Experimental Pulmonary Arterial Hypertension Research Articles

Imaging the TGFβ type I receptor in pulmonary arterial hypertension

Transforming growth factor β (TGFβ) activity is perturbed in remodelled pulmonary vasculature of patients with pulmonary arterial hypertension (PAH), cancer, vascular diseases and developmental disorders. Inhibition of TGFβ, which signals via activin receptor-like kinase 5 (ALK5), prevents progression and development of experimental PAH. The purpose of this study was to assess two ALK5 targeting positron emission tomography (PET) tracers ([11C]LR111 and [18F]EW-7197) for imaging ALK5 in monocrotaline (MCT)- and Sugen/hypoxia (SuHx)-induced PAH. Both tracers were subjected to extensive in vitro and in vivo studies. [11C]LR111 showed the highest metabolic stability, as 46 ± 2% of intact tracer was still present in rat blood plasma after 60 min. In autoradiography experiments, [11C]LR111 showed high ALK5 binding in vitro compared with controls, 3.2 and 1.5 times higher in SuHx and MCT, respectively. In addition, its binding could be blocked by SB431542, an adenosine triphosphate competitive ALK5 kinase inhibitor. However, [18F]EW-7197 showed the best in vivo results. 15 min after injection, uptake was 2.5 and 1.4 times higher in the SuHx and MCT lungs, compared with controls. Therefore, [18F]EW-7197 is a promising PET tracer for ALK5 imaging in PAH.

Novel Loss of Function KCNA5 Variants in Pulmonary Arterial Hypertension.

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in PAH patients, but their functional consequences and potential impact on the disease are largely unknown. Herein, we aimed to characterize the functional consequences of 7 KCNA5 variants found in a cohort of PAH patients. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with WT or mutant Kv1.5 cDNA. Flow cytometry, western blot and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells (hPASMC). KCNA5 variants found in PAH patients (namely, p.Arg184Pro and p.Gly384Arg) resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modelling analyses. The p.Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with p.Arg184Pro or p.Gly384Arg variants decreased apoptosis of hPASMCs compared with the WT, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1 %. Our data indicate that some KCNA5 variants found PAH patients have critical consequences for channel function supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

Sotatercept analog improves cardiopulmonary remodeling and pulmonary hypertension in experimental left heart failure.

Pulmonary hypertension due to left heart disease (PH-LHD) is the most frequent manifestation of PH but lacks any approved treatment. Activin receptor type IIA-Fc fusion protein (ActRIIA-Fc) was found previously to be efficacious in experimental and human pulmonary arterial hypertension (PAH). Here we tested the hypothesis that ActRIIA-Fc improves pulmonary vascular remodeling and alleviates PH in models of PH-LHD, specifically in subtypes of heart failure with reduced ejection fraction (PH-HFrEF) and preserved ejection fraction (PH-HFpEF). Treatment with murine ActRIIA-Fc reduced cardiac remodeling and improved cardiac function in two mouse models of left heart disease without PH, confirming that this inhibitor of activin-class ligand signaling can exert cardioprotective effects in heart failure. In a mouse model of PH-HFrEF with prolonged pressure overload caused by transverse aortic constriction, ActRIIA-Fc treatment significantly reduced pulmonary vascular remodeling, pulmonary fibrosis, and pulmonary hypertension while exerting beneficial structural, functional, and histological effects on both the left and right heart. Additionally, in an obese ZSF1-SU5416 rat model of PH-HFpEF with metabolic dysregulation, therapeutic treatment with ActRIIA-Fc normalized SMAD3 overactivation in pulmonary vascular and perivascular cells, reversed pathologic pulmonary vascular and cardiac remodeling, improved pulmonary and cardiac fibrosis, alleviated PH, and produced marked functional improvements in both cardiac ventricles. Studies in vitro revealed that treatment with ActRIIA-Fc prevents an abnormal, glucose-induced, activin-mediated, migratory phenotype in human pulmonary artery smooth muscle cells, providing a mechanism by which ActRIIA-Fc could exert therapeutic effects in experimental PH-HFpEF with metabolic dysregulation. Our results demonstrate that ActRIIA-Fc broadly corrects cardiopulmonary structure and function in experimental PH-LHD, including models of PH-HFrEF and PH-HFpEF, leading to alleviation of PH under diverse pathophysiological conditions. These findings highlight the important pathogenic contributions of activin-class ligands in multiple forms of experimental PH and support ongoing clinical evaluation of human ActRIIA-Fc (sotatercept) in patients with PH-HFpEF.

