/kg intraperitoneal injection). The expression level of CF6 was measured by immunohistochemistry, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The plasma level of 6-keto-PGF1α, a stable metabolite of PGI2, was measured by enzyme-linked immunosorbent assay. ResultsAfter MCT injection, although the plasma level of CF6 was markedly increased in a time-dependent manner, the level of 6-keto-PGF1α was decreased in the MCT rats as compared to that in the controls (P < 0.05). Reverse transcription polymerase chain reaction indicated that the lung tissue level of CF6 messenger RNA (mRNA) in the MCT rats was significantly upregulated compared to the level in the controls. There was an intense CF6 immunostain concentrated on the lung tissue of the MCT rats compared with the controls. Univariate analysis indicated that the plasma level of CF6 was not correlated with blood pressure, fasting blood glucose, cholesterol (high-density lipoprotein, triglycerides and total cholesterol) or C-reactive protein level. ConclusionsThese results demonstrated that CF6 was elevated in MCT-induced PAH rats and may play an important role in the development of PAH.

The role of tyrosine kinase inhibitor “Lapatinib” in pulmonary hypertension

IntroductionPulmonary Arterial Hypertension (PAH) and cancer share growth factor and protein kinase signaling pathways that result in smooth muscle cell proliferation and vasculopathy. There is little known about the impact of Lapatinib on the pulmonary vasculature. After reporting a case of Lapatinib-induced PAH we investigated the association of Lapatinib with the development of PAH in our institution. MethodsWe reviewed charts for all patients treated with Lapatinib at our institution between 2008 and 2013. Patients who had undergone 2D-echocardiogram both prior to and after treatment were included in the analysis. Increase in Pulmonary artery systolic pressure (PASP) was assessed. Patients were also evaluated in terms of risk factors for non-Group 1 PAH. ResultsA total of 27 patients were found to have 2-D echo done before and after starting treatment with Lapatinib. Six patients were found to have significant increase in their PASP after starting treatment. Right heart catheterization before and after stopping the medication was available in three patient, confirming the diagnosis of PAH with complete resolution after stopping the medication. The median pre-treatment and post treatment PASP in those 6 patients was 29 mmHg and 65.5 mmHg respectively (N = 6; p = 0.027). ConclusionLapatinib might be associated with the development of PAH. PASP should be evaluated in patients who become short of breath while on treatment, and stopping the drug in cases where no other reasons are identified could be associated with reversibility of the elevated pulmonary artery pressure.

Glycoprotein 130 Inhibitor Ameliorates Monocrotaline-Induced Pulmonary Hypertension in Rats

BackgroundPulmonary arterial hypertension (PAH) is characterized by vasoconstriction, vascular remodelling, and microthrombotic events. Inflammatory cytokine interleukin (IL-6) may be a key factor in the development of PAH, and glycoprotein 130 (Gp130) is an important signal-transducing subunit of IL-6. The aim of our study was to evaluate the effectiveness of Gp130 inhibitor in reducing inflammation and ameliorating PAH-related vascular remodelling in monocrotaline (MCT)-exposed rats. MethodsSprague-Dawley rats (n = 96; weight, 240-250 g) were randomly divided into 3 groups: control, MCT-exposed (MCT), and MCT-exposed plus Gp130 inhibitor (MCT-Gp) administered daily (5 mg/kg) from days 14-28. Eight rats were killed in each group at weeks 1 through 4, with the following measured variables compared across groups on day 28: hemodynamics, right ventricular hypertrophy, morphometric measurements, immunohistochemical results, levels of IL-6, phosphorylated signal transducer and activator of transcription 3, proliferating cell nuclear antigen (PCNA), bone morphogenetic protein receptor-2 (BMPR2), proangiogenic factor, vascular endothelial growth factor (VEGF), proproliferative kinase extracellular signal-regulated kinase (ERK), survivin, Bcl-2, and Bax. ResultsCompared with the MCT group, Gp130 inhibitor, after MCT exposure, improved hemodynamics and significantly reduced the severity of inflammation, as estimated by levels of IL-6 (P < 0.0001), and reversed pulmonary arterial remodelling, as assessed by medial wall thickness (P < 0.0001). Gp130 inhibitor upregulated BMPR2 expression in MCT-exposed lungs (P = 0.040) and decreased the expression of PCNA, VEGF, ERK, and survivin (all P < 0.05). ConclusionsGp130 inhibitor upregulated BMPR2 expression in MCT-exposed lungs, restored the BMPR2/IL-6 balance, reduced IL-6–associated inflammation, inhibited pulmonary artery smooth muscle cell proliferation, and ameliorated pulmonary vascular remodelling in MCT-induced PH in rats.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension

AimsPulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. Methods and ResultsBy Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. ConclusionOur data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.