Mechanisms Of Pulmonary Arterial Hypertension Research Articles

Pathogenic Mechanisms of Pulmonary Arterial Hypertension: Homeostasis Imbalance of Endothelium-Derived Relaxing and Contracting Factors.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease. Sustained pulmonary vasoconstriction and concentric pulmonary vascular remodeling contribute to the elevated pulmonary vascular resistance and pulmonary artery pressure in PAH. Endothelial cells regulate vascular tension by producing endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs). Homeostasis of EDRF and EDCF production has been identified as a marker of the endothelium integrity. Impaired synthesis or release of EDRFs induces persistent vascular contraction and pulmonary artery remodeling, which subsequently leads to the development and progression of PAH. In this review, the authors summarize how EDRFs and EDCFs affect pulmonary vascular homeostasis, with special attention to the recently published novel mechanisms related to endothelial dysfunction in PAH and drugs associated with EDRFs and EDCFs.

Future Perspectives of Pulmonary Hypertension Treatment.

Since the discovery of three major pathophysiological mechanisms of pulmonary arterial hypertension (PAH), including prostacyclin, endothelin and nitric oxide pathways, the therapeutic options for PAH have increased. Nevertheless, despite these advances, the prognosis remains unsatisfactory for many patients with PAH. With the progress of both pre-clinical and clinical research on PAH, several novel therapeutic targets have been identified for the treatment of PAH. In this study, we review updated information of novel pathophysiological pathways of pulmonary hypertension, mainly focusing on WHO Group I PAH. Drugs based on these pathways are currently under clinical or pre-clinical investigation, however they have been approved for clinical use. Large clinical trials are required to validate the clinical safety and effects of these novel therapies.

Senescence in pulmonary arterial hypertension: is there a link?

Cellular senescence has been recognized as a promising target in the treatment of many cardiovascular diseases. The pathways involved in the development of senescence share many similarities with pathobiological mechanisms of pulmonary arterial hypertension (PAH). But the potential of senolytics to improve pulmonary hemodynamics and to reduce vascular remodelling in PAH has thus far not been investigated in depth. PAH does not seem to be a disease of only young people since the mean age of PAH patients is constantly increasing. Changes in expression of senescence biomarkers related to cell cycle arrest, namely upregulation of the tumour suppressor protein p53 and the cell cycle inhibitors p16ink4A an p21cip1 as well as an increase in apoptosis resistance biomarker Bcl2 (B-cell lymphoma 2) and development of senescence-associated phenotype characterized by excessive production of matrix metalloproteinase 2 and interleukin 6 were demonstrated in PAH patients. Initiatives to link the senescence-modulating effect of certain compounds to clinically relevant outcomes in PAH are still limited. Further exploration of the role of senescence in the pathobiology of PAH may point to new relevant treatment strategies. Identification of the cell-specific senescence biomarkers which can be used in vivo, could further promote identification of clinically relevant pathways and design of clinical studies which will help to establish effective therapeutic use of senolytic compounds.

Integrated metabolomics and mechanism to reveal the protective effect of kaempferol on pulmonary arterial hypertension

Currently, there have been breakthroughs in the study of the underlying mechanisms of pulmonary arterial hypertension, but treatment is still challenging. The aim of this study was explore the effect of kaempferol in PAH and discover the mechanism of this process. First, we assessed the effect of kaempferol in animal models of PAH. The echocardiography and pressure-volume admittance catheter data showed that kaempferol could improve right ventricular function and alleviate the progression of PAH. Moreover, morphometric analysis of the lung vasculature indicated that pulmonary vascular remodeling could be reduced by kaempferol. The expression of autophagy-related proteins and autophagic processes were evaluated by western blotting and mRFP-GFP-LC3 fluorescence microscopy, the results indicated that autophagy played an important role in the process by which kaempferol prevents PAH. Moreover, we performed untargeted and targeted plasma metabolomics analysis on rat models of PAH and used multivariate analysis to reveal multiple pathways and targets. Consequently, we identified 247 plasma biomarkers that were tightly linked to the preventive effects of kaempferol, which were mainly related to amino acid metabolism and arachidonic acid metabolism. In conclusion, kaempferol could effectively alleviate the development of PAH by regulating abnormal autophagy and metabolic disorders, providing a novel perspective on the treatment of PAH.

