Treatment Of Pulmonary Arterial Hypertension Research Articles

Abstract 9359: Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca 2+ ) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca 2+ entry (SOCE) is involved in Ca 2+ homeostasis in PASMCs, but its properties in PAH are unclear. Methods: Using a combination of Ca 2+ imaging, molecular biology, in vitro , ex vivo , and in vivo approaches, we investigated the roles of SOCE channel Orai1 in PA remodeling in PAH, and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). Results: Human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs) showed upregulation of SOCE and Orai1 expression, in which ERK1/2, nuclear factor of activated T cells (NFAT), and nuclear factor-kappa B (NFκB) were contributing factors. Using siRNA and two Orai1 inhibitors, we found that Orai1 inhibition reduced SOCE, mitochondrial Ca 2+ entry, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic-hypoxia or monocrotaline or Sugen/hypoxia, administration of Orai1 inhibitors (BTP2, JPIII, or 5J4) protected against PH. Conclusions: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMC phenotype and attenuates PH in rat models, suggesting that Orai1 should be considered as a relevant therapeutic target for PAH.

Abstract 11414: Anti-Mir-7110-3p as a Therapeutic Molecule in Monocrotaline Induced Pulmonary Arterial Hypertension Rat Model

Introduction: Pulmonary arterial hypertension (PAH) is a chronic condition marked by unusually increased blood pressure in the pulmonary artery which transports blood from the heart to lungs. Advances in PAH treatment have improved PAH patients outcomes and prognosis, but still continues to be a life-threatening condition and also the current treatment strategies have failed to address the underlying cellular and molecular problems. MicroRNAs are short noncoding RNAs that suppress gene expression by binding to the 3' UTR of their target mRNA. According to our prior findings, there is evidence that miR-7110 has a prominent role in PAH. Hypothesis: In this study, we assessed the hypothesis to determine whether anti-miR-7110-3p can help to prevent PAH in monocrotaline-induced PAH (MCT-PAH) rat model via PKCδ/STAT3 signaling pathway. Methods: Pulmonary arterial smooth muscle cells (PASMCs) were transfected with anti-miR-7110-3p to investigate the effect of miR-7110-3p on PASMCs proliferation and apoptosis in in vitro condition. To induce PAH, male Sprague Dawley rats were given an intraperitoneal injection of 50 mg/kg monocrotaline. The expression of miR-7110-3p/PKCδ/STAT3 were assessed using RT-qPCR and western blotting analysis, while the activity of miR-7110-3p and its target STAT3 was determined using luciferase reporter assay. Systolic pulmonary arterial pressure was recorded using PowerLab data acquisition system and after obtaining the readings the rats were immediately sacrificed. The lower lobe of right lung tissue was collected, fixed in 4 % paraformaldehyde, sectioned and processed for histological analyses. Results: Our results demonstrated that miR-7110-3p has a proliferative and anti-apoptotic effect on PASMCs. Additionally, in vitro studies revealed that miR-7110-3p/ PKCδ/STAT3 axis in involved in PAH. Our histological analyses suggested that anti-miR-7110-3p was able to reduce the pulmonary vascular remodeling. As a result, the relationship between miR-7110-3p, STAT3 and anti-miR-7110-3p is an important step in gene expression analyses. Conclusions: In conclusion, our findings suggest that miR-7110-3p may be used as a biomarker and anti-miR-7110-3p can be used as a therapeutic molecule for the treatment of PAH.

Abstract 10715: Therapeutic Efficacy of Drpitor1a, a Novel Dynamin-Related Protein 1 (DRP1) Inhibitor, in Preclinical Pulmonary Arterial Hypertension

