Pulmonary Arterial Hypertension Animal Research Articles

Loss of Type 2 Bone Morphogenetic Protein Receptor Activates NOD-Like Receptor Family Protein 3/Gasdermin E-Mediated Pyroptosis in Pulmonary ArterialHypertension.

Pulmonary arterial hypertension (PAH) is an incurable disease initiated by endothelial dysfunction, secondary to vascular inflammation and occlusive pulmonary arterial vascular remodeling, resulting in elevated pulmonary arterial pressure and right heart failure. Previous research has reported that dysfunction of type 2 bone morphogenetic protein receptor (BMPR2) signaling pathway in endothelium is inclined to prompt inflammation in PAH models, but the underlying mechanism of BMPR2 deficiency-mediated inflammation needs further investigation. This study was designed to investigate whether BMPR2 deficiency contributes to pulmonary arterial hypertension via the NLRP3 (NOD-like receptor family protein 3)/GSDME (gasdermin E)-mediated pyroptosis pathway. NLRP3 knockout or short hairpin RNA interference of GSDME was performed in PAH animal models to investigate its effect on PAH progression. In addition, the effects of BMPR2 deficiency and restoration of BMPR2 by BMP9 (bone morphogenetic protein 9) or FK506 on pyroptosis were explored both in animal and cell models. Knockout of NLRP3 or short hairpin RNA interference of GSDME in animal models can alleviate the development of pyroptosis, accompanied with improved endothelial integrity, vascular remodeling, and right ventricular systolic pressure. Blocking BMPR2 is sufficient to induce NLRP3 upregulation and release of inflammatory factor IL-1β (interleukin-1β) in pulmonary arterial endothelial cells. Moreover, BMPR2 deficiency can induce GSDME-mediated pyroptosis through NLRP3 activation in 2 animal models, whereas activation of BMPR2 signaling by FK506 or BMP9 can reverse these phenotypes. These findings provide evidence that loss of BMPR2 signaling promotes endothelial cell pyroptosis by enhancing NLRP3/GSDME signaling in PAH. Our findings may provide new insights to explore the inflammatory mechanism of PAH treatment.

1,8-Cineole reduces pulmonary vascular remodelling in pulmonary arterial hypertension by restoring intercellular communication and inhibiting angiogenesis.

Pulmonary Arterial Hypertension (PAH) is characterized by pulmonary vascular remodelling, often associated with disruption of BMPR2/Smad1/5 and BMPR2/PPAR-γ signalling pathways that ultimately lead to right ventricle failure. Disruption of intercellular junctions and communication and a pro-angiogenic environment are also characteristic features of PAH. Although, current therapies improve pulmonary vascular tone, they fail to tackle other key pathological features that could prevent disease progression. In this scenario, aromatic plants emerge as promising sources of bioactive compounds, with 1,8-cineole standing out due to its hypotensive properties and cardioprotective effect in PAH. The present study aims to explore for the first time the effect of 1,8-cineole in pulmonary vascular remodelling associated with PAH. Resorting to the monocrotaline (MCT)-induced PAH animal model, the effect of 1,8-cineole on vascular remodelling including interstitial collagen accumulation, smooth muscle cell proliferation and protein levels of BMPR2 pathway-related proteins, was assessed by microscopy and western blot (WB) analysis. The integrity of gap junctions, pulmonary surfactant, mitochondrial structure and endothelial cell barrier were evaluated by transmission electron microscopy, confocal microscopy and WB analysis. Furthermore, the effect of 1,8-cineole on angiogenesis was determined on pulmonary artery endothelial cells (PAEC) submitted to hypoxia using the scratch wound and Matrigel angiogenesis assays, and the number of sprouts on isolated healthy and diseased pulmonary artery rings, treated with the compound, enabled the validation of these effects. 1,8-Cineole mitigated PAH-associated derailment of both BMPR2/Smad1/5 and BMPR2/PPAR-γ pathways and concomitantly reduced interstitial fibrosis and the arterial medial layer thickness in pulmonary arteries. The compound restored gap junction, lung surfactant and mitochondrial integrity and preserved endothelial barrier integrity. Furthermore, 1,8-cineole exerted an anti-angiogenic effect, by impairing the formation of vessel-like structures in PAEC and sprouting formation in isolated pulmonary arteries. The present study brings new insights about the mechanisms whereby 1,8-cineole impacts pulmonary vascular remodelling and demonstrates the potential of 1,8-cineole as a therapeutic strategy to hamper PAH progression.