Evaluation of heart rate variability in patients with different forms of pulmonary hypertension

Pulmonary hypertension (PH) is a severe pathology of the cardiovascular system with extremely poor prognosis, if unreated. Early diagnosis of PH is difficult, due to the absence pathognomonic symptoms, and at the initial stage the disease may be asymptomatic. An increase in pulmonary vascular resistance and pressure in the pulmonary artery cause severe dysfunction of the right ventricle, which affects the functional status of patients. The assessment of heart rate variability (HRV) parameters is used to predict the increased risk of sudden death in various diseases of the cardiovascular system and the functional status of the body. HRV is mainly regulated by the sympathetic and parasympathetic divisions of the autonomic nervous system. It is noted that pulmonary arterial hypertension is associated with increasing of sympathetic activity. The review, based on the analysis of the pathophysiological mechanisms of pulmonary arterial hypertension and chronic thromboembolic PH, presents the results of studies assessing the time and spectral parameters of HRV in patients with PH of various etiologies.

Untargeted metabolomics towards understanding molecular mechanisms of pulmonary arterial hypertension

Abstract Background Pulmonary hypertension constitutes a rare disease characterized by a severe development and a high risk of premature death. One of its main clinical types is pulmonary arterial hypertension (PAH), in which the highest percentage of patients are affected by idiopathic PAH. The pathogenesis of this disease has not completely been discovered and elucidated so far, and non-specific clinical symptoms make the diagnosis of PAH a serious problem. Currently, PAH is confirmed with invasive examination based on right heart catheterization. Purpose The main aim of the study was to evaluate and compare both plasma and urine fingerprints of PAH patients and control group with the use of an untargeted metabolomics approach. The study also focused on correlation analysis between the observed metabolic changes and the clinical parameters to select specific indicators of PAH disease. Methods An untargeted metabolomics approach was applied to the plasma and urine samples with the use of gas chromatography coupled to triple quadrupole mass spectrometry (GC-QqQ/MS) and advanced statistical tests were applied to evaluate the potential metabolic indicators of PAH. The PAH patients (n=40) and healthy controls (n=39) were matched for age, sex, BMI and included in the study. The obtained raw datasets were properly processed (data deconvolution, signal correction using QCSVR method and PQN normalization) and subsequently subjected to uni- and multivariate statistical tests (Student's t-test, Welch's test, U Mann-Whitney test, PCA and OPLS-DA). The identification of the statistically significant metabolites was performed using universal libraries, such as: Fiehn's and NIST11. Results The statistically significant metabolites (n=10 and 11 for urine and plasma samples, respectively)originate from various biochemical pathways associated with the carbohydrate, amino acid, lipid, fatty acid and pyrimidine metabolism. The metabolic changes observed in the urine samples of PAH patients (compared to the control group) included different concentrations in: threonic acid, hippuric acid, acetic acid, sorbitol, butanoic acid and propionic acid. The metabolic alterations in the plasma samples covered changes in the levels of valine, leucine, lactic acid, hydroxybutanoic acid, nonanoic acid, cholesterol and octadecanoic acid. Metabolites representing the highest correlation with mean pulmonary arterial pressure include, for instance: propionic acid (r=0.85), valine (r=0.75) and lactic acid (r=0.63) Conclusions The observed metabolic changes are related to various biological processes that are disturbed in the course of PAH, namely: proliferation of pulmonary vascular cells or the functions of cardiomyocytes and the right ventricle of the heart. The obtained results confirmed the potential of a metabolomics approach to uncover and explain the underlying molecular mechanisms of PAH. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science and High Education of Poland

Influence of atorvastatin on metabolic pattern of rats with pulmonary hypertension.