Introduction: Dynamin-related protein 1 (DRP1) is a large GTPase that mediates mitotic fission (the division of mitochondria which is coordinated with mitosis, ensuring equitable distribution of mitochondria to daughter cells). In pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells (PASMC), mitotic fission, and DRP1 activity are increased, contributing to its hyperproliferative phenotype. We investigated the therapeutic efficacy of Drpitor1a, a novel, small molecule, Drp1 GTPase inhibitor, in a rat PAH model. Methods: A single dose of monocrotaline (MCT, 60mg/kg, SC) or PBS was injected to female Sprague-Dawley rats on day 0 (n=20 for MCT and n=15 for PBS). On day 14, the development of PAH was confirmed by echocardiography and the rats were randomized for treatments. An indwelling catheter was implanted through the left jugular vein on day 15. Drpitor1a (1mg/kg) or normal saline (NS) was administered by IV every 48 hours from day 17 to day 27. Right ventricular (RV) structure and function were assessed on day 28 with echocardiography. Pulmonary hemodynamics was evaluated on day 29 using right heart catheterization (RHC). Results: MCT rats developed PAH with RV dysfunction on day 14. There was no statistical difference between the Drpitor1a and NS groups at randomization. At the endpoint, MCT+Drpitor1a rats, vs. MCT+NS rats, had a significantly reduced severity of pulmonary hypertension evident as longer pulmonary artery acceleration time (PAAT) and lower pulmonary vascular resistance index. Drpitor1a also improved RV function, evident as greater RV free wall thickening (RVFWT%), increased tricuspid annular plane systolic excursion (TAPSE), and increased cardiac index (CI). Drpitor1a regressed pulmonary artery medial thickening and inhibited RV hypertrophy without hepatological, renal or hepatic toxicities. Conclusion: Drpitor1a is a safe and effective treatment for preclinical PAH.

Abstract 11565: Improvement in Cardiopulmonary Function in a Rat Model of Pulmonary Arterial Hypertension Observed With RKER-012, a Novel Activin Receptor Type II Ligand Trap, was Associated With Reduced Markers of Inflammation and Fibrosis in the Right Ventricle

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary vascular resistance, impaired cardiac output, and right ventricle (RV) overload. PAH is associated with imbalanced TGF-ß signaling, marked by elevated SMAD2/3 signaling, which is associated with inflammation and fibrosis contributing to heart failure. Initially, the RV can compensate for increased pressure due to its adaptive contractility, but chronic pressure overload leads to maladaptive RV remodeling through inflammatory and fibrotic pathways. Current therapies for PAH are primarily vasodilators which do not treat the underlying pathology. RKER-012 is a research form of KER-012, an investigational modified ActRIIB ligand trap designed to inhibit signaling through SMAD2/3. We investigated RKER-012’s mechanism of prevention of cardiac pathology in a Sugen/Hypoxia (SH) PAH model. Rats received a subcutaneous (SQ) dose of 25 mg/kg Sugen 5416 under hypoxic conditions. For 3 weeks, SH rats received vehicle (SH-VEH) or 10 mg/kg RKER-012 (SH-RKER-012) twice weekly SQ. Control rats received VEH twice weekly in normoxic conditions. Systolic pulmonary arterial pressure (sPAP) and Fulton index (FI) were measured. qPCR of RV tissue was used to evaluate the mechanism of RKER-012 in this model. SH-VEH rats had increased FI and sPAP (both p≤0.0001) compared to NX, consistent with development of cardiac and pulmonary impairment. Relative to SH-VEH, RKER-012 reduced increased FIand prevented increased sPAP (both p≤0.001) in SH rats so that sPAP did not differ from NX. Relative to NX, SH-VEH had increased RV expression of fibrotic markers such as Timp1, Col1a1, Col3a1, Runx2, and LOX (all p≤0.05). In contrast, SH-RKER-012 rats had reduced expression of Timp1, Col1a1, Col3a1, Runx2 and LOX (all p≤0.05) relative to SH-VEH. Inflammation markers CD68 and MCP1 (both p≤0.05) were increased in SH-VEH compared to NX. CD68 expression was reduced in SH-RKER-012 (p≤0.01) compared to NX but MCP1 expression was not affected (p=0.54). These results support that RKER-012 attenuated upregulation of key inflammation and fibrosis markers in a PAH preclinical model and suggest that KER-012 has a distinct mechanism from vasodilation and could be developed as a cardioprotective treatment for PAH.

