PAH Model Research Articles

A novel complement C3 inhibitor CP40-KK protects against experimental pulmonary arterial hypertension via an inflammasome NLRP3 associated pathway

BackgroundPulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by complement dependent and proinflammatory activation of macrophages. However, effective treatment for complement activation in PAH is lacking. We aimed to explore the effect and mechanism of CP40-KK (a newly identified analog of selective complement C3 inhibitor CP40) in the PAH model.MethodsWe used western blotting, immunohistochemistry, and immunofluorescence staining of lung tissues from the monocrotaline (MCT)-induced rat PAH model to study macrophage infiltration, NLPR3 inflammasome activation, and proinflammatory cytokines (IL-1β and IL-18) release. Surface plasmon resonance (SPR), ELISA, and CH50 assays were used to test the affinity between CP40-KK and rat/human complement C3. CP40-KK group rats only received CP40-KK (2 mg/kg) by subcutaneous injection at day 15 to day 28 continuously.ResultsC3a was significantly upregulated in the plasma of MCT-treated rats. SPR, ELISA, and CH50 assays revealed that CP40-KK displayed similar affinity binding to human and rat complement C3. Pharmacological inhibition of complement C3 cleavage (CP40-KK) could ameliorate MCT-induced NLRP3 inflammasome activity, pulmonary vascular remodeling, and right ventricular hypertrophy. Mechanistically, increased proliferation of pulmonary arterial smooth muscle cells is closely associated with macrophage infiltration, NLPR3 inflammasome activation, and proinflammatory cytokines (IL-1β and IL-18) release. Besides, C3a enhanced IL-1β activity in macrophages and promoted pulmonary arterial smooth muscle cell proliferation in vitro.ConclusionOur findings suggest that CP40-KK treatment was protective in the MCT-induced rat PAH model, which might serve as a therapeutic option for PAH.

Abstract 18284: Inhibition of Sulfatase-1 Prevents Pulmonary Arterial Hypertension in Animal Models of Disease

Introduction: Sulfatase-1 (Sulf1) is an extracellular enzyme that removes 6-O-sulfate groups from heparan sulfate and is implicated in cardiovascular disease. In human pulmonary artery endothelial cells (HPAECs), Sulf1 mRNA and protein levels are regulated by the fibroproliferative protein NEDD9 involved in PAH. Hypothesis: Sulf1 regulates migration and profibrotic potential of HPAECs and its downregulation prevents the development of PAH. Methods: HPAECs were transfected with Sulf1 siRNA or adenovirus expressing Sulf1 and cell migration was analyzed by scratch assay. In a PAH disease prevention treatment protocol, rats received intratracheal injection of Sulf1 siRNA (si-Sulf1) (1mg/kg of body weight, once a week for 3 weeks) beginning one day following the administration of monocrotaline (MCT). In a disease reversal protocol, injection of si-Sulf1 (1mg/kg of body weight, once every 5 days for a total of 3 doses) began 14 days following initiation of SU-5416/Hypoxia (SU/Hypox) PAH model. Results: Overexpression of Sulf1 in HPAECs resulted in a significant reduction in migration compared to empty vector (wound size 506 ± 39 vs 353 ± 19 μm, P<0.01), whereas transfection with si-Sulf1 increased migration significantly compared to si-Scrambled control (wound size 212 ± 30 vs 295 ± 31 μm, P<0.01). Overexpression of Sulf1 also increased extracellular collagen III deposition in cultured HPAECs compared to empty vector (15.4 ± 5 vs. 7 ± 1 arb. units, P<0.01). Immunostaining of HPAECs for Sulf1 showed strong expression at focal adhesions. In both in vivo PAH models, si-Sulf1 treatment significantly decreased right ventricular (RV) systolic pressure compared to si-Scrambled control (MCT: 41 ± 5 vs 58 ± 6 mm Hg, P<0.05; SU/Hypox: 40 ± 5 vs 75 ± 15 mm Hg, P<0.05) and RV mass measured by Fulton index (MCT: 0.33 ± 0.06 vs 0.56 ± 0.06, P<0.01; SU/Hypox: 0.32 ± 0.02 vs 0.41 ± 0.02, P<0.05). Conclusions: These data demonstrate that pulmonary endothelial expression of Sulf1 is increased at focal adhesions and regulates cellular migration. Inhibition of Sulf1 significantly reduced the severity of PAH in vivo, which, taken together, identifies Sulf1 as a potential therapeutic target by which to improve proliferative vascular remodeling and pulmonary hypertension in PAH.