Background: Metabonomics has been widely used to analyze the initiation, progress, and development of diseases. However, application of metabonomics to explore the mechanism of pulmonary arterial hypertension (PAH) are poorly reported. This study aimed to investigate the influence of atorvastatin (Ato) on metabolic pattern of rats with pulmonary hypertension.Methods: PAH animal model was established using monocrotaline (MCT). The mean pulmonary artery pressure (mPAP) and right ventricular hypertrophy index (RVHI) were measured. The microstructure of pulmonary arterioles was observed by HE staining. Nuclear magnetic resonance was used to detect and analyze the serum metabolites. The levels of glycogen synthase kinase-3β (GSK-3β), hexokinase 2 (HK-2), sterol regulatory element-binding protein 1c (SREBP-1c), and carnitine palmitoyltransferase I (CPT-1) in the lung tissues were measured.Results: Ato significantly improved lung function by decreasing mPAP, RVHI, wall thickness, and wall area. Differences in metabolic patterns were observed among normal, PAH, and Ato group. The levels of GSK-3β and SREBP-1c were decreased, but HK-2 and CPT-1 were increased in the group PAH. Ato treatment markedly reversed the influence of MCT.Conclusion: Ato significantly improved the pulmonary vascular remodeling and pulmonary hypertension of PAH rats due to its inhibition on Warburg effect and fatty acid β oxidation.

Spermine promotes pulmonary vascular remodelling and its synthase is a therapeutic target for pulmonary arterial hypertension.

Pathological mechanisms of pulmonary arterial hypertension (PAH) remain largely unexplored. Effective treatment of PAH remains a challenge. The aim of this study was to discover the underlying mechanism of PAH through functional metabolomics and to help develop new strategies for prevention and treatment of PAH.Metabolomic profiling of plasma in patients with idiopathic PAH was evaluated through high-performance liquid chromatography mass spectrometry, with spermine identified to be the most significant and validated in another independent cohort. The roles of spermine and spermine synthase were examined in pulmonary arterial smooth muscle cells (PASMCs) and rodent models of pulmonary hypertension.Using targeted metabolomics, plasma spermine levels were found to be higher in patients with idiopathic PAH compared to healthy controls. Spermine administration promoted proliferation and migration of PASMCs and exacerbated vascular remodelling in rodent models of pulmonary hypertension. The spermine-mediated deteriorative effect can be attributed to a corresponding upregulation of its synthase in the pathological process. Inhibition of spermine synthase in vitro suppressed platelet-derived growth factor-BB-mediated proliferation of PASMCs, and in vivo attenuated monocrotaline-mediated pulmonary hypertension in rats.Plasma spermine promotes pulmonary vascular remodelling. Inhibiting spermine synthesis could be a therapeutic strategy for PAH.

Metabolomics reveals metabolite changes of patients with pulmonary arterial hypertension in China.

The specific mechanism of pulmonary arterial hypertension (PAH) remains elusive. The present study aimed to explore the underlying mechanism of PAH through the identity of novel biomarkers for PAH using metabolomics approach. Serum samples from 40 patients with idiopathic PAH (IPAH), 20 patients with congenital heart disease‐associated PAH (CHD‐PAH) and 20 healthy controls were collected and analysed by ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry (UPLC‐HRMS). Orthogonal partial least square‐discriminate analysis (OPLS‐DA) was applied to screen potential biomarkers. These results were validated in monocrotaline (MCT)‐induced PAH rat model. The OPLS‐DA model was successful in screening distinct metabolite signatures which distinguished IPAH and CHD‐PAH patients from healthy controls, respectively (26 and 15 metabolites). Unbiased analysis from OPLS‐DA identified 31 metabolites from PAH patients which were differentially regulated compared to the healthy controls. Our analysis showed dysregulation of the different metabolic pathways, including lipid metabolism, glucose metabolism, amino acid metabolism and phospholipid metabolism pathways in PAH patients compared to their healthy counterpart. Among these metabolites from dysregulated metabolic pathways, a panel of metabolites from lipid metabolism and fatty acid oxidation (lysophosphatidylcholine, phosphatidylcholine, perillic acid, palmitoleic acid, N‐acetylcholine‐d‐sphingomyelin, oleic acid, palmitic acid and 2‐Octenoylcarnitine metabolites) were found to have a close association with PAH. The results from the analysis of both real‐time quantitative PCR and Western blot showed that expression of LDHA, CD36, FASN, PDK1 GLUT1 and CPT‐1 in right heart/lung were significantly up‐regulated in MCT group than the control group.