Serum proteome profiling reveals HGFA as a candidate biomarker for pulmonary arterial hypertension

BackgroundIdentification and validation of potential biomarkers could facilitate the identification of pulmonary arterial hypertension (PAH) and thus aid to study their roles in the disease process.MethodsWe used the isobaric tag for relative and absolute quantitation approaches to compare the protein profiles between the serum of PAH patients and the controls. Bioinformatics analyses and enzyme-linked immunosorbent assay (ELISA) identification of PAH patients and the controls were performed to identify the potential biomarkers. The receiver operating characteristic curve (ROC) analysis was used to evaluate the diagnostic performance of these potential biomarkers. Mendelian randomization (MR) analysis further clarified the relationship between the potential biomarkers and PAH. Additionally, the expression levels of the potential biomarkers were further validated in two PAH animal models (monocrotaline-PH and Sugen5416 plus hypoxia-PH) using ELISA and reverse transcription-quantitative PCR (RT-qPCR).ResultsWe identified significant changes in three proteins including heparanase (HPSE), gelsolin (GSN), and hepatocyte growth factor activator (HGFA) in PAH patients. The ROC analysis showed that the areas under the curve of HPSE, GSN, and HGFA in differentiating PAH patients from controls were 0.769, 0.777, and 0.964, respectively. HGFA was correlated with multiple parameters of right ventricular functions in PAH patients. Besides proteomic analysis, we also used MR method to demonstrate the causal link between genetically reduced HGFA levels and an increased risk of PAH. In subsequent validation study in PAH animal models, the mRNA expression levels of HGFA in the lung tissues were significantly lower in PAH rat models than in controls. In the rat models, serum levels of HGFA were lower compared to the control group and showed a negative correlation with right ventricular systolic pressure.ConclusionThe study demonstrated that HGFA might be a promising biomarker for noninvasive detection of PAH.

Magnetic nanoradiotracers for targeted neutrophil detection in pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a severe disease characterized by elevated blood pressure in the pulmonary artery that can ultimately damage the right ventricle of the heart. PAH is pathophysiologically heterogeneous, which makes early diagnosis and treatment difficult. Inflammation is thought to be an important factor in the development and progression of this disease and may explain some of the observed interindividual differences. In the context of both acute and chronic inflammation, neutrophil recruitment to the lung has been suggested as a potential biomarker for studying PAH progression. However, there are currently no specific probes for its non-invasive in vivo detection. The imaging-based gold standard for assessing inflammation is [18F] fluorodeoxyglucose (18F-FDG), which is not cell specific. This highlights the urgent need for more specific molecular probes to support personalized medicine.MethodsThis study investigated the potential of magnetic nanoradiotracers based on ultrasmall iron oxide nanoparticles, functionalized with N-cinnamoyl-F-(D)L-F-(D)L-F peptide, to detect increased neutrophil infiltration in vivo in different PAH animal models via positron emission tomography. These nanoprobes target formyl peptide receptor 1, which is abundantly expressed in the cell membrane of neutrophils. To assess the benefit of these nanoprobes, their biodistribution was first assessed via magnetic resonance imaging and histology. Then, their lung uptake was compared by positron emission tomography with that of 18F-FDG in two types of PAH animal models with different profiles of inflammation and neutrophil infiltration: monocrotaline and double-hit Sugen-chronic hypoxia PAH rat models.ResultsOur targeted magnetic nanoradiotracer detected an increase in pulmonary neutrophil infiltration in both PAH models and distinguished between them, which was not possible with 18F-FDG PET.ConclusionsThis study underscores the importance of targeted imaging in providing an individualized and longitudinal evaluation of heterogeneous and multifactorial diseases such as PAH. The use of targeted multimodal nanoprobes, for magnetic resonance/positron emission tomography imaging has the potential to facilitate the diagnosis and monitoring of diseases, as well as the development of novel therapies.Graphical abstract

Colchicine as a therapeutic option for the treatment of pulmonary arterial hypertension (PAH) in a rat model