Pinocembrin attenuates susceptibility to atrial fibrillation in rats with pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling that triggers fibrosis and excessive myocardium apoptosis, ultimately facilitating atrial fibrillation (AF). In various rat models, Pinocembrin has anti-fibrotic and anti-apoptotic effects, reducing arrhythmia vulnerability. However, whether pinocembrin alleviates to AF in a PAH model remains unclear. The experiment aims to investigate how pinocembrin affects AF susceptibility in PAH rats and the possible mechanisms involved. MethodsThe PAH model was induced by monocrotaline (MCT; i. p. 60 mg/kg). Concurrently, rats received pinocembrin (i.p.50 mg/kg) or saline. Hemodynamics parameters, electrocardiogram parameters, lung H.E. staining, atrial electrophysiological parameters, histology, Western blot, and TUNEL assay were detected. ResultsCompared to the control rats, MCT-induced PAH rats possessed prominently enhancive mPAP (mean pulmonary artery pressure), pulmonary vascular remodeling, AF inducibility, HRV, right atrial myocardial fibrosis, apoptosis, atrial ERP, APD, and P-wave duration. Additionally, there were lowered protein levels of Cav1.2, Kv4.2, Kv4.3, and connexin 40 (CX40) in the MCT group in right atrial tissue. However, pinocembrin reversed the above pathologies and alleviated the activity of the Rho A/ROCKs signaling pathway, including the expression of Rho A, ROCK1, ROCK2, and its downstream MYPT-1, LIMK2, BCL-2, BAX, cleaved-caspase3 in right atrial and HL-1 cells. ConclusionPresent data exhibited pinocembrin attenuated atrial electrical, ion-channel, and autonomic remodeling, diminished myocardial fibrosis and apoptosis levels, thereby reducing susceptibility to AF in the MCT-induced PAH rats. Furthermore, we found that pinocembrin exerted inhibitory action on the Rho A/ROCK signaling pathway, which may be potentially associated with its anti-AF effects.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFβ1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1.In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

Effect of Pressure on Soot Formation and Properties in Laminar RP-3 Kerosene Diffusion Flames

ABSTRACT Obtaining quantitative experimental data on soot properties of aviation kerosene at elevated pressures is a significant concern for the surrogate fuel kinetic model of aviation kerosene. However, there are scarce studies on soot formation and properties of aviation kerosene in tractable flames at elevated pressures in literature. In this study, Chinese RP-3 aviation kerosene is used in relevant experiments and numerical simulations up to 3.5 atm to evaluate the effect of pressure on soot formation and properties. Soot morphology, nanostructure, and concentration are studied by transmission electron microscopy and laser extinction method. Then, an RP-3/PAH kinetic model containing the 5-aromatic rings (A5) growth mechanism is proposed to simulate the soot formation processes at elevated pressures. Quantitative experimental results show that pressure significantly affects the soot morphology, nanostructure parameters. The peak soot volume fractions in the RP-3 flame scale with the pressure as . The numerical results show that the RP-3/PAH model can depict the trends of soot particle size and soot volume fraction with pressure. At 1.0–3.5 atm, the rates of soot growth processes increase with pressure, especially the PAHs condensation. The significantly increased soot surface growth rate may be one of the main factors for the variation of soot properties with pressure. The quantitative experimental and numerical results contribute to the understanding of the kinetic mechanism of soot formation for RP-3 kerosene at elevated pressures.

Maresin-1 protects against pulmonary arterial hypertension by improving mitochondrial homeostasis through ALXR/HSP90α axis

AimsPulmonary arterial hypertension (PAH) is a progressive and lethal disease characterized by continuous proliferation of pulmonary arterial smooth muscle cell (PASMCs) and increased pulmonary vascular remodeling. Maresin-1 (MaR1) is a member of pro-resolving lipid mediators and exhibits protective effects on various inflammation-related diseases. Here we aimed to study the role of MaR1 in the development and progression of PAH and to explore the underlying mechanisms. Methods and resultsWe evaluated the effect of MaR1 treatment on PAH in both monocrotaline (MCT)-induced rat and hypoxia+SU5416 (HySu)-induced mouse models of pulmonary hypertension (PH). Plasma samples were collected from patients with PAH and rodent PH models to examine MaR1 production. Specific shRNA adenovirus or inhibitors were used to block the function of MaR1 receptors. The data showed that MaR1 significantly prevented the development and blunted the progression of PH in rodents. Blockade of the function of MaR1 receptor ALXR, but not LGR6 or RORα, with BOC-2, abolished the protective effect of MaR1 against PAH development and reduced its therapeutic potential. Mechanistically, we demonstrated that the MaR1/ALXR axis suppressed hypoxia-induced PASMCs proliferation and alleviated pulmonary vascular remodeling by inhibiting mitochondrial accumulation of heat shock protein 90α (HSP90α) and restoring mitophagy. ConclusionMaR1 protects against PAH by improving mitochondrial homeostasis through ALXR/HSP90α axis and represents a promising target for PAH prevention and treatment.

Astragaloside IV improves pulmonary arterial hypertension by increasing the expression of CCN1 and activating the ERK1/2 pathway.