Mir-455-3p-1 represses FGF7 expression to inhibit pulmonary arterial hypertension through inhibiting the RAS/ERK signaling pathway

ObjectiveTo analyze the effects of miR-455-3p-1 and its possible mechanisms in pulmonary arterial hypertension (PAH). MethodsA microarray assay was used to examine the expressed genes between normal and PAH. The expressed genes in PAH was assessed by qRT-PCR. The targeted interaction between miRNAs and FGF7 was confirmed using a dual luciferase reporter assay. A CCK-8 assay and cell count were used to analyze the pulmonary artery smooth muscle cells (PASMCs) activity and proliferation level, respectively. Apoptotic PASMCs were detected by flow cytometry. In addition, the mRNA and protein expression levels of RAS/ERK signaling pathway were determined by qRT-PCR and a Western blot assay, respectively. A PAH rat model was used to identify the effects of miR-455-3p-1 in vivo. ResultsFGF7 was upregulated in PAH. MiR-455-3p-1 was downregulated in PAH. MiR-455-3p-1 targeted FGF7. MiR-455-3p-1 decreased the expression of FGF7. Moreover, the effect of FGF7 on PASMCs was suppressed by miR-455-3p-1. MiR-455-3p-1 upregulation was associated with reduced mRNA and protein levels of core RAS/ERK signal genes, suggesting the inhibition of the RAS/ERK pathway. Furthermore, miR-455-3p-1 upregulation improved the RVSP, mPAP, ratio of RV/LV + S, CO and RV function of PAH rat model in vivo. ConclusionOur findings illustrate a role for miR-455-3p-1 in modulating FGF7-RAS/ERK signaling and suggest that an agomir of miR-455-3p-1 could inhibit the proliferation of PASMCs and mitigate PAH in vivo.

Abstract 17217: ActRIIA-Fc Rebalances Activin/GDF and BMP9 Signaling to Attenuate Experimental Pulmonary Hypertension

Introduction: Therapies targeting the underlying mechanisms of pulmonary arterial hypertension (PAH) might modify the natural history of disease. The genetics of heritable PAH implicate deficient BMP signaling as a pathogenetic driver. Lung tissues from experimental and human PAH exhibit deficient BMP & excessive TGFβ signaling. While previous studies suggest agonism of BMP9 signaling or inhibition of TGFβ signaling may prevent experimental PH, the roles of activin and growth and differentiation factor (GDF) ligand signaling are unclear. ACTRIIA-Fc (sotatercept) is an activin- and GDF8/11-specific ligand trap currently in Phase 2 studies for anemia and multiple myeloma. Hypothesis: ACTRIIA-Fc inhibits activin and GDF8/11 signaling in vascular cells to attenuate experimental PH and pulmonary vascular remodeling. Methods: Impact of ACTRIIA-Fc on BMP & TGFβ signaling was examined in human pulmonary microvascular endothelial (PMVEC) and pulmonary arterial smooth muscle cells (PASMC). ACTRIIA-Fc (15 mg/kg s.c. 2x/week), sildenafil (30 mg/kg p.o. BID) or vehicle were given prophylactically for 4 weeks to rats developing PH following a single injection of MCT (40 mg/kg s.c.), or via single injection of SU5416 (200 mg/kg s.c.) combined with hypoxia (FIO2=0.10, SU-Hx). Alternately, rats were allowed to develop PH following SU-Hx treatment for 3 weeks or MCT treatment for 4 weeks, and then treated with ACTRIIA-Fc (1, 3, or 10 mg/kg s.c. 2x/week) or isotype-matched control antibody (3 weeks under normoxia following SU-Hx, or 2 weeks following MCT). Invasive hemodynamics, RV mass and vascular remodeling were assessed. Results: In PMVEC, ACTRIIA-Fc enhanced BMP9-induced SMAD1/5/8 activation and transcriptional activity, and reversed inhibition of BMP9 signaling by GDF11. ACTRIIA-Fc inhibited GDF8-, GDF11- and activin A-mediated SMAD2 phosphorylation and myogenic differentiation of PASMC. In vivo , a standard dose of ACTRIIA-Fc prevented PH, RV hypertrophy and PV remodeling in MCT- and SU-Hx-rats, with more potency than sildenafil. All doses of ACTRIIA-Fc attenuated PH and RV hypertrophy when given in a delayed fashion after MCT- and SU-Hx-treatment, with graded effects on PV remodeling. Conclusions: ACTRIIA-Fc rebalances GDF11 vs. BMP9 signaling, with distinct effects in vascular endothelium vs. media, and potently inhibits experimental PH in several rodent models. Given its well-defined tolerability, dosing, and efficacy in previous human studies, ACTRIIA-Fc is an attractive candidate for PAH as a novel, mechanism-targeted therapy.