Abstract Introduction Pulmonary arterial hypertension (PAH) is a cardiovascular disease that is characterized by severe structural and functional damages, resulting from the obstructive remodeling of the pulmonary vasculature and consequent right ventricular pressure overload. The current therapy mainly focuses on the pulmonary vascular system, with uncertain efficacy. Therefore, it is crucial to explore new therapeutic approaches. The mechanism of colchicine is complex and not-fully understood, but it has been proven to exert cardiopulmonary protective effects. Methods PAH induction was carried out in 8-week-old male WKY rats with a single subcutaneous injection of 60 mg/kg monocrotaline. Monocrotaline, a pyrrolizidine alkaloid, is metabolized in liver and induces inflammatory processes, which can cause PAH in rats. Starting from day 14 after MCT injection, the animals received three times a week intraperitoneal colchicine treatment at a dose of 0.5 mg/kg for two weeks. Echocardiographic examinations were performed during the study. At the end of the study, besides histological examination of the right ventricles and lungs [arterial wall thickness (WT%) and area (WA%)], the expression and phosphorylation levels of the proteins involved in cardiac remodeling were evaluated using Western blot. We assessed the pyruvate dehydrogenase (PDH) enzyme activity in the heart, which plays an important role in energy metabolism. We also examined the production of cytokines and chemokines in the right ventricle. Results The VW/BW ratio in the colchicine-treated group was significantly reduced compared to the MCT group (p<0.01). Echocardiographic parameters, EF% and E/e' ratio – characterizing the left ventricular function – did not differ considerably between the groups during the treatment. The TAPSE and PAT values characterizing right ventricular function improved in the MCT+C group (p<0.05). Collagen deposition and TGFβ level were significantly reduced by colchicine (p<0.05) in PAH animals. In the MCT+C group, the vascular remodeling (WT% and WA%) significantly decreased compared to the MCT group (p<0.01). The expression of α-SMA in vessel walls was most pronounced in the MCT group (p<0.01), which was significantly reduced by colchicine treatment. The phosphorylation of prosurvival signaling factors (ERK1/2, Akt1, GSK3β) was significantly increased in the MCT+C group (p<0.01) compared to the MCT group. In the right ventricle, PDH enzyme activity increased in the MCT+C group compared to the MCT group (p<0.01). According to cytokine array, cytokines involved in mediating fibrosis and inflammatory processes (TIMP-1, VEGF, L-selectin, CXCL7) showed increased secretion in the MCT group, which was moderately attenuated by colchicine treatment. Conclusion Colchicine treatment reduced the extent of fibrosis and inflammation, improved the structure of the pulmonary vasculature, and enhanced right ventricular function and energy supply.

Unraveling the Impact of miR-146a in Pulmonary Arterial Hypertension Pathophysiology and Right Ventricular Function.

Pulmonary arterial hypertension (PAH) is a chronic disorder characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary vascular resistance and right ventricle (RV) overload and failure. MicroRNA-146a (miR-146a) promotes vascular smooth muscle cell proliferation and vascular neointimal hyperplasia, both hallmarks of PAH. This study aimed to investigate the effects of miR-146a through pharmacological or genetic inhibition on experimental PAH and RV pressure overload animal models. Additionally, we examined the overexpression of miR-146a on human pulmonary artery smooth muscle cells (hPASMCs). Here, we showed that miR-146a genic expression was increased in the lungs of patients with PAH and the plasma of monocrotaline (MCT) rats. Interestingly, genetic ablation of miR-146a improved RV hypertrophy and systolic pressures in Sugen 5415/hypoxia (SuHx) and pulmonary arterial banding (PAB) mice. Pharmacological inhibition of miR-146a improved RV remodeling in PAB-wild type mice and MCT rats, and enhanced exercise capacity in MCT rats. However, overexpression of miR-146a did not affect proliferation, migration, and apoptosis in control-hPASMCs. Our findings show that miR-146a may play a significant role in RV function and remodeling, representing a promising therapeutic target for RV hypertrophy and, consequently, PAH.

Hydroxy-Safflower Yellow A Mitigates Vascular Remodeling in Rat Pulmonary Arterial Hypertension.

The underlying causes of pulmonary arterial hypertension (PAH) often remain obscure. Addressing PAH with effective treatments presents a formidable challenge. Studies have shown that Hydroxysafflor yellow A (HSYA) has a potential role in PAH, While the mechanism underlies its protective role is still unclear. The study was conducted to investigate the potential mechanisms of the protective effects of HSYA. Using databases such as PharmMapper and GeneCards, we identified active components of HSYA and associated PAH targets, pinpointed intersecting genes, and constructed a protein-protein interaction (PPI) network. Core targets were singled out using Cytoscape for the development of a model illustrating drug-component-target-disease interactions. Intersection targets underwent analysis for Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Selected components were then modeled for target interaction using Autodock and Pymol. In vivo validation in a monocrotaline-induced PAH (MCT-PAH) animal model was utilized to substantiate the predictions made by network pharmacology. We associated HSYA with 113 targets, and PAH with 1737 targets, identifying 34 mutual targets for treatment by HSYA. HSYA predominantly affects 9 core targets. Molecular docking unveiled hydrogen bond interactions between HSYA and several PAH-related proteins such as ANXA5, EGFR, SRC, PPARG, PGR, and ESR1. Utilizing network pharmacology and molecular docking approaches, we investigated potential targets and relevant human disease pathways implicating HSYA in PAH therapy, such as the chemical carcinogenesis receptor activation pathway and the cancer pathway. Our findings were corroborated by the efficacious use of HSYA in an MCT-induced rat PAH model, confirming its therapeutic potential.