Shikonin improves pulmonary vascular remodeling in monocrotaline‑induced pulmonary arterial hypertension via regulation of PKM2.

Pulmonary arterial hypertension (PAH), a fatal disease with an insidious onset and rapid progression, shows characteristics such as increases in pulmonary circulatory resistance and pulmonary arterial pressure, and progressive right heart failure. Shikonin can reduce right ventricular systolic pressure in chronically hypoxic mice. However, the mechanisms underlying the protective effect of shikonin against PAH pathogenesis have only been sporadically identified. The present study evaluated whether inhibiting the expression of pyruvate kinase M2 (PKM2) contributed to the improvement of pulmonary vascular remodeling in PAH rats induced by monocrotaline (MCT) treatment. Hemodynamic parameters were assessed using echocardiography and right ventricular catheterization. Right ventricular hypertrophy index analysis and hematoxylin and eosin staining were used to evaluate the degree of pulmonary vascular and right heart remodeling. Moreover, PKM2, p‑PKM2, ERK, p‑ERK, glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA) protein expression levels were semi‑quantified using western blotting. The expression and distribution of PKM2 were assessed using immunofluorescence microscopy. The present study demonstrated that MCT treatment caused pulmonary arterial hypertension and pulmonary vascular remodeling in experimental rats. Shikonin improved hemodynamics and pulmonary vascular remodeling in MCT‑induced PAH rats, decreased aerobic glycolysis and downregulated PKM2, p‑PKM2, p‑ERK, GLUT 1 and LDHA protein expression levels. Shikonin improved experimental pulmonary arterial hypertension hemodynamics and pulmonary vascular remodeling at least partly through the inhibition of PKM2 and the resultant suppression of aerobic glycolysis. These results provide a novel understanding of possible new treatment targets for PAH.

β3 adrenergic agonism: A novel pathway which improves right ventricular-pulmonary arterial hemodynamics in pulmonary arterial hypertension.

Efficacy of therapies that target the downstream nitric oxide (NO) pathway in pulmonary arterial hypertension (PAH) depends on the bioavailability of NO. Reduced NO level in PAH is secondary to "uncoupling" of endothelial nitric oxide synthase (eNOS). Stimulation of β3 adrenergic receptors (β3 ARs) may lead to the recoupling of NOS and therefore be beneficial in PAH. We aimed to examine the efficacy of β3 AR agonism as a novel pathway in experimental PAH. In hypoxia (5 weeks) and Sugen hypoxia (hypoxia for 5 weeks + SU5416 injection) models of PAH, we examined the effects of the selective β3 AR agonist CL316243. We measured echocardiographic indices and invasive right ventricular (RV)-pulmonary arterial (PA) hemodynamics and compared CL316243 with riociguat and sildenafil. We assessed treatment effects on RV-PA remodeling, oxidative stress, and eNOS glutathionylation, an oxidative modification that uncouples eNOS. Compared with normoxic mice, RV systolic pressure was increased in the control hypoxic mice (p < 0.0001) and Sugen hypoxic mice (p < 0.0001). CL316243 reduced RV systolic pressure, to a similar degree to riociguat and sildenafil, in both hypoxia (p < 0.0001) and Sugen hypoxia models (p < 0.03). CL316243 reversed pulmonary vascular remodeling, decreased RV afterload, improved RV-PA coupling efficiency and reduced RV stiffness, hypertrophy, and fibrosis. Although all treatments decreased oxidative stress, CL316243 significantly reduced eNOS glutathionylation. β3 AR stimulation improved RV hemodynamics and led to beneficial RV-PA remodeling in experimental models of PAH. β3 AR agonists may be effective therapies in PAH.

Beneficial effects of berberine against pulmonary complications of experimental pulmonary arterial hypertension in rats and some relevant mechanisms.