Selenoprotein P Promotes the Development of Pulmonary Arterial Hypertension: Possible Novel Therapeutic Target.

Excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) are key mechanisms of pulmonary arterial hypertension (PAH). Despite the multiple combination therapy, a considerable number of patients develop severe pulmonary hypertension (PH) because of the lack of diagnostic biomarker and antiproliferative therapies for PASMCs. Microarray analyses were used to identify a novel therapeutic target for PAH. In vitro experiments, including lung and serum samples from patients with PAH, cultured PAH-PASMCs, and high-throughput screening of 3336 low-molecular-weight compounds, were used for mechanistic study and exploring a novel therapeutic agent. Five genetically modified mouse strains, including PASMC-specific selenoprotein P (SeP) knockout mice and PH model rats, were used to study the role of SeP and therapeutic capacity of the compounds for the development of PH in vivo. Microarray analysis revealed a 32-fold increase in SeP in PAH-PASMCs compared with control PASMCs. SeP is a widely expressed extracellular protein maintaining cellular metabolism. Immunoreactivity of SeP was enhanced in the thickened media of pulmonary arteries in PAH. Serum SeP levels were also elevated in patients with PH compared with controls, and high serum SeP predicted poor outcome. SeP-knockout mice ( SeP-/-) exposed to chronic hypoxia showed significantly reduced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling compared with controls. In contrast, systemic SeP-overexpressing mice showed exacerbation of hypoxia-induced PH. Furthermore, PASMC-specific SeP-/- mice showed reduced hypoxia-induced PH compared with controls, whereas neither liver-specific SeP knockout nor liver-specific SeP-overexpressing mice showed significant differences with controls. Altogether, protein levels of SeP in the lungs were associated with the development of PH. Mechanistic experiments demonstrated that SeP promotes PASMC proliferation and resistance to apoptosis through increased oxidative stress and mitochondrial dysfunction, which were associated with activated hypoxia-inducible factor-1α and dysregulated glutathione metabolism. It is important to note that the high-throughput screening of 3336 compounds identified that sanguinarine, a plant alkaloid with antiproliferative effects, reduced SeP expression and proliferation in PASMCs and ameliorated PH in mice and rats. These results indicate that SeP promotes the development of PH, suggesting that it is a novel biomarker and therapeutic target of the disorder.

Anti-apoptosis endothelial cell-secreted microRNA-195-5p promotes pulmonary arterial smooth muscle cell proliferation and migration in pulmonary arterial hypertension.

In the pathological mechanism of pulmonary arterial hypertension, the role of apoptosis-resistant pulmonary microvascular endothelial cells (PVECs/AR) has been emphasized on the pulmonary vascular remodeling. In the present study, we investigated whether PVECs/AR can promote the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), and to study the role of miR-195-5p in the crosstalk between these two types of cells. We confirmed that PVECs/AR can promote the proliferation and migration of PASMCs in a co-culture system of AR/PVECs and PASMCs. Additionally, after exposure to hypoxia for 12 or 24 h, AR/PVECs had a higher mature miR-195-5p level than PVECs (P < 0.05, 12 and 24 h). Luciferase reporter assays were used to validate indications of the existence of an HRE in the miR-195-5p promoter. Knocking down Smad7 can reverse the inhibition of Lv-S195 on TGF-β1-induced PASMCs remodeling. TGF-β1 promoted cell growth in PASMCs, and the supernatant of PVECs/AP infected with Lv-S195 inhibited TGF-β1 enhanced proliferation in PASMCs, which was also blocked by Lv-shRNA-Smad7. The result of this experiment confirmed the specificity of the HIF-1a/miR-195/Smad7 pathway. Our data indicate the possible function of PVECs/AR in the process of pulmonary vascular remodeling. MiRNA-195-5p played a role as an interacting paracrine factor between PVECs/AR and PASMC, which seemed to function through the HIF-1a/miRNA-195-5p/Smad7 pathway.

Endothelial to haematopoietic transition contributes to pulmonary arterial hypertension.

The pathogenic mechanisms of pulmonary arterial hypertension (PAH) remain unclear, but involve dysfunctional endothelial cells (ECs), dysregulated immunity and inflammation in the lung. We hypothesize that a developmental process called endothelial to haematopoietic transition (EHT) contributes to the pathogenesis of pulmonary hypertension (PH). We sought to determine the role of EHT in mouse models of PH, to characterize specific cell types involved in this process, and to identify potential therapeutic targets to prevent disease progression. When transgenic mice with fluorescence protein ZsGreen-labelled ECs were treated with Sugen/hypoxia (Su/Hx) combination to induce PH, the percentage of ZsGreen+ haematopoietic cells in the peripheral blood, primarily of myeloid lineage, significantly increased. This occurrence coincided with the depletion of bone marrow (BM) ZsGreen+ c-kit+ CD45- endothelial progenitor cells (EPCs), which could be detected accumulating in the lung upon PH-induction. Quantitative RT-PCR based gene array analysis showed that key transcription factors driving haematopoiesis were expressed in these EPCs. When transplanted into lethally irradiated recipient mice, the BM-derived EPCs exhibited long-term engraftment and haematopoietic differentiation capability, indicating these EPCs are haemogenic in nature. Specific inhibition of the critical haematopoietic transcription factor Runx1 blocked the EHT process in vivo, prevented egress of the BM EPCs and ultimately attenuated PH progression in Su/Hx- as well as in monocrotaline-induced PH in mice. Thus, myeloid-skewed EHT promotes the development of PH and inhibition of this process prevents disease progression in mouse models of PH. Furthermore, high levels of Runx1 expression were found in circulating CD34+ CD133+ EPCs isolated from peripheral blood of patients with PH, supporting the clinical relevance of our proposed mechanism of EHT. EHT contributes to the pathogenesis of PAH. The transcription factor Runx1 may be a novel therapeutic target for the treatment of PAH.

Proliferative Versus Degenerative Paradigms in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is believed to be a proliferative disease, triggered by endothelial cell injury and apoptosis and leading to the formation of occlusive vascular lesions caused by growth-dysregulated “cancer-like” cells. However, the current experimental and clinical evidence is not entirely consistent with this paradigm and suggests an alternate interpretation, specifically that microvascular rarefaction may be due to a degenerative process, driven by sustained endothelial cell apoptosis. The “degenerative” paradigm has important implications, not the least of which is that proliferative lesions may be a secondary manifestation of disease rather than the primary cause of microvascular rarefaction, and that regenerative strategies may be needed to restore the microcirculation in established disease . Although there remains much debate about the precise mechanisms of pulmonary arterial hypertension, it is generally accepted that there is functional microvascular rarefaction, resulting in a marked decrease in pulmonary vascular cross-sectional area. Two distinct, and seemingly mutually exclusive, mechanisms have been proposed for the loss of lung microvasculature. The first is a proliferative process that leads to arteriolar occlusion and obliteration, and the second is a degenerative process that leads to drop-out and loss of fragile lung vasculature. Let’s first consider the proliferative hypothesis. The so-called “cancer paradigm” of pulmonary arterial hypertension (PAH) is based on the idea that dysregulated endothelial cell (EC) growth can lead to disordered angiogenesis with the heaping up of intimal cells within the lumen causing arteriolar occlusion and obliteration. Indeed, dysregulated vascular cell growth is integrally linked to the development of plexiform lesions, which represent hallmark features of PAH. This is supported by evidence of changes in biochemical and molecular cellular activity in PAH, similar to those seen in cancer, including a shift in glucose metabolism from mitochondrial glucose oxidation to glycolysis (Warburg effect), activation of cell growth pathways, increased expression of …

New Biochemical Insights into the Mechanisms of Pulmonary Arterial Hypertension in Humans.

Diagnosis of pulmonary arterial hypertension (PAH) is difficult due to the lack of specific clinical symptoms and biomarkers, especially at early stages. We compared plasma metabolic fingerprints of PAH patients (n = 20) with matched healthy volunteers (n = 20) using, for the first time, untargeted multiplatform metabolomics approach consisting of high-performance liquid and gas chromatography coupled with mass spectrometry. Multivariate statistical analyses were performed to select metabolites that contribute most to groups’ classification (21 from liquid in both ionization modes and 9 from gas chromatography-mass spectrometry). We found metabolites related to energy imbalance, such as glycolysis-derived metabolites, as well as metabolites involved in fatty acid, lipid and amino acid metabolism. We observed statistically significant changes in threitol and aminomalonic acid in PAH patients, which could provide new biochemical insights into the pathogenesis of the disease. The results were externally validated on independent case and control cohorts, confirming up to 16 metabolites as statistically significant in the validation study. Multiplatform metabolomics, followed by multivariate chemometric data analysis has a huge potential for explaining pathogenesis of PAH and for searching potential and new more specific and less invasive markers of the disease.

Epigenetic mechanisms in pulmonary arterial hypertension: the need for global perspectives.

Pulmonary arterial hypertension (PAH) is a severe and progressive disease, characterised by high pulmonary artery pressure that usually culminates in right heart failure. Recent findings of alterations in the DNA methylation state of superoxide dismutase 2 and granulysin gene loci; histone H1 levels; aberrant expression levels of histone deacetylases and bromodomain-containing protein 4; and dysregulated microRNA networks together suggest the involvement of epigenetics in PAH pathogenesis. Thus, PAH pathogenesis evidently involves the interplay of a predisposed genetic background, epigenetic state and injurious events. Profiling the genome-wide alterations in the epigenetic mechanisms, such as DNA methylation or histone modification pattern in PAH vascular cells, may explain the great variability in susceptibility and disease severity that is frequently associated with pronounced remodelling and worse clinical outcome. Moreover, the influence of genetic predisposition and the acquisition of epigenetic alterations in response to environmental cues in PAH progression and establishment has largely been unexplored on a genome-wide scale. In order to gain insights into the molecular mechanisms leading to the development of PAH and to design novel therapeutic strategies, high-throughput approaches have to be adopted to facilitate systematic identification of the disease-specific networks using next-generation sequencing technologies, the application of these technologies in PAH has been relatively trivial to date.

Pulmonary vascular remodeling and right heart failure in pulmonary hypertension: future role of positron emission tomography in decoding the enigma.

Whereas the insights into the cellular and molecular mechanisms of pulmonary arterial hypertension (PAH) and associated right heart failure have increased in recent years, there is a lack of clinical tools to assess the pathobiological mechanisms in patients. Positron emission tomography (PET) provides an array of new possibilities to image and quantify relevant disease processes, including proliferation, angiogenesis, matrix remodeling, shifts in metabolism and neurohormonal signaling. Here we describe the first studies which were conducted to image pulmonary vascular remodeling and right heart failure in vivo and discuss additional targets for imaging which hold great promise for future use in PAH patients.Electronic supplementary materialThe online version of this article (doi:10.1186/2213-0802-1-16) contains supplementary material, which is available to authorized users.