Hypusine Signaling Promotes Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both invitro and invivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.

Characterization of PKCα-rutin interactions and their application as a treatment strategy for pulmonary arterial hypertension by inhibiting ferroptosis.

As a common plant-derived dietary flavonoid, rutin receives widespread attention because of its good antioxidant bioactivities. Protein kinase Cα (PKCα) is a serine/threonine kinase that is involved in uncountable cellular processes, among which ferroptosis, a novel form of cell death, is triggered by lipid peroxidation and has been reported to be associated with pulmonary arterial hypertension (PAH). But it is still not well appreciated how rutin inhibits ferroptosis in PAH and what function PKCα has in this process. In this study, we first observed whether rutin could prevent PAH by attenuating ferroptosis with a PAH animal model and pulmonary artery smooth muscle cells (PASMCs) under hypoxia. Mitochondrial metabolomics and network pharmacology were employed to clarify the metabolic alterations and screen target proteins, and the results showed that PKCα was a vital node in rutin regulating mitochondrial metabolism related to ferroptosis in PAH. Based on molecular docking and multispectral analysis, we found that rutin could directly interact with PKCα through hydrogen bonds, which could induce static quenching, and then influence the secondary structure of PKCα. In conclusion, these findings mainly point to a novel mechanism that rutin protects PAH rats by modifying the structure and altering the activity of PKCα, and thus suppressing ferroptosis. This work reveals that the interaction behaviors between small molecules and bio-macromolecules are a critical factor to develop natural biological active ingredients and gives an insight into the potential applications of flavonoids in health and disease.

Анализ роли нейтрофилов в развитии воспаления в монокроталининдуцированной легочной артериальной гипертензии

Pulmonary arterial hypertension (PAH) is a clinical syndrome with dismal outcome. This disease is associated with endothelial dysfunction or damage, pulmonary vascular remodeling, and increased vascular resistance and pulmonary artery blood pressure. All this provokes right ventricular failure and pulmonary edema, which leads to premature death of patients. PAH is accompanied by the inflammatory process deve­lopment, one of the main causes of which is the increased production of reactive oxygen and chlorine species by stimulated neutrophils. The purpose of our study was to study the role of these cells in the oxidative stress formation in experimental model of PAH. The studies were conducted on outbred white rats. Modeling of PAH was carried out by subcutaneous administration of a monocrotaline solution at a dose of 60 mg/kg. The control group consisted of 10 healthy animals. After monocrotaline injection, PAH animals were divided into 4 groups: 10 rats in the 2-week group, 10 rats in the 4-week group, 14 rats in the 6-week group, and 20 rats in the 8-week group. After collecting peripheral blood and isolating neutrophils, reactive oxygen and chlorine species production, secretory degranulation and the unbound calcium ions concentration in the cytosol were determined using spectral analysis methods. The results were compared with corresponding data obtained for 10 healthy animals. It has been established that the development of PAH in rats over 8 weeks is accompanied by neutrophil priming and oxidative stress formation as a result of “respiratory burst” mechanisms modification. Changes in reactive oxygen and chlorine species production, disruption of Ca2+-dependent intracellular signaling involving cyclooxygenase 1/2 and phosphatidylinositol 3-kinase, and increased secretion of myeloperoxidase from neutrophils were revealed.

Abstract 15967: Unravelling the Role of Deoxyhypusine Synthase-Mediated Hypusination of eIF5A in Pulmonary Arterial Hypertension