Pulmonary arterial hypertension (PAH) is a severe disease that leads to pulmonary vascular remodeling characterized by a rise in pulmonary vascular resistance and pressure. We assessed the effects of an herbal compound, berberine (BB), and some related mechanisms on PAH in rats. Male Wistar rats were assigned to seven groups: control, monocrotaline (MCT), MCT+vehicle, and MCT+BB (with doses of 10, 20, 30, and 40 mg/kg) groups. Three weeks after induction of PAH by MCT, treatment groups received daily intraperitoneal injections of vehicle or BB for 3 weeks. On Day 43, the right ventricular systolic pressure (RVSP, as an index of pulmonary arterial pressure) and the ratio of RV to LV+septum weight (as RV hypertrophy index, right ventricle hypertrophy [RHVI]) were measured. Inflammatory and oxidative stress indices and histopathology of the lungs were also assessed. RVSP (89.4 ± 8.2 vs. 23 ± 3.3), RVHI (0.63 ± 0.08 vs. 0.26 ± 0.04), and lung inflammatory cytokines TNF-α (2.03 ± 0.25 vs. 1.21 ± 0.3) and IL-6 (8.8 ± 0.59 vs. 6.3 ± 0.95) significantly increased in the MCT group compared to the control group. MCT also raised the level of Malondialdehyde (0.11 ± 0.01 vs. 0.09 ± 0.01) and diminished total antioxidant capacity (6.5 ± 0.51 vs. 8.3 ± 0.62), the activity of superoxide dismutase (1.19 ± 0.22 vs. 1.93 ± 0.2), glutathione peroxidase (0.02 ± 0.002 vs. 0.03 ± 0.005), catalase (2.1 ± 0.29 vs. 2.8 ± 0.20) and Bax/Bcl-2 ratio (0.41 ± 0.07 vs. 0.61 ± 0.09) in the lungs. Treatment with BB significantly recovered all of these alterations. BB ameliorated pulmonary vascular remodeling by decreasing inflammation and fibrosis and increasing apoptosis and antioxidant/oxidant balance. Therefore, this herbal derivative may be considered a therapeutic goal against PAH.

The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction. The effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM). In the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM. This study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions.

Necroptosis, unlike pyroptosis mediates right ventricular damage in experimental pulmonary arterial hypertension

Necroptosis, unlike pyroptosis mediates right ventricular damage in experimental pulmonary arterial hypertension

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.

Abstract 15069: The Rna M 6 A Demethylase, Fto, Regulates Vascular Remodeling in the Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative pulmonary vascular remodeling and progressive elevation of pulmonary vascular resistance that leads to right heart failure and premature death. RNA modifications such as N6-methyladenosine (m 6 A) has have recently been discovered as essential regulators of gene expression but its function in regulating the development of PAH remain unclear. Hypothesis: Fat mass and obesity-associated protein (FTO), as a major m 6 A eraser, acts as a key regulator of endothelial cell (EC) dysfunction and pulmonary vascular remodeling and, thus, is a novel therapeutic target for PAH. Methods: To determine the FTO expression on the idiopathic PAH (IPAH) patients, we performed Western blotting and immunofluorescence on lung tissue from IPAH patients. A novel mouse model with Tie2-Cre-mediated deletion of Fto in endothelial cells was generated and treated with hypoxia for 3 weeks to induce experimental PAH. 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. To evaluate the translational potential of targeting FTO in PAH, rats were exposed to monocrotaline to induce pulmonary hypertension and then were treated with FTO inhibitor FB23-2. RVSP, RV/(LV+S) ratio and histology assessment were measured subsequently. Results: FTO expression level is markedly elevated in the endothelial cells of IPAH patients. The Tie2-Cre-Fto (CKO) mice exhibited inhibited PAH under hypoxia treatment as evident by reduced RVSP and lower RV/(LV+S) ratio. Histological and immunofluorescent staining showed that media layer of pulmonary arteries had less cell proliferation and reduced pulmonary vascular remodeling. Moreover, preclinical MCT-rat PAH model showed attenuated PAH phenotype as well as pulmonary vascular remodeling with pharmacological inhibition of FTO. Conclusions: These studies demonstrate that FTO expression markedly elevates in the EC of IPAH patients. Tie2-Cre mediated loss of FTO protects mice from hypoxia-induced PAH. Thus, targeting FTO is a promising therapeutic strategy for PAH.

Abstract 10909: A Novel ATP Citrate Lyase-Dependent Metabolic-Transcriptional Axis in Pulmonary Arterial Hypertension

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary artery smooth muscle (PASMC) proliferation and suppressed apoptosis. In cancer, the sustainability of this phenotype required metabolic adaptation (Warburg effect / lipid synthesis) influencing chromatin structure and gene expression. By producing Acetyl coA the ATP Citrate Lyase (ACLY) has emerged as a key player in cancer by favoring Warburg effect, lipid synthesis and histone acetylation. We hypothesized that ACLY is upregulated in PAH and supports PAH-PASMC proliferation and contributes to PA remodelling. METHODS/RESULTS: The implication of ACLY in PAH was demonstrated by 1) an upregulation (n=10 p&lt;0.05) of its expression, activation (P-ACLY) and nuclear localization in human PAH lungs, PASMCs and experimental PAH models (sugen/hypoxia, Su/Hx rats and mice) compared to control (immunoblot/immunofluorescence); 2) mice with ACLY deleted in SMCs did not develop Su/Hx-induced PA remodelling and PAH (n=20 p&lt;0.05). Mechanistically, ACLY inhibition (siRNA/BMS) decreased proliferation (Ki67/PCNA) and increased apoptosis (AnnexinV / Survivin) in PAH-PASMCs (n=8 p&lt;0.05). These effects were associated with the inhibition of multiple genes linked to cell cycle, metabolism and lipid synthesis (RNAseq p&lt;0.05 n=6) leading to a decrease (p&lt;0.05) in Warburg effect (LDH, PKM2, P-PDH) and an increase in OCR/ECAR (seahorse). These findings were coupled with an inhibition of the histone acetyl-transferase P300 and histones H3/H4 acetylation. Motif enrichment analyses of transcriptomic datasets identified NFY, NFAT and FOXM1 (all involved in PAH and P300 regulated) as putative transcription factors implicated in ACLY-dependent gene regulation in PAH. This will be confirmed by CHIPseq/ATACseq. In vivo, effects of ACLY inhibitors (BMS/ETC1002) were assessed in rats with Su/Hx-PAH. Both decreased PA pressure, resistance, wall thickness and improved right ventricular functions (n=20; p&lt;0.05) (Echo/catheterization). CONCLUSION: We uncovered a novel ACLY-dependent metabolic-transcriptional axis explaining how PASMCs proliferate and survive in PAH. ACLY inhibition is an attractive therapeutic strategy to improve PAH as ETC1002 is clinically advanced.

Therapeutic effects of nebulization-based delivery of anti-miR-146a in experimental pulmonary arterial hypertension

Abstract Pulmonary arterial hypertension (PAH), is a chronic disorder characterized by excessive pulmonary vascular remodelling, resulting in elevated pulmonary vascular resistance and right ventricle (RV) overload and failure. PAH remains incurable, and new therapeutic approaches are required. The microRNA-146a (miR-146a) promotes vascular smooth muscle cell proliferation and vascular neointimal hyperplasia, both important hallmarks of PAH. This study aimed to investigate the role of miR-146a in the pathophysiology of PAH and in the progression to RV hypertrophy and failure. Sprague Dawley rats received a subcutaneous injection of monocrotaline (MCT group) or vehicle (CTRL group) and, after fourteen days they were treated, by intratracheal nebulization, with an anti-miR-146a, forming four distinct groups: CTRL-treated; CTRL-untreated, MCT-treated, and MCT-untreated. Twenty-seven days after MCT injection, echocardiographic and invasive hemodynamic evaluations were performed in all animal groups. Also, wild-type (WT) and miR-146a knock-out (KO) (miR-146a−/−) mice were submitted to either 3 weeks of chronic hypoxia-induced PAH with weekly Sugen 5416 administration (SuHx). Compared to MCT-untreated rats, the MCT-treated rats showed decreased RV hypertrophy, (as measured by the Fulton index),decreased RV end-diastolic diameter/left ventricle end-diastolic diameter (RVEDD/LVEDD) ratio, and decreased in RV diastolic pressures. KO-SuHx group, when compared to WT-SuHx group, showed decreased RV hypertrophy (as measured by the Fulton index), and decreased RV systolic pressures and dilation, as well as lower RV end end-diastolic diameter (RVEDDi) and right atria area (RAAi). Our findings show that miR-146a pharmacological or genetic inhibition reduces RV remodelling and improves cardiac function. Thus, miR-146a represents a promising therapeutic target in PAH. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This research was supported by the Portuguese Foundation for Science and Technology (FCT).

Mineralocorticoid Receptor Antagonism by Finerenone Attenuates Established Pulmonary Hypertension in Rats.

We studied the ability of the nonsteroidal MR (mineralocorticoid receptor) antagonist finerenone to attenuate vascular remodeling and pulmonary hypertension using two complementary preclinical models (the monocrotaline and sugen/hypoxia rat models) of severe pulmonary hypertension. We first examined the distribution pattern of MR in the lungs of patients with pulmonary arterial hypertension (PAH) and in monocrotaline and sugen/hypoxia rat lungs. Subsequent studies were performed to explore the effect of MR inhibition on proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. To validate the functional importance of MR activation in the pulmonary vascular remodeling characteristic of pulmonary hypertension, mice overexpressing human MR (hMR+) were studied, and curative treatments with finerenone (1 mg/kg per day by gavage), started 2 weeks after monocrotaline injection or 5 weeks after Sugen injection were realized. We demonstrated that MR is overexpressed in experimental and human PAH and that its inhibition following small interfering RNA-mediated MR silencing or finerenone treatment attenuates proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. In addition, we obtained evidence that hMR+ mice display increased right ventricular systolic pressure, right ventricular hypertrophy, and remodeling of pulmonary arterioles. Consistent with these observations, curative treatments with finerenone partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and vascular remodeling. Finally, we found that continued finerenone treatment decreases inflammatory cell infiltration and vascular cell proliferation in monocrotaline and sugen/hypoxia rat lungs. Finerenone treatment appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.

Exercise training counteracts the cardiac metabolic remodelling induced by experimental pulmonary arterial hypertension

Exercise training provides several cardiovascular benefits in both physiological and pathological conditions; however, its use as a therapeutic tool for pulmonary arterial hypertension (PAH) has been poorly explored. This study aimed to extend the comprehension of the cardioprotective effects of exercise training in the set of PAH focusing on the metabolic changes promoted by exercise in the right ventricle (RV). The monocrotaline animal model of PAH was used and male Wistar rats were submitted to two weeks of treadmill exercise training (5 days/week, 60 min/day, 25 m/min) following disease establishment. Trained rats showed an improved diastolic function (lower end-diastolic pressure and tau) despite the presence of cardiac overload (increased peak systolic pressure, end-diastolic pressure and arterial elastance). This enhanced hemodynamic response was paralleled by an increased uptake of glucose to cardiomyocytes through glucose transporter type 4 (GLUT4) followed by increased lactate dehydrogenase (LDH) activity. Exercise did not reverse the decrease of fatty acid oxidation related to PAH but increased the content of the transcription factors peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPAR-γ). Two weeks of exercise did not modulate the changes in amino acid metabolism secondary to PAH. Our work suggests that continuous aerobic exercise of moderate intensity, despite its short-term duration and application in a late stage of the disease, supports the RV response to PAH by promoting a shift in the cardiac metabolic phenotype.

Pirfenidone ameliorates pulmonary arterial pressure and neointimal remodeling in experimental pulmonary arterial hypertension by suppressing NLRP3 inflammasome activation.

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased pulmonary arterial pressure, inflammation, and neointimal remodeling of pulmonary arterioles. Serum levels of interleukin (IL)‐1β and IL‐18 are elevated in PAH patients and may enhance proinflammatory neointimal remodeling. NLRP3 inflammasome activation induces cleavage of the cytokines IL‐1β and IL‐18, required for their secretion. Pirfenidone (PFD), an antifibrotic and anti‐inflammatory drug, has been suggested to inhibit NLRP3 inflammasome activation. We hypothesized that PFD delays the progression of PAH by suppressing NLRP3 inflammasome activation. We assessed the effects of PFD treatment in a rat model for neointimal PAH induced by monocrotaline and aortocaval shunt using echocardiographic, hemodynamic, and vascular remodeling parameters. We measured inflammasome activation by NLRP3 immunostaining, Western blots for caspase‐1, IL‐1β, and IL‐18 cleavage, and macrophage IL‐1β secretion. PFD treatment ameliorated pulmonary arterial pressure, pulmonary vascular resistance, and pulmonary vascular remodeling in PAH rats. In PAH rats, immunostaining of NLRP3 in pulmonary arterioles and caspase‐1, IL‐1β, and IL‐18 cleavage in lung homogenates were increased compared to controls, reflecting NLRP3 inflammasome activation in vivo. PFD decreased IL‐1β and IL‐18 cleavage, as well as macrophage IL‐1β secretion in vitro. Our studies show that PFD ameliorates pulmonary hemodynamics and vascular remodeling in experimental PAH. Although PFD did not affect all NLRP3 inflammasome parameters, it decreased IL‐1β and IL‐18 cleavage, the products of NLRP3 inflammasome activation that are key to its downstream effects. Our findings thus suggest a therapeutic benefit of PFD in PAH via suppression of NLRP3 inflammasome activation.

Loss of cAbl Tyrosine Kinase in Pulmonary Arterial Hypertension Causes Dysfunction of Vascular Endothelial Cells.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. cAbl (non-receptor tyrosine kinase c-Abelson) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is downactivated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from patients with PAH (PAH-ECs). We found cAbl and phosphorylated cAbl concentrations to be lower in the endothelium of remodeled pulmonary vessels in the lungs of patients with PAH than in control subjects. Similar observations were obtained for the lungs of Sugen + hypoxia and monocrotaline rats with established pulmonary hypertension. These in situ abnormalities were also replicated invitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA interference in control ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with 5-(1,3-diaryl-1H-pyrazol-4-yl) hydantoin reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of cross-talk between cAbl and bone morphogenetic protein receptor type II. This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension Kosmas Kosmas1,2, Zoe Michael2,3, Fotios Spyropoulos1,2, Jeffrey Widrick2,4, Ravi Jasuja2, Aimilia Papathanasiou1,2, Helen Christou 1,21Department of Pediatric Newborn Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA 2Harvard Medical School, Boston, MA 02215, USA 3Department of Pediatrics, Boston Children’s Hospital, Boston, MA 02215, USA 4Department of Genetics, Boston Children’s Hospital, Boston, MA 02215,

BackgroundPulmonary arterial hypertension (PAH), is a progressive disease that seriously compromises quality of life due to dyspnea and fatigue on exertion that worsens over time such that patients are ultimately immobile. Although hemodynamic compromise is the main contributor to exercise intolerance in PAH, accumulating evidence supports that intrinsic skeletal muscle dysfunction also occurs and may contribute to this symptomatology. We previously showed that treatment with the carbonic anhydrase inhibitor Acetazolamide (ACTZ) ameliorates the hemodynamic components of experimental PAH but its effects on skeletal muscle function and exercise tolerance are unknown. Our objective is to characterize the mechanisms of skeletal muscle dysfunction in a preclinical model of severe PAH and evaluate the effect of ACTZ treatment.HypothesesWe hypothesized that reduced exercise endurance in experimental PAH is associated with increased proportion of fast glycolytic (Type II) fibers to slow oxidative (Type I) fibers, and sarcomeric dysfunction in skeletal muscles and that treatment with ACTZ, alters muscle fiber specificity, improves skeletal muscle contractile properties and exercise tolerance.MethodsWe used the adult rat Sprague Dawley sugen/hypoxia (Su/Hx) model of severe pulmonary hypertension and performed hemodynamic and exercise tolerance studies (treadmill). We evaluated the abundance of Type I and Type II fiber specific markers and sarcomeric organization in muscles from Su/Hx and ACTZ treated rats and measured twitch and tetanic forces, contraction and relaxation times in diaphragm, soleus and extensor digitorum longus (EDL). We used the rat myoblast cell line (L6) to evaluate the molecular mechanisms of muscle fiber switch using immunofluorescence, RNA and protein expression studies.ResultsWe found that diaphragm and soleus muscle from the adult rat sugen/hypoxia (Su/Hx) model of severe PAH have increased abundance of type II fiber markers and thick sarcomeric aggregates and these changes are accompanied by decreased twitch and tetanic forces, contraction and half‐relaxation time and exercise endurance despite absence of muscle atrophy. Acetazolamide (ACTZ), treatment restored the above derangements. In vitro rat myotube differentiation was associated with a predominance of type II fiber markers in conjunction with increased abundance of the transcription factor FoxO1. This pattern of increased abundance of FoxO1 and increased type II markers was also seen in Su/Hx soleus muscles and was reversed in response to ACTZ treatment.ConclusionsWe conclude that a shift in skeletal muscle fiber type specification may underlie skeletal muscle dysfunction in experimental PAH and that treatment with ACTZ may ameliorate skeletal muscle dysfunction through an inhibitory effect on FoxO1‐mediated fiber type specification.

PDZ‐Binding Kinase Drives Pulmonary Artery Smooth Muscle Proliferation and Vascular Remodeling in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive disease that is caused by high blood pressure in the lungs that originates from increased resistance of small pulmonary arteries (PA). A defining characteristic of PAH is vascular smooth muscle cell (VSMC) proliferation and remodeling of PA, that eventually results in right ventricular failure and death. The hyperproliferative nature of vascular cells in PAH shares many characteristics with cancer cells such as sustained proliferative signaling, changes in hypoxia signaling and cellular metabolism. Using microarray analysis, we discovered a novel gene, PDZ‐Binding Kinase (PBK) that is upregulated in hypertensive PA. PBK is a serine/threonine kinase that has important roles in mitosis and cell proliferation. Also, elevated expression of PBK is associated with numerous aggressive cancers suggesting that it may be an important contributor to PAH. Our hypothesis is that PBK has a functional role in the pathogenesis of PAH but the therapeutic utility of targeting PBK in PAH and the mechanisms by which PBK promotes pulmonary vascular remodeling have not been studied. To investigate the role of PBK in PAH, we performed immunostaining, and found that PBK was highly upregulated in pulmonary arterial smooth muscle in the lungs of two experimental PAH rat models (MCT; Su/H), in the Su/H mouse model, as well as in human PAs and lungs with PAH. Furthermore, human pulmonary arterial smooth cells (HPASMCs) overexpressing PBK showed increased proliferation, while silencing PBK or in vitro pharmacological inhibition significantly reduced HPASMC proliferation. To determine whether PBK plays an important role in PA remodeling and PAH in vivo, we next utilized PBK inhibitor (OTS‐514) to treat MCT‐induced PAH rats in a reversal treatment method and Su/H Rats and Mouse in a preventative treatment. In all of the PAH animal models, right ventricle (RV) thickness, velocity time integral (VTI, index of PA stiffness), right ventricular systolic pressure (RVSP), and the Fulton Index were significantly improved in the presence of the PBK inhibitor. To complement our pharmacological studies with a genetic approach, we have generated PBK KO rats to investigate whether genetic ablation of PBK protects against the development of PAH. Taken together, these data support the hypothesis, that PBK drives pulmonary artery smooth muscle proliferation leading to abnormal vascular remodeling and the development of PAH. Our findings advance the utility of novel therapeutic approaches targeting PBK to improve the morbidity and mortality associated with PAH.

Altered gut microbiota and its association with inflammation in patients with chronic thromboembolic pulmonary hypertension: a single-center observational study in Japan

BackgroundThe pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) is considered to be associated with chronic inflammation; however, the underlying mechanism remains unclear. Recently, altered gut microbiota were found in patients with pulmonary arterial hypertension (PAH) and in experimental PAH models. The aim of this study was to characterize the gut microbiota in patients with CTEPH and assess the relationship between gut dysbiosis and inflammation in CTEPH.MethodsIn this observational study, fecal samples were collected from 11 patients with CTEPH and 22 healthy participants. The abundance of gut microbiota in these fecal samples was assessed using 16S ribosomal ribonucleic acid (rRNA) gene sequencing. Inflammatory cytokine and endotoxin levels were also assessed in patients with CTEPH and control participants.ResultsThe levels of serum tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and macrophage inflammatory protein (MIP)-1α were elevated in patients with CTEPH. Plasma endotoxin levels were significantly increased in patients with CTEPH (P < 0.001), and were positively correlated with TNF-α, IL-6, IL-8, and MIP-1α levels. The 16S rRNA gene sequencing and the principal coordinate analysis revealed the distinction in the gut microbiota between patients with CTEPH (P < 0.01) and control participants as well as the decreased bacterial alpha-diversity in patients with CTEPH. A random forest analysis for predicting the distinction in gut microbiota revealed an accuracy of 80.3%.ConclusionThe composition of the gut microbiota in patients with CTEPH was distinct from that of healthy participants, which may be associated with the elevated inflammatory cytokines and endotoxins in CTEPH.

Analysis of necroptosis and its association with pyroptosis in organ damage in experimental pulmonary arterial hypertension.

In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group—sacrificed 4 weeks after MCT administration and ptMCT group—prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr231/Ser232‐RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr231/Ser232‐RIP3 levels in both PAH subgroups were associated rather with GSDMD‐mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH‐induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr231/Ser232‐RIP3 upregulation resulting in two different necrosis‐like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.