Abstract 10523: Involvement of Sur2/Kir6.1 In the Physiopathology of Pulmonary Arterial Hypertension

Introduction: The ATP-sensitive K+ channels (KATP) regulatory subunit ABCC8 may be considered a new therapeutic target for pulmonary arterial hypertension (PAH). Hypothesis: We hypothesised that the second KATP regulatory subunit (ABCC9) contributes to PAH pathogenesis. ABCC9 gene encodes for two regulatory subunits of KATP channels: the SUR2A and SURB proteins. In the KATP channel, the SUR2 subunits are associated with the K+ channel Kir6.1. We investigated how the SUR2/Kir6.1 channel contributes to PAH pathogenesis and its potential as a therapeutic target against PAH. Methods: Using in vitro, ex vivo, and in vivo approaches, we analysed the localisation and expression of SUR2A, SUR2B, and Kir6.1 in pulmonary vasculature of controls and patients with PAH as in experimental PH rat models and its contribution to PAH physiopathology. Finally, we deciphered the consequences of in vivo activation of SUR2/Kir6.1 in the monocrotaline (MCT)-induced PH model. Results: We demonstrated that SUR2A, SUR2B, and Kir6.1 were expressed in the lungs of controls and patients with PAH and MCT-induced PH rat model. Myograph experiments showed that activation of SUR2 by pinacidil induced pulmonary arterial relaxation. In vitro experiments on human pulmonary arterial smooth muscle cells and endothelial cells (hPASMCs and hPAECs) in controls and PAH patients showed decreased cell proliferation and migration after SUR2 activation. Using patch-clamp, we demonstrated that SUR2 activation in rat right ventricular (RV) cardiomyocytes reduced RV action potential duration. Chronic pinacidil administration in control rats increased heart rate without changes in hemodynamic parameters. Finally, in vivo pharmacological activation of SUR2 on MCT-induced-PH rats showed an improvement of PH during the preventive and curative protocol. Conclusions: We demonstrated that SUR2A, SUR2B, and Kir6.1 were expressed in hPASMCs and hPAECs of controls and PAH patients. In vivo SUR2 activation reduced MCT-induced PH phenotype, suggesting that SUR2 activation should be considered for treatment of PAH.

Pulmonary Arterial Hypertension Patients Have a Proinflammatory Gut Microbiome and Altered Circulating Microbial Metabolites.

Rationale: Inflammation drives pulmonary arterial hypertension (PAH). Gut dysbiosis causes immune dysregulation and systemic inflammation by altering circulating microbial metabolites; however, little is known about gut dysbiosis and microbial metabolites in PAH. Objectives: To characterize the gut microbiome and microbial metabolites in patients with PAH. Methods: We performed 16S ribosomal RNA gene and shotgun metagenomics sequencing on stool from patients with PAH, family control subjects, and healthy control subjects. We measured markers of inflammation, gut permeability, and microbial metabolites in plasma from patients with PAH, family control subjects, and healthy control subjects. Measurements and Main Results: The gut microbiome was less diverse in patients with PAH. Shannon diversity index correlated with measures of pulmonary vascular disease but not with right ventricular function. Patients with PAH had a distinct gut microbial signature at the phylogenetic level, with fewer copies of gut microbial genes that produce antiinflammatory short-chain fatty acids (SCFAs) and secondary bile acids and lower relative abundances of species encoding these genes. Consistent with the gut microbial changes, patients with PAH had relatively lower plasma concentrations of SCFAs and secondary bile acids. Patients with PAH also had enrichment of species with the microbial genes that encoded the proinflammatory microbial metabolite trimethylamine. The changes in the gut microbiome and circulating microbial metabolites between patients with PAH and family control subjects were not as substantial as the differences between patients with PAH and healthy control subjects. Conclusions: Patients with PAH have proinflammatory gut dysbiosis, in which lower circulating SCFAs and secondary bile acids may facilitate pulmonary vascular disease. These findings support investigating modulation of the gut microbiome as a potential treatment for PAH.

Recommendations for the diagnosis and treatment of connective tissue disease associated pulmonary arterial hypertension in China

Pulmonary arterial hypertension (PAH) is a clinicopathological syndrome caused by the increase of pulmonary artery, and it is the most serious complication of connective tissue disease (CTD). In recent years, a lot of progress has been made in the diagnosis, treatment and evaluation of PAH. Chinese Rheumatology Association formulated this recommendation on the basis of current experience and guidelines, in order to promote early screening, early diagnosis and early intervention of CTD-PAH, as well as patient follow-up and management, to improve the prognosis of CTD-PAH patients.