The aim of the present study was to investigate the underlying mechanism of AS-IV and CCN1 in PAH and to evaluate whether the protective effect of AS-IV against PAH is associated with CCN1 and its related signalling pathway. In vivo, male SD rats were intraperitoneally injected with monocrotaline (MCT, 60 mg/kg) or exposed to hypoxia (10% oxygen) and gavaged with AS-IV (20, 40 and 80 mg/kg/day) to create a PAH model. In vitro, human pulmonary artery endothelial cells (hPAECs) were exposed to hypoxia (3% oxygen) or monocrotaline pyrrole (MCTP, 60 μg/mL) and treated with AS-IV (10, 20 and 40 μM), EGF (10nM, ERK agonist), small interfering CCN1 (CCN1 siRNA) and recombinant CCN1 protein (rCCN1, 100 ng/mL). We identified the differences in the expression of genes in the lung tissues of PAH rats by proteomics. At the same time, we dynamically detected the expression of CCN1 by Western blot both in vivo and in vitro. The Western blot experimental results showed that the expression of CCN1 increased in the early stage of PAH and decreased in the advanced stage of PAH. The results showed that compared with the control group, MCT- and hypoxia-induced increased the hemodynamic parameters and apoptosis. AS-IV can improve PAH, as characterized by decreased hemodynamic parameters, vascular wall area ratio (WA%), vascular wall thickness ratio (WT%) and α-SMA expression and inhibition of cell apoptosis. Moreover, the improvement of PAH by AS-IV was accompanied by increased CCN1 expression, which activated the ERK1/2 signalling pathway. Meanwhile, CCN1 and p-ERK1/2 were inhibited by siCCN1 and promoted by rCCN1. EGF not only activated the ERK1/2 signalling pathway but also induced the expression of CCN1. In conclusion, AS-IV improves PAH by increasing the expression of CCN1 and activating the ERK1/2 signalling pathway. The results of our study provide a theoretical basis for additional study on the protective effect of AS-IV against PAH.

Involvement of SUR2/Kir6.1 channel in the physiopathology of pulmonary arterial hypertension.

We hypothesized that the ATP-sensitive K+ channels (KATP) regulatory subunit (ABCC9) contributes to PAH pathogenesis. ABCC9 gene encodes for two regulatory subunits of KATP channels: the SUR2A and SUR2B 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 in PAH. Using in vitro, ex vivo, and in vivo approaches, we analyzed the localization and expression of SUR2A, SUR2B, and Kir6.1 in the pulmonary vasculature of controls and patients with PAH as in experimental pulmonary hypertension (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. We found that SUR2A, SUR2B, and Kir6.1 were expressed in the lungs of controls and patients with PAH and MCT-induced PH rat models. Organ bath studies showed that SUR2 activation by pinacidil induced relaxation of pulmonary arterial in rats and humans. 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. We demonstrated that SUR2 activation in rat right ventricular (RV) cardiomyocytes reduced RV action potential duration by patch-clamp. Chronic pinacidil administration in control rats increased heart rate without changes in hemodynamic parameters. Finally, in vivo pharmacological activation of SUR2 on MCT and Chronic-hypoxia (CH)-induced-PH rats showed improved PH. We showed that SUR2A, SUR2B, and Kir6.1 are presented in hPASMCs and hPAECs of controls and PAH patients. In vivo SUR2 activation reduced the MCT-induced and CH-induced PH phenotype, suggesting that SUR2 activation should be considered for treating PAH.

Mitochondrial PKD Activates Mitochondrial Fission and Proliferative Signaling in Cardiac Fibroblasts