Subcellular Mechanisms in Pulmonary Arterial Hypertension: Combinatorial Modalities that Inhibit Anterograde Trafficking and Cause Bone Morphogenetic Protein Receptor Type 2 Mislocalization

Abstract The natural history of familial pulmonary arterial hypertension (PAH) typically involves mutations in and/or haploinsuffciency of BMPR2 (gene for bone morphogenetic protein receptor type 2) but with low penetrance (10%-15%), delayed onset (in the third or fourth decade), and a gender bias (two- to fourfold more prevalent in postpubertal women). Thus, investigators have sought an understanding of "second-hit" modalities that might affect BMPR2 anterograde trafficking and/or function. Indeed, vascular lung lesions in PAH have been reported to contain enlarged "vacuolated" endothelial and smooth muscle cells with dilated endoplasmic reticulum (ER) cisternae, increased ER structural protein reticulon 4 (also called Nogo-B), and enlarged and fragmented Golgi apparatus. We recently replicated this cellular phenotype in primary human pulmonary arterial endothelial cells and human pulmonary arterial smooth muscle cells in culture by acute knockdown of the estradiol 17β (E2)-responsive proteins signal transducer and activator of transcription 5a (STAT5a) and STAT5b using small interfering RNAs (siRNAs). We have now investigated whether functional haploinsufficiences of these molecules, alone or in combination with other modalities, might interfere with anterograde membrane trafficking using (a) the quantitative tsO45VSV-G-GFP trafficking assay and (b) assays for cell-surface localization of Flag-tagged BMPR2 molecules. The G glycoprotein of the vesicular stomatitis virus (VSV-G) trafficking assay was validated in EA.hy926 endothelial cells by showing that cells exposed to monocrotaline pyrrole displayed reduced anterograde trafficking. Thereafter, the combinatorial knockdowns of STAT5a, STAT5b, BMPR2, and/or endothelial nitric oxide synthase as well as exposure to E2 or 2-methoxyestradiol were observed to significantly inhibit VSV-G trafficking. These combinations also led to intracellular trapping of wild-type Flag-tagged BMPR2. Overexpression of the PAH disease-derived F14 and KDF mutants of BMPR2, which were trapped in the ER/Golgi, also inhibited VSV-G trafficking in trans. Moreover, probenecid, a chemical chaperone in clinical use today, partially restored cell-surface localization of the KDF but not the F14 mutant. These data identify several combinatorial modalities that inhibit VSV-G anterograde trafficking and cause mislocalization of BMPR2. These modalities merit consideration in defining aspects of the late-developing and gender-biased natural history of human PAH.

Vascular Histomolecular Analysis by Sequential Endoarterial Biopsy in a Shunt Model of Pulmonary Hypertension

The molecular mechanisms of pulmonary arterial hypertension (PAH) remain ill-defined. The aims of this study were to obtain sequential endoarterial biopsy samples in a surgical porcine model of PAH and assess changes in histology and mRNA expression during the disease progression. Differentially expressed genes were then analyzed as potential pharmacological targets. Four Yucatan micro-pigs underwent surgical anastomosis of the left pulmonary artery to the descending aorta. Endovascular samples were obtained with a biopsy catheter at baseline (before surgery) and from the left lung 7, 60, and 180 days after surgery. RNA was isolated from biopsy samples, amplified and analyzed. Dysregulated genes were linked to drugs with potential to treat or prevent PAH. With the development of PAH in our model, we identified changes in histology and in the expression of several genes with known or investigational inhibitors and several novel genes for PAH. Gene dysregulation displayed time-related variations during disease progression. Endoarterial biopsy provides a new method of assessing pulmonary vascular histology and gene expression in PAH. This analysis could identify novel applications for existing and new PAH drugs. The detection of stage- and disease-specific variation in gene expression could lead to individualized therapies.