Upregulated ClC3 Channels/Transporters Elicit Swelling-Activated Cl- Currents and Induce Excessive Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling of the pulmonary artery, which is mainly attributed to the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) comprising the medial layer of pulmonary arteries. The activity of ion channels associated with cytosolic Ca2+ signaling regulates the pathogenesis of PAH. Limited information is currently available on the role of Cl- channels in PASMCs. Therefore, the functional expression of ClC3 channels/transporters was herein investigated in the PASMCs of normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Expression analyses revealed the upregulated expression of ClC3 channels/transporters at the mRNA and protein levels in IPAH-PASMCs. Hypoosmotic perfusion (230 mOsm) evoked swelling-activated Cl- currents (ICl-swell) in normal-PASMCs, whereas 100 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) exerted the opposite effects. The small interfering RNA (siRNA) knockdown of ClC3 did not affect ICl-swell. On the other hand, ICl-swell was larger in IPAH-PASMCs and inhibited by DIDS and the siRNA knockdown of ClC3. IPAH-PASMCs grew more than normal-PASMCs. The growth of IPAH-PASMCs was suppressed by niflumic acid and DIDS, but not by 9-anthracenecarboxylic acid or T16Ainh-A01. The siRNA knockdown of ClC3 also inhibited the proliferation of IPAH-PASMCs. Collectively, the present results indicate that upregulated ClC3 channels/transporters are involved in ICl-swell and the excessive proliferation of IPAH-PASMCs, thereby contributing to the pathogenesis of PAH. Therefore, ClC3 channels/transporters have potential as a target of therapeutic drugs for the treatment of PAH.

Scientific substantiation of the concept of pharmacoeconomic analysis of technologies for early diagnosis and pharmacotherapy of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare, progressive disorder characterized by high blood pressure (hypertension) in the arteries of the lungs (pulmonary artery) for no apparent reason.
 Because symptoms are nonspecific and physical signs may be subtle, the disease is often diagnosed at late stages. Over the past few decades, significant progress has been made in the field of pulmonary arterial hypertension. Technological progress allows to diagnose the disease in the early stages, as well as to better assess the severity of the disease.
 Today, screening studies are becoming increasingly important to establish a clinical diagnosis and minimize lost opportunities without timely diagnosis, especially in patients with idiopathic PAH.
 Therefore, the issue of early diagnosis of patients with suspected PAH and confirmation of the diagnosis is a very important and urgent issue today.
 The objective of the paper: to study the methods of implementation of technologies for early diagnosis and pharmacotherapy of pulmonary arterial hypertension and conceptual presentation of the results of their implementation.
 The work used available scientific sources of information on the results of the implementation of diagnostic screening for early detection of PAH and pharmacotherapy in the world. The analysis of input data was carried out using systematic, documentary, informational and graphical research methods.
 The result of the analysis of information materials showed that over the past two decades, significant progress has been made in the treatment of PAH and more than doubled the survival rate of patients.
 It has been found that diagnostic screening is a relatively unique medical technology in relation to other medical interventions and can be defined as the systematic use of diagnostic technologies in individuals at risk to detect the disease before the onset of symptoms.
 The study proposes a systematic concept of economic evaluation of the diagnostic screening program for PAH to determine its optimal design.
 At the same time, this concept embodies the complex effect of the introduction of technologies for early diagnosis and pharmacotherapy of PAH, which is expressed in the positive dynamics of clinical indicators, reduced mortality and improved quality of life.
 It should be noted that a number of new tools and approaches for diagnostic screening give hope that advances in the diagnosis of PAH will also affect the effectiveness of medical care.

A New Spectrophotometric Method for The Determination of Macitentan in Human Plasma

Macitentan is an endothelin receptor antagonist drug used in the treatment of pulmonary arterial hypertension (PAH). PAH is defined as a serious disease of the arteries (pulmonary arteries) that connect the lungs to the heart. The aim of our study is to develop and validate a new, sensitive, simple, accurate and reliable UV spectrophotometric method for the quantification of macitentan in human plasma. Using a wavelength of 269 nm, macitentan was quantitatively analyzed. For standard solution concentrations of 1–30 μg/mL and for human plasma concentrations of 2–30 μg/mL, respectively, the method was linear. By computing the correlation coefficient, linearity was determined. In standard solution and human plasma, these values were found to be 0.9996 and 0.9991, respectively. In terms of accuracy, sensitivity, precision, and repeatability, the developed spectrophotometric method was deemed satisfactory. In addition to this, this method could be directly and easily applied to both bulk and human plasma.