IntroductionCardiac fibrosis, characterized by wall stiffening, reduced contractility, and impaired overall heart performance, is a self‐reinforcing process in response to injury (e.g., myocardial infarction) and pressure overload (e.g., systemic hypertension and pulmonary arterial hypertension [PAH]). The severity of cardiac fibrosis is associated with cardiac dysfunction, increased risk of arrhythmia, and mortality. However, there is a lack of effective therapies designed to inhibit or reverse cardiac fibrosis. Hyperproliferation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts are the key contributors to cardiac fibrosis. We previously showed that mitochondrial reactive oxygen species (mROS) are one of the regulators for activating proliferative signaling in rat neonatal CFs. Moreover, we reported that phosphorylation of dynamin‐related protein 1 (DRP1) by a stress‐responsive protein kinase D (PKD) at the outer mitochondrial membrane (OMM) promotes mROS generation via increased mitochondrial fission in H9c2 cardiac myoblasts.HypothesisMitochondrial PKD‐DRP1 signaling increases mitochondrial fission leading to increased mROS levels, which activates proliferative signaling.MethodsPrimary adult CFs from human ventricles and the right ventricles (RVs) of a SU5415/hypoxia‐induced rat PAH model were used. Mitochondrial morphology and mROS levels were measured by confocal microscopy.ResultsOverexpression of PKD1 increased DRP1 phosphorylation at Ser637, and activates proliferative signaling pathways including ERK1/2 and p38 in human CFs, but did not promote their differentiation into myofibroblasts, assessed by the amount of a‐smooth muscle actin. To specifically identify the involvement of OMM‐localized PKD activity in these processes, we generated an OMM‐targeted dominant‐negative PKD1 (termed mt‐PKD‐DN) by adding an OMM‐target sequence (amino acid 1‐33 from human TOM20) at the N‐terminus of PKD1‐K612W. Importantly, mt‐PKD‐DN was able to inhibit mitochondrial fission, decrease mROS, hyperpolarize mitochondrial membrane potential, and ultimately decrease proliferation in human CFs. To further understand the role of PKD‐DRP1 signaling in vivo, we employed a preclinical PAH animal model. Immunofluorescence staining of RVs from PAH rats revealed that PKD activity and DRP1 phosphorylation increased only in CFs, but not in ventricular myocytes. RV‐CFs isolated from PAH rats exhibited increased mitochondrial fission, mROS levels, and proliferative signaling compared to RV‐CFs from control rats, and these changes were abolished by adenoviral expression of mt‐PKD‐DN.ConclusionMitochondrial mROS elevation via PKD‐DRP1‐dependent mitochondrial fission promotes CF proliferation. Targeting mitochondrial PKD may be a potential therapeutic strategy to reduce cardiac fibrosis under pathological conditions such as PAH.

Preventive Effect of MG-132 on Monocrotaline Induced Pulmonary Hypertension in Rat Pulmonary Artery Hypertension Model

Aim of Study: To Prevent the Monocrotaline induced extracellular matrix remodeling of pulmonary artery by inhibiting Ubiquitin Proteasome System in rat pulmonary artery hypertension model. Study Design: Experimental Study Place and Duration of Study: Xi’an Jiaotong University, Animal experiment center Xi’an China, from January 2016 to July 2018. Materials and Methods: Thirty-three male Sprague-Dawley rats were divided in to three groups namely control group, Monocrotaline (M.C.T) induced group and M.G-132 (a proteasome inhibitor) treated group. The pulmonary artery hypertension (P.A.H) model was established by giving intraperitoneal injection of Monocrotaline (M.C.T) furthermore the M.G-132 was given to hold back proteasome function. The right ventricular systolic pressure (R.V.S.P) and the right ventricular hypertrophy index (R.V.H.I) were used to assess the progress developing P.A.H. Vascular remodeling was determined by H&E staining and the level of ubiquitinated-PTEN protein was measured by Immunoblotting. Results: The final results revealed that the M.C.T increased R.V.S.P and R.V.H.I in rats, whereas these alterations were concealed in P.A.H induced rats which were treated by M.G-132. Also the Zymograghy results in control vs. experimental group revealed that the activity of Matrix metalloproteinase (MMP2/9) in the PAH model group was considerably elevated at (P<0.05), while treatment of M.G-132 in M.C.T-induced P.A.H-rats decreased the activity of MMP2/9 at (p<0.05). Conclusions: However, further in-depth investigations are need of a time to explore the transformations of ubiquitin proteasome & MMP activity in chronic MCT induced-pulmonary artery hypertension model. Keywords: P.A.H, Ubiquitin, Monocrotaline, Extra-cellular remodeling, M.M.P (Matrix metalloproteinases)

Targeted treprostinil delivery inhibits pulmonary arterial remodeling

IntroductionPulmonary arterial hypertension (PAH) is a fatal disease caused by the progressive remodeling of pulmonary arteries (PAs). Treprostinil (TPS) is a tricyclic benzidine prostacyclin clinically used for PAH treatment. However, due to low bioavailability, short half-times, and severe systemic side effects, TPS efficacy remains limited. MethodsIn this study, glucuronic acid (GlcA)-modified liposomes were developed to improve the site-specific delivery of TPS to pulmonary arterial smooth muscle cells (PASMCs) by targeting the glucose transporter-1 (GLUT-1) in vitro and in vivo. ResultsNon-GlcA-modified and GlcA-modified liposomes encapsulating TPS were 106 ± 1.12 nm in diameter. The drug encapsulation efficiency (EE) was 92%. Data from rat PASMCs showed that GlcA-liposomes enhanced the inhibitory effects of TPS on PASMC proliferation and migration by suppressing growth factor expression, including transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), and cAMP, which was possibly mediated by the cAMP-C/EBP-α p42-p21 signaling pathway. In PAH model rats, GlcA-modified liposomes significantly improved TPS bioavailability and sustained its release over time. Most importantly, the selective inhibition of pulmonary arterial pressure, rather than systemic arterial pressure, indicated the increased pulmonary-specific accumulation of TPS. Of the three TPS formulations, TPS-loaded GlcA-modified liposomes exhibited the most potent activity by inhibiting PA remodeling and muscularization, decreasing PA medial thickening, suppressing collagen deposition in PAs, and attenuating right ventricle hypertrophy (RVH) in sugen-5416-induced PAH rats. ConclusionsThe GLUT-1-targeted delivery of TPS increased pulmonary specificity and enhanced TPS anti-PAH activities in vivo and in vitro.