Introduction: Pulmonary Arterial Hypertension (PAH) is characterized by progressive distal pulmonary arteries (PAs) obstruction leading to heart failure and death. PA smooth muscle cells (PASMCs) from PAH patients display a “cancer-like” phenotype that contributes to PA remodeling. Appreciation of the pivotal role of translational control in hyperproliferating diseases is steadily increasing. In this regard, eukaryotic translation initiation factor 5A (eIF5A) was shown to provide cancer cells with a competitive advantage by increasing translation of mRNAs with oncogenic proprieties. Strikingly, eIF5A is the only protein containing the unique spermidine-derived amino acid hypusine required for its function. Hypusine formation is catalyzed by the sequential actions of deoxyhypusine synthase (DHPS) and deoxyhypusine hydrolase (DOHH). We hypothesized that increased Hyp eIF5A in PAH-PASMCs is required to promote translational efficiency of a set of factors conferring a higher survival and proliferative capacity, leading to pulmonary vascular remodeling. Methods/Results: As assessed by LC-MSMS and WB, expression levels of DHPS, DOHH and both total and hypusinated forms of eIF5A were found increased in PAs and PASMCs from PAH patients and animal models. In vitro , both molecular and pharmacological inhibition of DHPS and DOHH significantly attenuated PAH-PASMCs survival (WB Survivin; Annexin V labeling) and proliferation (WB MCM2, PLK1; Ki67 labeling and EdU incorporation). Hypusine signaling promoted the expression of a broad array of proteins involved in oxidative phosphorylation, supporting the bioenergetic requirements of cell survival and proliferation (LC-MSMS, WB, Seahorse). In vivo , smooth muscle cells-targeted inactivation of one allele of Dhps conferred partial protection against the development of Sugen/Hypoxia (Su/Hx)-induced PAH in mice. Accordingly, pharmacological inhibition of DHPS using GC7 improved hemodynamics (RVSP, mPAP, CO) and vascular remodeling (EVG) in monocrotaline and Su/Hx rats with established PAH. Conclusion: We showed for the first time that hypusine signaling is implicated in PAH development and represents a new promising therapeutic target.

Untangling the mechanisms of pulmonary arterial hypertension-induced right ventricular stiffening in a large animal model.

Pulmonary hypertension (PHT) is a devastating disease with low survival rates. In PHT, chronic pressure overload leads to right ventricle (RV) stiffening; thus, impeding diastolic filling. Multiple mechanisms may contribute to RV stiffening, including wall thickening, microstructural disorganization, and myocardial stiffening. The relative importance of each mechanism is unclear. Our objective is to use a large animal model to untangle these mechanisms. Thus, we induced pulmonary arterial hypertension (PAH) in sheep via pulmonary artery banding. After eight weeks, the hearts underwent anatomic and diffusion tensor MRI to characterize wall thickening and microstructural disorganization. Additionally, myocardial samples underwent histological and gene expression analyses to quantify compositional changes and mechanical testing to quantify myocardial stiffening. Finally, we used finite element modeling to disentangle the relative importance of each stiffening mechanism. We found that the RVs of PAH animals thickened most at the base and the free wall and that PAH induced excessive collagen synthesis, increased cardiomyocyte cross-sectional area, and led to microstructural disorganization, consistent with increased expression of fibrotic genes. We also found that the myocardium itself stiffened significantly. Importantly, myocardial stiffening correlated significantly with collagen synthesis. Finally, our computational models predicted that myocardial stiffness contributes to RV stiffening significantly more than other mechanisms. Thus, myocardial stiffening may be the most important predictor for PAH progression. Given the correlation between myocardial stiffness and collagen synthesis, collagen-sensitive imaging modalities may be useful for estimating myocardial stiffness and predicting PAH outcomes. STATEMENT OF SIGNIFICANCE: Ventricular stiffening is a significant contributor to pulmonary hypertension-induced right heart failure. However, the mechanisms that lead to ventricular stiffening are not fully understood. The novelty of our work lies in answering this question through the use of a large animal model in combination with spatially- and directionally sensitive experimental techniques. We find that myocardial stiffness is the primary mechanism that leads to ventricular stiffening. Clinically, this knowledge may be used to improve diagnostic, prognostic, and therapeutic strategies for patients with pulmonary hypertension.

NAMPT mediates PDGF-induced pulmonary arterial smooth muscle cell proliferation by TLR4/NF-κB/PLK4 signaling pathway

Nicotinamide phosphoribosyltransferase (NAMPT), a pleiotropic protein, promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which is associated with the genesis and progression of pulmonary arterial hypertension (PAH). NAMPT is highly increased in PAH patient's plasma and highly relevant to PAH severity. The mRNA and protein levels of NAMPT are elevated in PAH animal models. However, the underlying molecular mechanisms how NAMPT mediated platelet-derived growth factor (PDGF)-induced PASMCs proliferation are still unclear. The present study aimed to address these issues. Primary cultured PASMCs were attained from male Sprague-Dawley (SD) rats. Western blotting, RT-PCR, ELISA, cell transfection, Cell Counting Kit-8 (CCK-8) and EdU incorporation assays were used in the experiments. We showed that PDGF upregulated NAMPT expression through the activation of signal transducers and activators of transcription 5 (STAT5), and elevated extracellular NAMPT further promoted the activation of NF-κB through Toll-like receptor 4 (TLR4), which ultimately upregulated polo-like kinase 4 (PLK4) expression leading to PASMCs proliferation. Knockdown of STAT5, NAMPT or PLK4, and inhibition of TLR4 or NF-κB suppressed PDGF-induced PASMCs proliferation. Our study suggests that NAMPT plays an essential role in PDGF-induced PASMCs proliferation via TLR4/NF-κB/PLK4 pathway, suggesting that targeting NAMPT might be valuable in ameliorating pulmonary arterial hypertension.