Comprehensive Review of Pulmonary Hypertension and Treatment Options in the Paediatric Population

Pulmonary hypertension (PH) is a complex condition that can occur as a result of a wide range of disorders, including left heart disease, lung disease, and chronic pulmonary thromboembolism. Multiple improvements have been made in the diagnosis and treatment of pulmonary arterial hypertension (PAH)including a greater understanding of the involvement of extrapulmonary vascular organ systems, validated point of care, clinical assessment tools, and a focus on the initial exposure of numerous pharmacotherapeutics in the appropriate level of care. To achieve a minimal symptom burden, improve the patient's biochemical, hemodynamic, and functional profile, and reduce adverse impact, early diagnosis of PAH is a key objective today. The preferred method of management for thromboembolic PH, which is chronic, is pulmonary endarterectomysince the majority of affected patients are operable. The timing of pulmonary endarterectomy should never be delayed for medical reasons, and risk stratification can enable us to select patients who have a high chance of success. Patients who are not qualified for endarterectomy should be referred for drug trials. Even though there are more effective ways to guarantee a sufficient, long-lasting septostomy, atrial septostomy is promising but undervalued. The procedure's indications remain the sameand needto be taken into account more frequently. Class III or IV patients who are not improving need to be consulted at a transplant centre as soon as possible as they may be candidates for potential recipients of bilateral sequential lung or heart-lung transplants, which is a significant choice for some people. PH is rarely linked to other conditions like connective tissue or thromboembolic disease. It is either idiopathic or linked to congenital heart disease. Infants and children with PH are more frequently recognised in conjunction with a congenital diaphragmatic hernia and developmental lung diseases like bronchopulmonary dysplasia. Although the underlying disease has not yet been treated and advanced structural changes have not yet been reversed, the value of natural life and survival have suggestively increased. Children's haemodynamic and functional outcomes have improved as a result of endothelin receptor antagonists, prostacyclin analogues, and phosphodiesterase type 5 inhibitors, which are examples of targeted pulmonary vasodilator therapies. The health maintenance of paediatric PH is still difficult because treatment decisions are largely based on the findings of adult studies that have been supported by evidence and the clinical expertise of paediatric specialists.

Pharmacological counseling in hepatotoxicity induced by macitentan and selexipag: a case report

BackgroundPulmonary arterial hypertension is a progressive, debilitating condition characterized by increased resistance in the pulmonary arterial circulation. Current treatments for pulmonary arterial hypertension include endothelin receptor antagonists such as bosentan, sitaxentan, ambrisentan, macitentan, and oral prostacyclin receptor agonists such as selexipag. Endothelin receptor antagonists have been associated with liver injury, while hepatotoxicity was not reported for selexipag. Although genetic variability has been indisputably associated with variability in drug response, no study has been designed until now to assess its effects on the pharmacokinetics of endothelin receptor antagonists or selexipag.Case presentationWe report the case of a 58-year-old female Caucasian patient with a dramatic increase in plasma levels of transaminases after treatment with macitentan and selexipag, drugs whose risk of causing liver injury has so far been considered limited. After therapy discontinuation, plasma levels of transaminases returned to baseline, thus suggesting a role of these drugs in the observed hepatotoxicity. After pharmacological counseling, we decided to introduce ambrisentan for the patient’s treatment. After 7 months of treatment, no liver injury has been reported. To evaluate the role of genetic factors in the observed hepatotoxicity, we genotyped the patient for single-nucleotide polymorphisms previously associated with macitentan, ambrisentan, or selexipag metabolism. We found a genetic profile associated with a poor metabolizer (PM) phenotype for CYP2C8 and CYP2C9, key enzymes for elimination of both macitentan and selexipag. The reported results suggest that an allelic profile associated with low activity for CYP2C8 and CYP2C9 enzyme could be a potential risk factor for macitentan and selexipag-induced liver injury and could provide a possible marker for early identification of subjects at higher risk of developing hepatotoxicity.ConclusionsA multidisciplinary approach based on clinical evaluation, as well as pharmacological counseling and evaluation of the patient’s genetic profile, might be useful for identification of patients with a high chance of drug-induced liver injury, avoiding unnecessary risks in therapy selection and prescription.

Recent Advances in the Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a disease in which stenosis or obstruction of the pulmonary arteries (PAs) causes an increase in PA pressure, leading to right-sided heart failure and death. Basic research has revealed a decrease in the levels of endogenous vasodilators, such as prostacyclin, and an increase in the levels of endogenous vasoconstrictors, such as endothelin, in patients with PAH, leading to the development of therapeutic agents. Currently, therapeutic agents for PAH target three pathways that are selective for PAs: the prostacyclin, endothelin, and nitric oxide pathways. These treatments improve the prognosis of PAH patients. In this review, we introduce new drug therapies and provide an overview of the current therapeutic agents.

The Role and Mechanism of Gut Microbiota in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a malignant pulmonary vascular disease characterized by increased pulmonary vascular resistance, pulmonary vasoconstriction, and right ventricular hypertrophy. Recent developments in genomics and metabolomics have gradually revealed the roles of the gut microbiota (GM) and its metabolites in cardiovascular diseases. Accumulating evidence reveals that the GM plays important roles in the occurrence and development of PAH. Gut microbiota dysbiosis directly increases the gut permeability, thereby facilitating pathological bacterial translocation and allowing translocation of bacterial products such as lipopolysaccharides from the gut into circulation. This process aggravates pulmonary perivascular inflammation and exacerbates PAH development through the endothelial–mesenchymal transition. Additionally, a shift in the composition of PAH also affects the gut metabolites. Changes in gut metabolites, such as decreased short-chain fatty acids, increased trimethylamine N-oxide, and elevated serotonin, contribute to pulmonary perivascular inflammation and pulmonary vascular remodeling by activating several signaling pathways. Studies of the intestinal microbiota in treating pulmonary hypertension have strengthened linkages between the GM and PAH. Probiotic therapy and fecal microbiota transplantation may supplement existing PAH treatments. In this article, we provide new insight for diagnosing, preventing and treating PAH by adding to the current knowledge of the intestinal flora mechanisms and its metabolites efficacy involved in PAH.

MircoRNA in Extracellular Vesicles from Patients with Pulmonary Arterial Hypertension Alters Endothelial Angiogenic Response.

Pulmonary arterial hypertension (PAH) is characterized by a progressive elevation of pulmonary pressure leading to right ventricular dysfunction and is associated with a poor prognosis. Patients with PAH have increased numbers of circulating extracellular vesicles (EVs) and altered expression of circulating microRNAs (miRs). The study aimed to evaluate the miR profile contained within purified EVs derived from the plasma of PAH patients as compared to healthy controls (HC). Circulating EVs, purified from platelet-free plasma were analyzed using flow cytometry, western blot, and electron microscopy. Total RNA isolated from EVs was subjected to Microarray analysis using GeneChip miRNA 4.0 Array and bioinformatics tools. Overexpression and inhibition of miRs were conducted in human pulmonary artery endothelial cells (hPAECs) that had been incubated previously with either PAH- or HC-derived EVs. Cell proliferation (MTT assay) and angiogenesis (tube formation assay) were tested in hPAECs to determine miR functionality. MiR profiling revealed 370 heats while comparing PAH and HC groups, 22 of which were found to be down-regulated and 6 were up-regulated in the PAH EVs. Among the altered miRs, miR-486-5p was overexpressed, while miR-26a-5p was downregulated in PAH EVs compared to HC EVs. Inhibition of mir-486-5p or overexpression of miR-26a-5p in hPAECs post-exposure of PAH EVs abrogated proangiogenic and proliferative effects posed by PAH EVs contrary to HC EVs. The angiogenic and proliferative effects of the miRs from PAH EVs were observed to be mediated through nuclear factor (NF)-κB activation. PAH EVs carry and present an altered miR profile that can be targeted to restrict angiogenesis and reduce pulmonary endothelium activation. Further studies concerning miRs from circulating heterogeneous EVs in PAH patients are warranted to understand their potential as targets for treatment in PAH.

Sox17 Deficiency Promotes Pulmonary Arterial Hypertension via HGF/c-Met Signaling.

In large-scale genomic studies, Sox17, an endothelial-specific transcription factor, has been suggested as a putative causal gene of pulmonary arterial hypertension (PAH); however, its role and molecular mechanisms remain to be elucidated. We investigated the functional impacts and acting mechanisms of impaired Sox17 (SRY-related HMG-box17) pathway in PAH and explored its potential as a therapeutic target. In adult mice, Sox17 deletion in pulmonary endothelial cells (ECs) induced PAH under hypoxia with high penetrance and severity, but not under normoxia. Key features of PAH, such as hypermuscularization, EC hyperplasia, and inflammation in lung arterioles, right ventricular hypertrophy, and elevated pulmonary arterial pressure, persisted even after long rest in normoxia. Mechanistically, transcriptomic profiling predicted that the combination of Sox17 deficiency and hypoxia activated c-Met signaling in lung ECs. HGF (hepatocyte grow factor), a ligand of c-Met, was upregulated in Sox17-deficient lung ECs. Pharmacologic inhibition of HGF/c-Met signaling attenuated and reversed the features of PAH in both preventive and therapeutic settings. Similar to findings in animal models, Sox17 levels in lung ECs were repressed in 26.7% of PAH patients (4 of 15), while those were robust in all 14 non-PAH controls. HGF levels in pulmonary arterioles were increased in 86.7% of patients with PAH (13 of 15), but none of the controls showed that pattern. The downregulation of Sox17 levels in pulmonary arterioles increases the susceptibility to PAH, particularly when exposed to hypoxia. Our findings suggest the reactive upregulation of HGF/c-Met signaling as a novel druggable target for PAH treatment.

Biomarker-based approach to determine etiology and severity of pulmonary hypertension: Focus on microRNA.

Pulmonary arterial hypertension (PAH) is characterized by remodeling of the pulmonary arteries, and defined by elevated pulmonary arterial pressure, measured during right heart catheterization. There are three main challenges to the diagnostic and therapeutic process of patients with PAH. First, it is difficult to differentiate particular PAH etiology. Second, invasive diagnostic is required to precisely determine the severity of PAH, and thus to qualify patients for an appropriate treatment. Third, the results of treatment of PAH are unpredictable and remain unsatisfactory. MicroRNAs (miRNAs) are small non-coding RNAs that regulate post transcriptional gene-expression. Their role as a prognostic, and diagnostic biomarkers in many different diseases have been studied in recent years. MiRNAs are promising novel biomarkers in PAH due to their activity in various molecular pathways and processes underlying PAH. Lack of biomarkers to differentiate between particular PAH etiology and evaluate the severity of PAH, as well as paucity of therapeutic targets in PAH open a new field for the possibility to use miRNAs in these applications. In our article, we discuss the potential of miRNAs use as diagnostic tools, prognostic biomarkers and therapeutic targets in PAH.

Monocrotaline-Induced Pulmonary Arterial Hypertension and Bosentan Treatment in Rats: Focus on Plasma and Erythrocyte Parameters.

The objective of our study was to contribute to the characterization of monocrotaline-induced pulmonary arterial hypertension (PAH) in a rat model, with emphasis on the renin–angiotensin–aldosterone system, parameters of oxidative stress, the activity of matrix metalloproteinases, and erythrocyte parameters. Moreover, we aimed to analyze the effects of bosentan. Experiments were performed on 12-week-old male Wistar rats randomly assigned to 3 groups: control, monocrotaline-treated (60 mg/kg), and monocrotaline combined with bosentan (300 mg/kg/day). Our study confirmed the well-known effects of monocrotaline administration on lungs and the right ventricle, as well as pulmonary arterial pressure. In addition, we observed activation of the alternative pathway of the renin–angiotensin system, namely an increase in angiotensin (Ang) 1–7 and Ang 1-5 together with an increase in Ang I, but without any change in Ang II level, and downregulation of aldosterone 4 weeks after monocrotaline administration. For the first time, modifications of erythrocyte Na,K-ATPase enzyme kinetics were demonstrated as well. Our observations do not support data obtained in PAH patients showing an increase in Ang II levels, increase in oxidative stress, and deterioration in RBC deformability. Although bosentan primarily targets the vascular smooth muscle, our study confirmed its antioxidant effect. The obtained data suggest that besides the known action of bosentan, it decreases heart rate and increases erythrocyte deformability, and hence could have a beneficial hemodynamic effect in the PAH condition.

Riociguat and the right ventricle in pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are progressive diseases that can lead to right heart failure and death. Right ventricular dysfunction, hypertrophy and maladaptive remodelling are consequences of increased right ventricular (RV) afterload in PAH and CTEPH and are indicative of long-term outcomes. Because RV failure is the main cause of morbidity and mortality in PAH and CTEPH, successful treatments should lead to improvements in RV parameters. Riociguat is a soluble guanylate cyclase stimulator approved for the treatment of PAH and inoperable or persistent/recurrent CTEPH after pulmonary endarterectomy. This review examines the current evidence showing the effect of riociguat on the right ventricle, with particular focus on remodelling, function and structural parameters in preclinical models and patients with PAH or CTEPH.

Change in REVEAL Lite 2 and COMPERA 2.0 risk status in patients with pulmonary arterial hypertension initiating oral treprostinil on dual background therapy: a retrospective chart review

Abstract Background Post-hoc analyses of the FREEDOM-EV study demonstrated that addition of oral treprostinil (ORE) to background monotherapy improves risk in patients with pulmonary arterial hypertension (PAH). Evidence is needed to understand the incremental effect of ORE added to dual background therapy with an endothelin receptor antagonist (ERA) and phosphodiesterase-5 inhibitor (PDE5)/soluble guanylate cyclase stimulator (sGC). Purpose To describe longitudinal changes in REVEAL Lite 2 and COMPERA 2.0 risk status in patients initiating ORE on dual background therapy. Methods A multisite, double-blind retrospective chart review of PAH patients was conducted by US PAH-treating physicians and included patients meeting prior diagnostic PAH criteria (mPAP ≥25 mmHg/PAWP ≤15 mmHg/PVR >3 WU), age ≥18 at diagnosis, began a PAH treatment 01/2014–03/2017, and had NT-proBNP/BNP, WHO functional class (FC), and 6-minute walk distance (6MWD) at baseline visit. Baseline was most recent assessment prior to ORE initiation. This analysis included a subset of patients on dual background therapy at time of ORE initiation without prior prostacyclin use. Follow-up was evaluated in 0–6 and 6–12 months after ORE initiation. Results 30 (61.2%) of 49 ORE patients were on dual background therapy at time of ORE start; 23 had baseline assessments while on dual background therapy and were included in this analysis. At baseline, mean (SD) 6MWD was 140.9 (49.8) m and 21 (91.3%) patients were FC III. Prior to ORE, median (IQR) time on ERA was 6.6 (5.5–7.6) months and PDE5/sGC was 3.7 (3.3–5.7) months. After ORE start, median (IQR) time to follow-up was 4.3 (3.4–5.6) months in 0–6 months and 10.9 (9.6–11.6) months in 6–12 months. Using REVEAL Lite 2, baseline mean (SD) REVEAL Lite 2 risk score was 7.9 (0.8); 1 (4.4%), 2 (8.7%), and 20 (87%) patients were in the low, intermediate, and high risk strata, respectively. In 0–6 months (n=22), 13 (59.1%) patients improved and 9 (40.9%) stabilized REVEAL Lite 2 risk strata from baseline. In 6–12 months (n=22), mean (SD) REVEAL Lite 2 risk score was 6.5 (1.1); 15 patients (68.2%) improved, 6 (27.3%) stabilized, and 1 (4.6%) worsened REVEAL Lite 2 risk strata from baseline. Using COMPERA 2.0, at baseline, 1 (4.4%) patient was in the intermediate-low and 22 (95.7%) patients were in the intermediate-high risk strata. In 0–6 months (n=15), 10 (66.7%) patients improved and 5 (33.3%) patients stabilized COMPERA 2.0 risk strata from baseline. In 6–12 months (n=17), 15 (88.2%) patients improved and 2 (11.8%) stabilized COMPERA 2.0 risk strata from baseline. Conclusion Of patients initiating oral treprostinil in this study, most were on dual background therapy and at high risk by REVEAL Lite 2 or intermediate-high risk by COMPERA 2.0. This analysis suggests adding ORE to dual background therapy can improve risk status in patients, with clinical benefit attained within 6 months and continued incremental improvement through first year of therapy. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): United Therapeutics Corporation