BMPR2 promoter methylation and its expression in valvular heart disease complicated with pulmonary artery hypertension.

Valvular heart disease (VHD) is a common heart disease that affects blood flow. It usually requires heart surgery. Valvular heart disease complicated with pulmonary artery hypertension (VHD-PAH) may be lethal due to heart failure that results from increased heart burden. It is important for these patients to seek early treatment in order to minimize the heart damage. However, there is no reliable diagnosis method in VHD. In this study, we found DNA methylation was increased at the promoter of BMPR2 gene in the VHD patients compared with the healthy controls. This finding was confirmed by an independent cohort study of VHD patients and healthy controls. In addition, BMPR2 mRNA levels were reduced in the plasma of the VHD patients. There is strong correlation between BMPR2 promoter DNA methylation and the severity of VHD. Indeed, we found that both BMPR2 promoter DNA methylation and BMPR2 mRNA levels in the plasma are good biomarkers of VHD by themselves, with the respective AUC value of 0.879 and 0.725, respectively. When they were used in combination, the diagnostic value was even better, with the AUC value of 0.93. Consistent with the results in the VHD patients, we observed decreased BMPR2 and increased fibrosis in the lung of a PAH model mouse. BMPR2 was also decreased in the hearts of the PAH mice, whereas BMP4 was increased. Furthermore, BMPR2 was reduced in the heart valve tissue samples of human VHD patients after valve replacement with moderate/severe PAH compared with those with mild PAH. There was also increased apoptosis in the hearts of the PAH mice. BMPR2 promoter DNA methylation and its expression appear to be good biomarkers for VHD. Our results also suggest that DNA methylation may cause PAH through deregulation of BMP signaling and increased apoptosis.

Pulmonary artery radiofrequency ablation in an acute porcine model of pulmonary arterial hypertension

Abstract Introduction Pulmonary hemodynamics improvement after pulmonary artery denervation (PADN) was demonstrated in PAH. Questions arise regarding PADN perioperative effectiveness and the accuracy of the target nerves damage. The aim of the study was to evaluate whether PADN decreases pulmonary artery pressure (PAP) in acute thromboxane A2 (U46619)–induced PAH, and damages PA perivascular nerve fibers. Materials and methods 10 male Landrace swine (34.7±5.1 kg). In 6 swine acute reversible target mean PAP of 40 mm Hg was induced with synthetic thromboxane A2 infusion (U46619). Control group: 4 swine with PADN. Hemodynamics was assessed throughout the study, PAH modeling was done before and 20 min after PADN (radiofrequency energy, 40 Watts), followed by pathology and immunohistochemical studies. Results The mean number of RF applications was 17.5±3.6. Pulmonary embolism (PE) was observed after PADN in 3 swine with U46619 infusion, which were excluded. There was no differences in mPAP, PVR and U46619 dosage after PADN in PAH model (12.3±3.5 vs 12.1±1 mm Hg, p=0.2; 150.4±48.7 vs 129.2±64.1 dynes s cm–5; p=0.2; 24.9±3.3 vs 22.4±4.1 mcg; p=0.18; respectively). Similar hemodynamic results were observed in the control group after PADN (mPAP; p=0.3; PVR; p=0.58). S100 expression was evident in the majority of RFA PA species and in some species loss in tyrosine hydroxylase and M1 acetylcholine receptors expression was detected with no hemodynamic correlation. Conclusions PADN using an electrophysiological catheter with unipolar energy does not lead to an acute PA perivascular nerve fibers destruction and detectable mPAP changes in U46619-induced PAH. Delayed nerve damage might be attributable to PADN effects observed in previous studies. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant from the Ministry of Science and Higher Education of the Russian Federation (agreement #075-15-2020-800).