Therapeutic effects of hypoxia-preconditioned bone marrow-derived mesenchymal stromal cells and their extracellular vesicles in experimental pulmonary arterial hypertension

AimsTo evaluate BM-MSCs and their extracellular vesicles (EVs) preconditioned with hypoxia or normoxia in experimental pulmonary arterial hypertension (PAH). Main methodsBM-MSCs were isolated and cultured under normoxia (MSC-N, 21%O2) or hypoxia (MSC-H, 1%O2) for 48 h. EVs were then isolated from MSCs under normoxia (EV-N) or hypoxia (EV-H). PAH was induced in male Wistar rats (n = 35) with monocrotaline (60 mg/kg); control animals (CTRL, n = 7) were treated with saline. On day 14, PAH animals received MSCs or EVs under normoxia or hypoxia, intravenously (n = 7/group). On day 28, right ventricular systolic pressure (RVSP), pulmonary acceleration time (PAT)/pulmonary ejection time (PET), and right ventricular hypertrophy (RVH) index were evaluated. Perivascular collagen content, vascular wall thickness, and endothelium-mesenchymal transition were analyzed. Key findingsPAT/PET was lower in the PAH group (0.26 ± 0.02, P < 0.001) than in CTRLs (0.43 ± 0.02) and only increased in the EV-H group (0.33 ± 0.03, P = 0.014). MSC-N (32 ± 6 mmHg, P = 0.036), MSC-H (31 ± 3 mmHg, P = 0.019), EV-N (27 ± 4 mmHg, P < 0.001), and EV-H (26 ± 5 mmHg, P < 0.001) reduced RVSP compared with the PAH group (39 ± 4 mmHg). RVH was higher in the PAH group than in CTRL and reduced after all therapies. All therapies decreased perivascular collagen fiber content, vascular wall thickness, and the expression of endothelial markers remained unaltered; only MSC-H and EV-H decreased expression of mesenchymal markers in pulmonary arterioles. SignificanceMSCs and EVs, under normoxia or hypoxia, reduced right ventricular hypertrophy, perivascular collagen, and vessel wall thickness. Under hypoxia, MSCs and EVs were more effective at improving endothelial to mesenchymal transition in experimental PAH.

Effect of Induced Pulmonary Arterial Hypertension on Testicular Parameters of Wistar Rats Subjected to Resistance Exercise Training.

Pulmonary arterial hypertension (PAH) is characterized by elevated arterial pressure and vascular resistance. PAH may cause alterations in the microcirculation of several organs, including the kidney, liver, brain, and testes. However, it remains unclear whether monocrotaline-induced PAH exerts detrimental effects on animal testes. Thus, we analyzed the impact of PAH on testicular morphology and function. Additionally, we investigated the effect of resistance exercise training (RT) on testicular parameters in PAH rats. Eight healthy Wistar rats and eight PAH rats were subjected to RT training for 30 days; the other PAH and healthy rats (n = 8/group) did not exercise. PAH rats had lower reproductive organ weight, serum testosterone levels, testicular glucose, and nitric oxide (NO) levels, Leydig cell parameters, tubular morphometry, germ cell counts, and daily sperm production than healthy animals did. The practice of RT attenuated the negative impact of PAH on the relative weights of the testes and epididymides, Leydig cell number, nuclear volume, testicular NO levels, and seminiferous epithelium architecture. Moreover, RT positively influenced testosterone levels in PAH animals. We conclude that PAH exerts deleterious effects on testicular histology and function. However, RT can be beneficial to the PAH-affected testicular parameters.

Temporal Changes in Key Signal Transduction Pathways Mediating Muscle Protein Synthesis with Adaptive and Maladaptive Right Ventricular Hypertrophy in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive disease characterized by occlusive remodeling of pulmonary arteries &lt; 500 micron and increased pulmonary vascular resistance. With the onset of PAH, the right ventricle (RV) of the heart adapts to the increased afterload pressure by undergoing adaptive hypertrophic remodeling to maintain adequate blood flow. However, for unknown reasons, maladaptive influences ensue, resulting in impaired RV function with progressive decompensation and right heart failure. Using a rodent model of PAH, key signaling pathways mediating cardiac muscle protein synthesis in the RV were evaluated during both the adaptive hypertrophy phase, with preserved right heart function, and the decompensated maladaptive phase, in which right heart failure (RHF) was present. Analysis of protein and gene expression changes in PAH animals identified three key signaling pathways involved in the shift toward maladaptive right heart failure: i) PI3K/Akt/mTOR; ii) GSK-3; iii) MAPK/ERK, as well as IGF-1 regulation. During adaptive hypertrophy, significant increments of phosphorylated proteins in the three signaling pathways were observed with increases in RV fibrosis and decreased capillarity found. In the maladaptive phase, mTORC1 and its downstream effector p-70S6K were significantly activated, contributing to the decreased LC3-I/II ratio, a marker of autophagy inhibition together with further significant RV muscle fibrosis and greater capillary rarefaction. We propose that autophagy inhibition in conjunction with other maladaptive processes reported in the decompensated RV muscle contributes to the genesis of overt RHF in PAH, and that a continuum of changes characterizes the adaptive and maladaptive phases in the RV muscle Keywords: Pulmonary Arterial Hypertension (P

Abstract 14938: Endothelial Cell-Targeted Nanoparticle Delivery of Crispr/Cas9 Plasmid Dna Disrupts Endothelial Epas1 in Adult Rats and Inhibits Experimental Pah

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling that leads to right heart failure and death. HIF-2α (hypoxia-inducible factor-2α) activation in lung endothelial cells is obligatory to obliterative pulmonary vascular remodeling in PAH patients and animal PAH models. Hypothesis: Endothelial cell-targeted nanoparticle can deliver plasmid DNA expressing the CRISPR/Cas9 system to disrupt Epas1 (encoding HIF-2α) selectively in endothelial cells in adult rats and inhibit PAH in monocrotaline rats. Methods: To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether- block -poly(lactide-co-glycolide) (PEG- b -PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by rat Cdh5 promoter and guide RNAs targeting Epas1 driven by U6 promoter induced highly efficient genome editing in endothelial cells in 6-weeks old SD rats. Rat Cdh5 -Cas9 plasmid DNA with scramble guide RNA were injected as a control group. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, hemodynamic measurement of right ventricular systolic pressure RVSP and weight ratio of right ventricular versus left ventricular plus septum (RV/LV+S), an indicator of RV hypertrophy were determined. Results: Single administration of mixture of nanoparticles and CRISPR Cdh5 plasmid DNA caused diminished HIF-2α protein expression selectively in endothelial cells indicating highly efficient genome editing. Genomic editing-mediated disruption of Epas1 in endothelial cells reduced RVSP and right ventricular hypertrophy as well as obliterative pulmonary vascular remodeling in MCT-induced PAH rats. Conclusions: These findings demonstrate robust genome editing of Epas1 via EC-targeted nanoparticle delivery of CRISPR-Cas9 plasmid DNA in adult rats leading to attenuated PAH in MCT rats, suggesting EC-targeted nanoparticle delivery of genome editing system to disrupt PAH-causing gene(s) in ECs is a promising precision gene therapy of PAH in patients.

Abstract 10900: Implication of the Post-Translational Formation of Hypusine in Eif5a in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is characterized by progressive pulmonary arteries (PAs) obstruction leading to heart failure and death. PA smooth muscle cells (PASMCs) of PAH patients display a “cancer-like” phenotype that contributes to PA remodeling. Eukaryotic translation initiation factor 5A (eIF5A) was shown to provide cancer cells with a competitive advantage by increasing translation of mRNAs with oncogenic proprieties, many of them containing proline/glycine-rich patterns. Strikingly, eIF5A is the only protein containing the unique, polyamine-derived amino acid hypusine, which is required for its function. Hypusine formation is catalyzed by the sequential actions of deoxyhypusine synthase (DHPS) and deoxyhypusine hydrolase (DOHH). We hypothesized that increased eiF5A Hyp in PAH-PASMCs is required to promote translational efficiency of a set of factors conferring a higher survival and fibroproliferative capacity, leading to pulmonary vascular remodeling. Data derived from a comparative proteomic analysis (LC-MSMS) between normal and PAH-PASMCs and confirmed by Western blot indicate that DHPS and DOHH are overexpressed in PAH-PASMCs compared to controls (p&lt;0.05). Consistently, both total and hypusinated forms of eIF5A were found up-regulated in dissected PAs and PASMCs from PAH patients and animal models (MCT and Su/Hx rats, p&lt;0.05). In vitro , inhibition of DHPS and DOHH, using GC7 and ciclopirox, respectively, significantly attenuates PAH-PASMCs survival and proliferation, (Annexin V; Ki67 labeling and EdU incorporation, p&lt;0.01). These effects were confirmed by a reduced expression eiF5A Hyp , MCM2, PCNA and Survivin (p&lt;0.01) and a downregulation of BRD4, EP300 and COL1, three factors implicated in PAH containing proline/glycine-rich patterns. In vivo , inhibition of DHPS using GC7 in MCT rats with established PAH improves hemodynamics (RVSP, mPAP, CO, p&lt;0.05) and vascular remodeling (EVG, p&lt;0.05). Furthermore, preliminary data indicate that smooth muscle cells-targeted inactivation of one allele of Dhps tends to confer protection against Su/Hx-induced PAH in mice. We showed for the first time that hypusine signaling is implicated in PAH development and represents a new promising therapeutic target.