The thromboxane receptor antagonist NTP42 improves haemodynamics, inhibits remodelling, and restores function within the cardiopulmonary system in preclinical pulmonary arterial hypertension models

Abstract Background Pulmonary arterial hypertension (PAH) is a rare and fatal disease characterised by a progressive increase of pulmonary artery pressure leading to right heart failure. While current PAH therapies provide symptomatic relief and some improvement in quality of life, they show limited benefit in preventing disease progression and often have severe side effects. Recent studies by us reported that a novel thromboxane A2 receptor (TP) antagonist NTP42 attenuated multiple disease hallmarks of PAH in two preclinical models of the disease. Purpose The aim of this study was to assess the efficacy of a novel oral formulation of NTP42 developed for pharmaceutical use on monocrotaline (MCT)-induced PAH in rats across a range of cardiopulmonary parameters, and when compared with standard of care (SOC) PAH therapies. It also aimed to evaluate the expression of the TP, the target receptor for NTP42, in tissues from the MCT-induced PAH model and related disease models. Methods PAH was induced by subcutaneous injection of 60 mg/kg MCT. Rats were randomly assigned to 7 groups: (1) No MCT; (2) MCT Only; (3) MCT+NTP42 Formulation (1 mg/kg); (4) MCT+Sildenafil (50 mg/kg); (5) MCT+Macitentan (30 mg/kg), (6) MCT+Selexipag (1 mg/kg), and (7) MCT+Riociguat (5 mg/kg), where in all cases twice-daily oral dosing was initiated 7 days post-MCT and continued for 22 days. Results The results show that the formulated NTP42 reduced the severity of MCT-induced PAH as determined from haemodynamic measurements of mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP), Fulton's Index of cardiac hypertrophy and histological assessments of pulmonary vascular remodelling, pulmonary oedema, inflammation and fibrosis, and right ventricular fibrosis. In addition, these assessments showed that NTP42 inhibited these effects at least to a similar extent relative to the SOC drugs Sildenafil, Macitentan, Selexipag and Riociguat. Notably, unlike most of the PAH SOCs, NTP42 had no adverse effects on liver weight and or on hepatic histopathology. Importantly, NTP42 had no effects on either the systemic mean arterial pressure (mAP) or heart rate (HR). Furthermore, while abundant expression of the target TP was noted in numerous cell and tissue types of both cardiac and pulmonary tissue, there were highly significant upregulations of TP expression observed following PAH-induction (RV, >2-fold, p=0.0002). Conclusions Taken together, these data demonstrate that NTP42 significantly attenuates MCT-induced PAH and to a similar or greater extent relative to market leader PAH SOC drugs. Notably, we reveal that expression of the TP, the target receptor for NTP42, was shown to be highly significantly upregulated throughout the cardiopulmonary system, validating the potential importance of this largely ignored PAH target ripe for pharmaceutical intervention. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EU Horizon 2020 SME Instrument

Progression of liver fibrosis in pulmonary arterial hypertension

Abstract Background Pulmonary arterial hypertension (PAH) causes congestive liver due to right heart failure. There are few cases of PAH that lead to liver cirrhosis, and little attention is paid to liver function in PAH patients. However, it is certain that long-term congestion due to right heart failure in PAH causes a gradual exacerbation of liver dysfunction and affects metabolic function. Purpose The purpose of this study is to investigate liver fibrosis associated with the severity and prognosis of PAH. Methods This retrospective observational study was included 57 PAH patients and 22 control subjects. PAH patients were assigned to three risk variables according to the simplified risk stratification proposed at the 6thWSPH 2018 after measuring hemodynamic parameters using right heart catheterization, WHO functional class, 6- minutes walking distance (6MWD), and BNP plasma levels. The Fibrosis-4 (FIB4)-index, a liver fibrosis marker, was calculated using the formula: FIB-4 = Age (years) × AST (U/L) / [PLT (109/L) × ALT1/2 (U/L)], and assessed for association with severity of PAH. PAH patients were followed up for 12 years to assess the occurrence of major adverse event, such as death or lung transplantation. Next, adult 8-week-old C57BL/6 mice were exposed to chronic hypoxia (10% O2) or normoxia for 6 weeks. Then, mice were anesthetized and performed right heart catheterization. Liver tissue was collected for histological assessment by Hematoxylin and eosin and Azan staining, and evaluated RNA expression involved in liver fibrosis by real-time PCR. Results The levels of FIB4-index in intermediate and high risk groups of PAH patients had significantly increased compared to those in control group. In PAH patients, FIB4-index was not obviously correlated with hemodynamic parameters, BNP, or 6MWD. Major adverse events occurred in 18 PAH patients (32%): death in 18 (100%) and lung transplant in none (0%). Kaplan-Meier curves for PAH patients with and without major adverse events were constructed based on a cut-off frequency of 2.001 for FIB4-index. During the 12-years follow-up period, major-event-free survival was significantly better in PAH patients with FIB4-index <2.001 than in patients with FIB4-index >2.001 (hazard ratio, 3.3; P=0.038). In a PAH model mice, hemodynamic parameters showed that chronic hypoxia significantly increased the right ventricular systolic pressure. In histological analysis, there was no significantly difference in liver fibrosis in hypoxia or normoxia group. However, the RNA expression such as αSMA and TGFβ1 associated with liver fibrosis in PAH model mice was increased compared to control mice. Conclusion This study showed that the liver fibrosis gradually progressed subsurfacely with severity of PAH. Even the slight liver dysfunction may affect metabolism and cause exacerbation of PAH, so it might be necessary to pay attention to liver fibrosis as one of the risk factors of PAH. Funding Acknowledgement Type of funding sources: None.

PAH Spectroscopy from 1 to 5 μm

Abstract The PAH model predicts many weak emission features in the 1–5 μm region that can resolve significant questions that it has faced since its inception in the mid-80s. These features contain fundamental information about the PAH population that is inaccessible via the much stronger PAH bands in the 5–20 μm region. Apart from the 3.3 μm band and plateau, PAH spectroscopy across most of the 1–5 μm region has been unexplored due to its low intrinsic intensity. ISO and Akari covered some of this wavelength range, but lacked the combined sensitivity and resolution to measure the predicted bands with sufficient fidelity. The spectroscopic capabilities of the NIRSpec instrument on board JWST will make it possible to measure and fully characterize many of the PAH features expected in this region. These include the fundamental, overtone and combination C–D and C ≡ N stretching bands of deuterated PAHs, cyano-PAHs (PAH-C≡N), and the overtones and combinations of the strong PAH bands that dominate the 5–20 μm region. These bands will reveal the amount of D tied up in PAHs, the PAH D/H ratio, the D distribution between PAH aliphatic and aromatic subcomponents, and delineate key stages in PAH formation and evolution on an object-by-object basis and within extended objects. If cyano-PAHs are present, these bands will also reveal the amount of cyano groups tied up in PAHs, determine the N/C ratio within that PAH subset, and distinguish between the bands near 4.5 μm that arise from CD versus C ≡ N.

Osthole improves pulmonary artery hypertension by inducing apoptosis in pulmonary artery smooth muscle cells

The objectives of this study were to explore the effect of Osthole (Ost) on apoptosis in pulmonary artery smooth muscle cells (PASMCs) and investigate the potential mechanism of this effect. Rats were injected subcutaneously with monocrotaline (MCT) to establish a PAH model, and Ost were intragastrically administrated from day 1 to day 35. After 35 days administration, the mean pulmonary artery pressure and lung weight index were measured. HE and TUNEL staining were used to observe the morphology of pulmonary artery and the apoptosis of PASMCs. In addition, the apoptosis of PASMCs were detected by flow cytometry in cultured PASMCs. The proteins of Bax and Bcl-2, and the levels of p-ASK1 and cleaved caspase 3 were measured by Western blot. Ost decreased the mean pulmonary artery pressure and lung weight index in MCT-induced rats, and promoted apoptosis in PASMCs in MCT-induced rats and PDGF-BB stimulated PASMCs. Ost increased the ratio of Bax/Bcl-2 and the levels of p-ASK1, cleaved caspase 3 in MCT-induced rats and PDGF-BB stimulated PASMCs. Ost promoted apoptosis in PASMCs in vivo and in vitro, and the mechanism may be associated with upregulation of ASK1 and the Bax/Bcl-2-caspase 3 signalling pathway.

Abstract 15168: Circulating Cell-free DNA Correlates With Markers of Disease Severity in the Rat Sugen/hypoxia Model of Pulmonary Arterial Hypertension

Introduction: Cell-free DNA (cfDNA) is released into circulation following tissue injury and cell turnover. In cancer and allograft rejection, cfDNA serves as a valuable biomarker for monitoring disease severity, progression, and response to therapy. The goal of the current study is to determine the temporal relationship between plasma cfDNA and disease severity in a PAH rodent model. Methods: Blood collection was performed weekly (weeks 4-11) in both sugen/hypoxia PAH rats (n=12) and age-matched, normoxia controls (n=12). cfDNA was isolated from plasma and concentration was determined by qRT-PCR. Cardiac MRI, cardiac catheterization and necropsy were performed at the end of study. Log-transformed concentrations of cfDNA were compared using linear mixed models. Pearson correlation coefficients were calculated to identify associations between rates of change in cfDNA and measures of disease severity. Results: Plasma cfDNA concentrations were significantly higher in PAH rats beginning at week 5 and continuing through week 10 (P < 0.05 for the comparison at each week; Figure 1). From weeks 4 to 11, there was an overall trend toward increasing cfDNA concentration over time in PAH rats (P = 0.07), whereas concentrations in control rats remained unchanged (P = 0.52). In PAH rats, the rate of change in log-transformed cfDNA concentration correlated inversely with RVEF (r = -0.80, P = 0.003), stroke volume index (r = -0.67, P = 0.02), and positively with total pulmonary resistance index (r = 0.62, P = 0.04). There were non-significant trends between the rate of change in cfDNA concentration and LV end-diastolic volume index (r = -0.54, P = 0.09), and Fulton index (r = 0.55, P = 0.08). Conclusions: Plasma cfDNA is elevated in the rat sugen/hypoxia PAH model and significantly correlated with markers of disease severity. Circulating cfDNA may reflect ongoing pulmonary vascular injury and remodeling, RV strain or both and thus may serve as a novel biomarker of PAH disease progression.

Pulmonary hypertension: reduction of vascular and cardiac remodeling by a new inhibitor of p38-alpha MAPK

Abstract Background Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling with subsequent right ventricular (RV) failure. This work investigated the effects of a new p38-alpha mitogen-activated protein kinase (p38-α MAPK) inhibitor, named LASSBio-1824, on PAH model in rats. Methods All experiments were in accordance with the Animal Care and Use Committee at our university (039/19). Male Wistar rats was exposed to hypoxia (10% O2) during 3 weeks plus i.p. injection of an antagonist of vascular endothelial growth factor receptor (SU5416; 20 mg/kg/week) to induce PAH. Control rats were kept in normoxia (21% O2). After 21 days of protocol, echocardiography images confirmed PAH using the pulmonary artery (PA) outflow waveform. After disease onset, animals were randomly divided into three groups: control + vehicle (DMSO), HAP + vehicle and HAP + LASSBio-1824 (50 mg/kg/day). Vehicle and LASSBio-1824 were administered by oral gavage during two weeks. Results Pulmonary acceleration time (PAT; ms) was reduced from 33.2±2.7 (control) to 22.7±1.1 in PAH + vehicle group (p<0.05) and restored to 29.6±1.9 after treatment with LASSBio-1824 (p<0.05). RV afterload was detected in HAP group because of the increase of systolic pressure (mmHg) from 22.2±1.5 (control) to 47.2±3.6 (p<0.05) and reduced to 18.0±2.9 with LASSBio-1824. Medial wall thickness (%) of distal PA (<50 μm) was measured by using immunohistochemistry for alpha smooth muscle actin (α-SMA) which was increased from 62.5±5.6 (control) to 78.0±7.9 in PAH group (p<0.05), but it was significantly reduced to 54.4±2.3 in PAH + LASSBio-1824 group (p<0.05). Vascular reactivity was evaluated comparing the acetylcholine (ACh)-induced maximal relaxation (%) in PA from normal and PAH rats, which was reduced from 67.5±2.1 to 49.63±5.6 (PAH group; p<0.05). Therefore, the treatment with the p38 inhibitor normalized the ACh-induced response to 73.7±4.1%. The immunohistochemistry for c-fos protein, a product of the p38 pathway related to cell proliferation, showed an increased ratio of stained and total myocyte nuclei (%) in PAH animals of 38.0±0.5 compared to normal group of 20.2±4.2. LASSBio-1824 significantly reduced ratio to 24.0±2.3% (p<0.05). PAH increased inflammatory condition because the expression of inducible nitric oxide synthase increased from 0.31±0.01 (control) to 0.57±0.02 and it was recovered by the treatment with LASSBio-1824 to 0.31±0.10 (p<0.05). Conclusion LASSBio-1824, an inhibitor of p38-alpha MAPK represents an important approach for the future treatment of PAH which improves the underlying remodeling and inflammation processes in the cardiopulmonary system. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES; Conselho Nacional de Desenvolvimento Científico e Tecnolόgico - CNPq

Pyruvate kinase M2 activation protects against the proliferation and migration of pulmonary artery smooth muscle cells.

Pyruvate kinase M2 (PKM2), which is encoded by PKM, is a ubiquitously expressed intracellular protein and is associated with proliferation cell phenotype. In PAH patients and PAH models, we found higher levels of PKM2 tyrosine 105 phosphorylation (phospho-PKM2 (Y105)) than in controls, both in vivo and in vitro. Here, we demonstrate that PKM2 stimulates inflammatory and apoptosis signalling pathways in pulmonary artery smooth muscle cells (PASMCs) and promotes PASMC migration and proliferation. PKM2 phosphorylation promoted the dimerization activation and nuclear translocation of STAT3, a transcription factor regulating proliferation, growth, and apoptosis. TLR2, a transmembrane protein receptor involved in both innate and adaptive immune responses, promoted PKM2 phosphorylation in hypoxia-induced PASMCs. Therefore, we hypothesized that PKM2 also affects the proliferation and migration of PASMCs. The proliferation of hypoxia-induced normal human pulmonary artery smooth muscle cells (normal-HPASMCs) was found to be inhibited by TEPP-46 (PKM2 agonist) and PKM2 siRNA using wound healing, 5-ethynyl-2'-deoxyuridine (EdU), and immunofluorescence (Ki67) assays. PASMCs isolated from PAH patients (PAH-HPASMCs) and hypoxia-treated rats (PAH-RPASMCs) also confirmed the above results. TEPP-46 treatment was found to improve hypoxia-induced pulmonary artery remodelling and right heart function in mice, and the link between PKM2 and STAT3 was also confirmed in vivo. In conclusion, PKM2 plays crucial roles in the proliferation and migration of PASMCs.