Abstract 12700: Post-GWAS Functional SNP Analysis to Define Genetic and Acquired Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an enigmatic and morbid disease where insights are emerging regarding genetic susceptibility. Genome-wide Association Studies (GWAS) have identified genomic SNPs associated with increased PAH risk. It has been challenging to define the mechanisms underlying the contribution of non-coding PAH-associated functional SNPs (fSNPs) to the pathogenesis of this disease. Methods: We developed a post-GWAS functional genomics strategy to define the causative fSNPs and the associated biological mechanisms. It includes Reel-seq (Regulatory element-sequencing), an EMSA-based technique to identify fSNPs in a synthetic SNP library; SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify proteins that specifically bind to fSNPs; and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to show allele-imbalanced binding of these proteins to fSNPs. The regulation of risk gene expression by fSNP-binding proteins and their pathogenicity were determined in human pulmonary arterial endothelial cells (PAECs) and confirmed in PAH animal models and patients. Results: An intergenic SNP rs4738801 was identified as a fSNP by Reel-seq. This non-coding fSNP resides in a remote upstream enhancer region of SOX17, an endothelial effector increasingly associated with PAH pathogenesis. Using SDCP-MS and AIDP-Wb, we found that transcription factor FUBP1 binds to rs4738801 risk allele C with lower affinity than non-risk allele G, resulting in a decrease in SOX17 expression. FUBP1 and target gene SOX17 controlled PAH-associated pathophenotypes in PAECs, including proliferation, apoptosis, and angiogenesis. Downregulated by major acquired PAH trigger hypoxia, FUBP1 and SOX17 were decreased in lungs and pulmonary ECs isolated from PAH patients and mouse models. A 3.8-fold enrichment of risk allele C was found in patients with PAH induced by hypoxia, but not in PAH associated with connective tissue disease or other etiology. CONCLUSION: Our functional genomics findings identify a novel role of FUBP1 in the functional regulation of SOX17 locus and elucidate a pathogenic mechanism that combines the acquired PAH-triggering factors and altered genetic susceptibility.

Abstract 10202: DNA-PKcs: A Novel Therapeutic Target in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vascular obliteration of small pulmonary arteries (PAs), notably due to excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). Accumulating evidence indicates that PAH-PASMCs have acquired an over-efficient DNA damage response (DDR) ensuring sensing, signaling, and repair of DNA damages, accounting for their enhanced capacity to survive and proliferate under stressful conditions. DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery which plays an instrumental role in the overall survival and proliferation of cancer cells. Based on similarities between PAH and cancer cells, we thus hypothesized that increased expression of DNA-PK contributes to vascular remodeling in PAH. Methods and Results: Using Western blot (WB) and immunohistochemistry, we found that expression and activity of DNA-PK were increased in PAH-PASMCs (n=6) compared to controls (n=6, p&lt;0.01). Similarly, DNA-PK was significantly up-regulated in distal PAs from PAH animal models (i.e. MCT rats and Su/Hx rats and mice, n=5/group, p&lt;0.01). In vitro, the pharmacological inhibition of DNA-PK using AZD-7648 or Nedisertib attenuates PAH-PASMC proliferation (Ki67 and EdU labeling, MCM2, PLK1, and PNCA by WB; p&lt;0.05) and resistance to apoptosis (AnnexinV labeling, Survivin by WB)(n=5 cell lines; p&lt;0.05). These effects were associated with reduced expression of gH2AX. As assessed by echocardiography and right heart catheterization, treatment with AZD7648 improved pulmonary hemodynamics (RVSP, mPAP, and TPR) and right ventricular function (CO, PAT/PET, TAPSE) in Su/Hx mice with established PAH (n=10/group; all p&lt;0.01). Accordingly, pulmonary vascular remodeling (EVG) and the proportion of PCNA-positive PASMCs were reduced in AZD7648-treated Su/Hx mice when compared to vehicle-treated Su/Hx mice (p&lt;0.001). Conclusions: We showed for the first time that DNA-PK is overexpressed in human and experimental PAH and contributes to the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs.