Unraveling the Mfn2-Warburg effect nexus: a therapeutic strategy to combat pulmonary arterial hypertension arising from catch-up growth after IUGR.

BACKGROUND: The interplay between intrauterine and early postnatal environments has been associated with an increased risk of cardiovascular diseases in adulthood, including pulmonary arterial hypertension (PAH). While emerging evidence highlights the crucial role of mitochondrial pathology in PAH, the specific mechanisms driving fetal-originated PAH remain elusive. METHODS AND RESULTS: To elucidate the role of mitochondrial dynamics in the pathogenesis of fetal-originated PAH, we established a rat model of postnatal catch-up growth following intrauterine growth restriction (IUGR) to induce pulmonary arterial hypertension (PAH). RNA-seq analysis of pulmonary artery samples from the rats revealed dysregulated mitochondrial metabolic genes and pathways associated with increased pulmonary arterial pressure and pulmonary arterial remodeling in the RC group (postnatal catch-up growth following IUGR). In vitro experiments using pulmonary arterial smooth muscle cells (PASMCs) from the RC group demonstrated elevated proliferation, migration, and impaired mitochondrial functions. Notably, reduced expression of Mitofusion 2 (Mfn2), a mitochondrial outer membrane protein involved in mitochondrial fusion, was observed in the RC group. Reconstitution of Mfn2 resulted in enhanced mitochondrial fusion and improved mitochondrial functions in PASMCs of RC group, effectively reversing the Warburg effect. Importantly, Mfn2 reconstitution alleviated the PAH phenotype in the RC group rats. CONCLUSIONS: Imbalanced mitochondrial dynamics, characterized by reduced Mfn2 expression, plays a critical role in the development of fetal-originated PAH following postnatal catch-up growth after IUGR. Mfn2 emerges as a promising therapeutic strategy for managing IUGR-catch-up growth induced PAH.

Mitochondria as a primary determinant of angiogenic modality in pulmonary arterial hypertension.

Impaired pulmonary angiogenesis plays a pivotal role in the progression of pulmonary arterial hypertension (PAH) and patient mortality, yet the molecular mechanisms driving this process remain enigmatic. Our study uncovered a striking connection between mitochondrial dysfunction (MD), caused by a humanized mutation in the NFU1 gene, and severely disrupted pulmonary angiogenesis in adult lungs. Restoring the bioavailability of the NFU1 downstream target, lipoic acid (LA), alleviated MD and angiogenic deficiency and rescued the progressive PAH phenotype in the NFU1G206C model. Notably, significant NFU1 expression and signaling insufficiencies were also identified in idiopathic PAH (iPAH) patients' lungs, emphasizing this study's relevance beyond NFU1 mutation cases. The remarkable improvement in mitochondrial function of PAH patient-derived pulmonary artery endothelial cells (PAECs) following LA supplementation introduces LA as a potential therapeutic approach. In conclusion, this study unveils a novel role for MD in dysregulated pulmonary angiogenesis and PAH manifestation, emphasizing the need to correct MD in PAH patients with unrecognized NFU1/LA deficiency.

The Impact of Highly Selective Thoracic Sympathectomy on the Progression of Monocrotaline-induced Pulmonary Arterial Hypertension in Rats.

ABSTRACT: Pulmonary arterial hypertension (PAH) is characterized by persistently elevated pulmonary artery pressure and vascular resistance. Sympathetic overactivity in hypertension participates in pulmonary vascular remodeling and heart failure. The present study aims to explore the efficacy of highly selective thoracic sympathectomy (HSTS) on lowering pulmonary artery pressure, reversing pulmonary vascular remodeling, and improving right ventricular function in rats. A total of 24 Sprague-Dawley rats were randomly assigned into the control group ( n = 8) and experimental group ( n = 16). Rats in the control group were intraperitoneally injected with 0.9% normal saline, and those in the experimental group were similarly administered with received monocrotaline (MCT) injections at 60 mg/kg. Two weeks later, rats in the experimental group were further subdivided randomly into the MCT-HSTS group ( n = 8) and MCT-sham group ( n = 8), and they were surgically treated with HSTS and sham operation, respectively. Two weeks later, significantly lowered mean pulmonary artery pressure (mPAP), pulmonary artery systolic pressure (sPAP), and the ratio of sPAP to femoral artery systolic pressure (sFAP) were detected in the MCT-HSTS group than those of the MCT-sham group. In addition, rats in the MCT-HSTS group presented a significantly lower ratio of vascular wall area to the total vascular area (WT%), right ventricular hypertrophy index, and degrees of right ventricular fibrosis and lung fibrosis in comparison to those of the MCT-sham group. HSTS significantly downregulated protein levels of inflammasomes in pulmonary artery smooth muscle cells (PASMCs). Collectively, HSTS effectively reduces pulmonary artery pressure, pulmonary arteriolar media hypertrophy, and right ventricular hypertrophy in MCT-induced PAH rats. It also exerts an anti-inflammatory effect on PASMCs in PAH rats by suppressing inflammasomes and the subsequent release of inflammatory cytokines.

The protective mechanism of sevoflurane in pulmonary arterial hypertension via downregulation of TRAF6.

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy that, if not promptly treated, culminates in right heart failure. Therefore, pre-clinical studies are needed to support and optimize therapeutic approaches of PAH. Here, we explore a prospective function of sevoflurane in experimental PAH through regulating TRAF6. Monocrotaline (MCT)-induced PAH rats were subjected to sevoflurane inhalation and intratracheal instillation of lentivirus overexpressing TRAF6. Platelet-derived growth factor (PDGF)-treated pulmonary artery smooth muscle cells (PASMCs) were exposed to sevoflurane and genetically manipulated for TRAF6 overexpression. It was found that MCT and PDGF challenge upregulated the levels of TRAF6 in rat lung tissues and PASMCs, but sevoflurane treatment led to reduced TRAF6 expression. Sevoflurane inhalation in MCT-induced rats resulted in alleviative pulmonary vascular remodeling, mitigated right ventricular dysfunction and hypertrophy, improved mitochondrial function and dynamics, and inactivation of NF-κB pathway. In vitro studies confirmed that exposure to sevoflurane repressed PDGF-induced proliferation, migration, and phenotype switching of PASMCs, and suppressed mitochondrial dysfunction and NF-κB activation in PDGF-stimulated PASMCs. The beneficial impact of sevoflurane on pathological changes of lung and cell phenotype of PASMCs were reversed by overexpression of TRAF6. In summary, our study suggested the protective properties of sevoflurane in targeting PAH by downregulating TRAF6 expression, providing a novel avenue for the management of PAH.

MFN2-dependent mitochondrial dysfunction contributes to Relm-ß-induced pulmonary arterial hypertension via USP18/Twist1/miR-214 pathway.

Induction of resistin-like molecule ß (Relm-ß) and mitofusin 2 (MFN2) mediated aberrant mitochondrial fission have been found to be involved in the pathogenesis of pulmonary arterial hypertension (PAH). However, the molecular mechanisms underlying Relm-ß regulation of MFN2 therefore mitochondrial fission remain unclear. This study aims to address these issues. Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. The results showed that Relm-ß promoted cells proliferation in PASMCs, this was accompanied with the upregulation of USP18, Twist1 and miR-214, and downregulation of MFN2. We found that Relm-ß increased USP18 expression which in turn raised Twist1 by suppressing its proteasome degradation. Elevation of Twist1 increased miR-214 expression and then reduced MFN2 expression and mitochondrial fragmentation leading to PASMCs proliferation. In vivo study, we confirmed that Relm-ß was elevated in MCT-induced PAH rat model, and USP18/Twist1/miR-214/MFN2 axis was altered similar as in vitro. Targeting this cascade by Relm-ß receptor inhibitor Calhex231, proteasome inhibitor MG-132, Twist1 inhibitor Harmine or miR-214 antagomiR prevented the development of pulmonary vascular remodeling and therefore PAH in MCT-treated rats. In conclusion, we demonstrate that Relm-ß promotes PASMCs proliferation and vascular remodeling by activating USP18/Twist1/miR-214 dependent MFN2 reduction and mitochondrial fission, suggesting that this signaling pathway might be a promising target for management of PAH.

Animal models of pulmonary arterial hypertension associated with atrial septal defect.

Pulmonary arterial hypertension (PAH) is a well-known complication of congenital heart disease (CHD). The lack of a satisfactory animal model for PAH associated with CHD (PAH-CHD) has limited progress in understanding the pathogenesis of PAH and the development of therapeutic agents. The development of a rat model for PAH associated with atrial septal defect (ASD) was achieved through atrial septal puncture and thermal ablation. Two and 4 weeks after modeling, hematoxylin and eosin staining showed that the vascular thickness, vascular thickness index, vascular area, and vascular area index in pulmonary arteries with an outer diameter of 50-300 µm in the PAH-ASD 2 and 4 weeks group were higher than those in the sham group (all P < 0.05). Alpha-smooth muscle actin (ɑ-SMA) staining showed that the medial thickness, medial thickness index, medial area, and medial area index in pulmonary arteries with an outer diameter of 50-300 µm at 2 and 4 weeks after modeling were significantly higher than those in the sham group (all P < 0.05). Additionally, mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR) in the PAH-ASD 2 and 4 weeks groups were significantly higher than those in the sham group (both P < 0.05). Elastin van Gieson staining showed that the vascular obstruction score in the PAH-ASD 2 and 4 weeks group was significantly higher than that in the sham group (both P < 0.05). The PAH-ASD rats were successfully generated. These findings suggest that our model would be useful for further research into the pathogenesis, prevention, and treatment of PAH-ASD.

Cell Death in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by increased pulmonary vascular resistance because of vascular remodeling and vasoconstriction. Subsequently, PAH leads to right ventricular hypertrophy and heart failure. Cell death mechanisms play a significant role in development and tissue homeostasis, and regulate the balance between cell proliferation and differentiation. Several basic and clinical studies have demonstrated that multiple mechanisms of cell death, including pyroptosis, apoptosis, autophagy, ferroptosis, anoikis, parthanatos, and senescence, are closely linked with the pathogenesis of PAH. This review summarizes different cell death mechanisms involved in the death of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs), the primary target cells in PAH. This review summarizes the role of these cell death mechanisms, associated signaling pathways, unique effector molecules, and various pro-survival or reprogramming mechanisms. The aim of this review is to summarize the currently known molecular mechanisms underlying PAH. Further investigations of the cell death mechanisms may unravel new avenues for the prevention and treatment of PAH.

The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease.

BACKGROUND: Due to a special hemodynamic feature, pulmonary vascular disease in pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD) has two stages: reversible and irreversible. So far, the mechanism involved in the transition from reversible to irreversible stage is elusive. Moreover, no recognized and reliable assessments to distinguish these two stages are available. Furthermore, we found that compared with control and reversible PAH, thrombospondin-4 (THBS4) was significantly upregulated in irreversible group by bioinformatic analysis. Hence, we further verify and investigate the expression and role of THBS4 in PAH-CHD. METHODS: We established the monocrotaline plus aorto-cava shunt-induced (MCT-AV) rat model. We measured the expression of THBS4 in lung tissues from MCT-AV rats. Double immunofluorescence staining of lung tissue for THBS4 and α-SMA (biomarker of smooth muscle cells) or vWF (biomarker of endothelial cells) to identify the location of THBS4 in the pulmonary artery. Primary pulmonary artery smooth muscle cells (PASMCs) were cultivated, identified, and used in this study. THBS4 was inhibited and overexpressed by siRNA and plasmid, respectively, to explore the effect of THBS4 on phenotype transformation, proliferation, apoptosis, and migration of PASMCs. The effect of THBS4 on pulmonary vascular remodeling was evaluated in vivo by adeno-associated virus which suppressed THBS4 expression. Circulating level of THBS4 in patients with PAH-CHD was measured by ELISA. RESULTS: THBS4 was upregulated in the lung tissues of MCT-AV rats, and was further upregulated in severe pulmonary vascular lesions. And THBS4 was expressed mainly in PASMCs. When THBS4 was inhibited, contractile markers α-SMA and MYH11 were upregulated, while the proliferative marker PCNA was decreased, the endothelial-mensenchymal transition marker N-cad was downregulated, proapototic marker BAX was increased. Additionally, proliferation and migration of PASMCs was inhibited and apoptosis was increased. Conversely, THBS4 overexpression resulted in opposite effects. And the impact of THBS4 on PASMCs was probably achieved through the regulation of the PI3K/AKT pathway. THBS4 suppression attenuated pulmonary vascular remodeling. Furthermore, compared with patients with simple congenital heart disease and mild PAH-CHD, the circulating level of THBS4 was higher in patients with severe PAH-CHD. CONCLUSIONS: THBS4 is a promising biomarker to distinguish reversible from irreversible PAH-CHD before repairing the shunt. THBS4 is a potential treatment target in PAH-CHD, especially in irreversible stage.

Cellular senescence in the pathogenesis of pulmonary arterial hypertension: the good, the bad and the uncertain.

Senescence refers to a cellular state marked by irreversible cell cycle arrest and the secretion of pro-inflammatory and tissue-remodeling factors. The senescence associated secretory phenotype (SASP) impacts the tissue microenvironment and provides cues for the immune system to eliminate senescent cells (SCs). Cellular senescence has a dual nature; it can be beneficial during embryonic development, tissue repair, and tumor suppression, but it can also be detrimental in the context of chronic stress, persistent tissue injury, together with an impairment in SC clearance. Recently, the accumulation of SCs has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), a progressive condition affecting the pre-capillary pulmonary arterial bed. PAH is characterized by endothelial cell (EC) injury, inflammation, and proliferative arterial remodeling, which leads to right heart failure and premature mortality. While vasodilator therapies can improve symptoms, there are currently no approved treatments capable of reversing the obliterative arterial remodeling. Ongoing endothelial injury and dysfunction is central to the development of PAH, perpetuated by hemodynamic perturbation leading to pathological intimal shear stress. The precise role of senescent ECs in PAH remains unclear. Cellular senescence may facilitate endothelial repair, particularly in the early stages of disease. However, in more advanced disease the accumulation of senescent ECs may promote vascular inflammation and occlusive arterial remodeling. In this review, we will examine the evidence that supports a role of endothelial cell senescence to the pathogenesis of PAH. Furthermore, we will compare and discuss the apparent contradictory outcomes with the use of interventions targeting cellular senescence in the context of experimental models of pulmonary hypertension. Finally, we will attempt to propose a framework for the understanding of the complex interplay between EC injury, senescence, inflammation and arterial remodeling, which can guide further research in this area and the development of effective therapeutic strategies.

Ubiquitination of angiotensin-converting enzyme 2 contributes to the development of pulmonary arterial hypertension mediated by neural precursor cell-expressed developmentally down-regulated gene 4-Like.

OBJECTIVES: In this study, we investigated whether neural precursor cell-expressed developmentally down-regulated gene 4-like (NEDD4L) is the E3 enzyme of angiotensin-converting enzyme 2 (ACE2) and whether NEDD4L degrades ACE2 via ubiquitination, leading to the progression of pulmonary arterial hypertension (PAH). METHODS: Bioinformatic analyses were used to explore the E3 ligase that ubiquitinates ACE2. Cultured pulmonary arterial smooth muscle cells (PASMCs) and specimens from patients with PAH were used to investigate the crosstalk between NEDD4L and ACE2 and its ubiquitination in the context of PAH. RESULTS: The inhibition of ubiquitination attenuated hypoxia-induced proliferation of PASMCs. The levels of NEDD4L were increased, and those of ACE2 were decreased in lung tissues from patients with PAH and in PASMCs. NEDD4L, the E3 ligase of ACE2, inhibited the expression of ACE2 in PASMCs, possibly through ubiquitination-mediated degradation. PAH was associated with upregulation of NEDD4L expression and downregulation of ACE2 expression. CONCLUSIONS: NEDD4L, the E3 ubiquitination enzyme of ACE2, promotes the proliferation of PASMCs, ultimately leading to PAH.

Autophagy and ubiquitin-dependent proteolysis processes in left ventricular mass loss in pulmonary arterial hypertension.

The goal of this study was to evaluate the intensity of autophagy and ubiquitin-dependent proteolysis processes occurring in myocardium of left ventricle (LV) in subsequent stages of pulmonary arterial hypertension (PAH) to determine mechanisms responsible for LV mass loss in a monocrotaline-induced PAH rat model. LV myocardium samples collected from 32 Wistar rats were analyzed in an early PAH group (n = 8), controls time-paired (n = 8), an end-stage PAH group (n = 8), and their controls (n = 8). Samples were subjected to histological analyses with immunofluorescence staining, autophagy assessment by western blotting, and evaluation of ubiquitin-dependent proteolysis in the LV by immunoprecipitation of ubiquitinated proteins. Echocardiographic, hemodynamic, and heart morphometric parameters were assessed regularly throughout the experiment. Considerable morphological and hemodynamic remodeling of the LV was observed over the course of PAH. The end-stage PAH was associated with significantly impaired LV systolic function and a decrease in LV mass. The LC3B-II expression in the LV was significantly higher in the end-stage PAH group compared to the early PAH group (p = 0.040). The measured LC3B-II/LC3B-I ratios in the end-stage PAH group were significantly elevated compared to the controls (p = 0.039). Immunofluorescence staining showed a significant increase in the abundance of LC3 puncta in the end-stage PAH group compared to the matched controls. There were no statistically significant differences in the levels of expression of all ubiquitinated proteins when comparing both PAH groups and matched controls. Autophagy may be considered as the mechanism behind the LV mass loss at the end stage of PAH.

Melatonin effects on oxidative stress and on TLR4/NF-kß inflammatory pathway in the right ventricle of rats with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure and a compromised the right ventricle (RV), together with progression to heart failure and premature death. Studies have evaluated the role of melatonin as a promising therapeutic strategy for PAH. The objective of this study was to evaluate melatonin's effects on oxidative stress and on the TLR4/NF-kß inflammatory pathway in the RV of rats with PAH. Male Wistar rats were divided into the following groups: control, monocrotaline (MCT), and monocrotaline plus melatonin groups. These two last groups received one intraperitoneal injection of MCT (60 mg/kg) on the first day of experimental protocol. The monocrotaline plus melatonin group received 10 mg/kg/day of melatonin by gavage for 21 days. Echocardiographic analysis was performed, and the RV was collected for morphometric analysis oxidative stress and molecular evaluations. The main findings of the present study were that melatonin administration attenuated the reduction in RV function that was induced by monocrotaline, as assessed by TAPSE. In addition, melatonin prevented RV diastolic area reduction caused by PAH. Furthermore, animals treated with melatonin did not show an increase in ROS levels or in NF-kß expression. In addition, the monocrotaline plus melatonin group showed a reduction in TLR4 expression when compared with control and monocrotaline groups. To our knowledge, this is the first study demonstrating a positive effect of melatonin on the TLR4/NF-kß pathway in the RV of rats with PAH. In this sense, this study makes it possible to think of melatonin as a possible ally in mitigating RV alterations caused by PAH.

Investigation into the role of H2-Ab1 in vascular remodeling in pulmonary arterial hypertension via Bioinformatics.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease of vascular remodeling characterized by persistent pulmonary arterial pressure elevation, which can lead to right heart failure and premature death. Given the complex pathogenesis and poor prognosis of PAH, the identification and investigation of biomarkers become increasingly critical for advancing further understanding of the disease. METHODS: PAH-related datasets, GSE49114, GSE180169 and GSE154959, were downloaded from the publicly available GEO database. By performing WGCNA on the GSE49114 dataset, a total of 906 PAH-related key module genes were screened out. By carrying out differential analysis on the GSE180169 dataset, a total of 576 differentially expressed genes were identified. Additionally, the GSE154959 single-cell sequencing dataset was also subjected to differential analysis, leading to the identification of 34 DEGs within endothelial cells. By taking intersection of the above three groups of DEGs, five PAH-related hub genes were screened out, namely Plvap, Cyp4b1, Foxf1, H2-Ab1, and H2-Eb1, among which H2-Ab1 was selected for subsequent experiments. RESULTS: A SuHx mouse model was prepared using the SU5416/hypoxia method, and the successful construction of the model was evaluated through Hematoxylin-Eosin staining, hemodynamic detection, fulton index, and Western Blot (WB). The results of WB and qRT-PCR demonstrated a significant upregulation of H2-Ab1 expression in SuHx mice. Consistent with the results of bioinformatics analysis, a time-dependent increase was observed in H2-Ab1 expression in hypoxia-treated mouse pulmonary artery endothelial cells (PAECs). To investigate whether H2-Ab1 affects the development and progression of PAH, we knocked down H2-Ab1 expression in PAECs, and found that its knockdown inhibited the viability, adhesion, migration, and angiogenesis, while concurrently promoted the apoptosis of PAECs. CONCLUSION: H2-Ab1 could regulate the proliferation, apoptosis, adhesion, migration, and angiogenesis of PAECs.

A novel PDGFR inhibitor WQ-C-401 prevents pulmonary vascular remodeling in rats with monocrotaline-induced pulmonary arterial hypertension.

Platelet-derived growth factor (PDGF) is one of the most important cytokines associated with pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). PDGF receptor (PDGFR) inhibition exerted therapeutic effects on PAH in clinical trials, but serious side effects warrant the withdrawal of existing drugs. In this study, a novel highly selective PDGFR inhibitor WQ-C-401 was developed, and its effects on PDGFR signaling pathway and pulmonary vascular remodeling in PAH were investigated. Cell proliferation assays and Western blot analysis of PDGFRα/ß phosphorylation showed that WQ-C-401 inhibited PDGFR-mediated cell proliferation assay and suppressed PDGFR phosphorylation in a concentration-dependent manner. DiscoverX's KinomeScanTM technology confirmed the good kinome selectivity of WQ-C-401 (S score (1) of PDGFR = (0.01)). In monocrotaline (MCT)-induced PAH rats, intragastric administration of WQ-C-401 (25, 50, 100 mg/kg/d) or imatinib (50 mg/kg/d, positive control) significantly decreased right ventricular systolic pressure (RVSP). Histological analysis demonstrated that WQ-C-401 inhibited pulmonary vascular remodeling by reducing muscularization and fibrosis, as well as alleviated right ventricular hypertrophy in MCT-treated rats. In addition, WQ-C-401 suppressed MCT-induced cell hyperproliferation and CD68+ macrophage infiltration around the pulmonary artery. In vitro, WQ-C-401 inhibited PDGF-BB-induced proliferation and migration of human pulmonary arterial smooth muscle cells (PASMCs). Moreover, Western blot analysis showed that WQ-C-401 concertration-dependently inhibited PDGF-BB-induced phosphorylation of ERK1/2 and PDGFRß Y751, decreased collagen Ⅰ synthesis and increased alpha smooth muscle actin (α-SMA) expression in PASMCs. Collectively, our results suggest that WQ-C-401 is a selective and potent PDGFR inhibitor which could be a promising drug for the therapeutics of PAH by preventing pulmonary vascular remodeling.

Upregulation of Angiomotin-Like 2 Ameliorates Experimental Pulmonary Arterial Hypertension by Inactivating YAP1 Signaling.

ABSTRACT: Angiomotin-like 2 (AMOTL2) is related to numerous physiological and pathological conditions by affecting signal transduction. However, whether AMOTL2 is linked to pulmonary arterial hypertension (PAH) has not been addressed. This work aimed to investigate the potential role of AMOTL2 in PAH. A decrease in AMOTL2 abundance was observed in the lungs of PAH rats. The upregulation of AMOTL2 significantly decreased right ventricle systolic pressure and right ventricular hypertrophy in PAH rats. Overexpression of AMOTL2 also led to a noteworthy decrease in vascular wall thickness, pulmonary artery area, and collagen deposition in rats with PAH. AMOTL2 was downregulated in hypoxia-stimulated pulmonary arterial smooth muscle cells (PASMCs). Moreover, AMOTL2 overexpression impeded hypoxia-evoked proliferation, migration, and phenotypic transformation in rat PASMCs. Mechanistic investigation revealed that Yes-associated protein 1 (YAP1) activation in PAH rats or hypoxia-stimulated PASMCs was markedly inhibited by AMOTL2 overexpression, which was associated with increased large tumor suppressor 1/2 phosphorylation. The inhibition of large tumor suppressor 1/2 reversed the AMOTL2-mediated inactivation of YAP1. Restoring the activity of YAP1 reversed the inhibitory effect of AMOTL2 on hypoxia-evoked proliferation, migration, and phenotypic transformation of PASMCs. Collectively, these results suggest that AMOTL2 can ameliorate PAH in a rat model by interfering with pulmonary arterial remodeling via the inactivation of YAP1 signaling. Our work indicates that AMOTL2 may be a candidate target for novel drug development for the treatment of PAH.

Fluorinated perhexiline derivative attenuates vascular proliferation in pulmonary arterial hypertension smooth muscle cells.

Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKTSer473, ERK 1/2Thr202/Tyr204 and PDH-E1αSer293, indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKTSer473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1αSer293), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology.

Novel Hemodynamic, Vascular Lesion, and Cytokine/Chemokine Differences Regarding Sex in a Pulmonary Arterial Hypertension Model.

Sex differences are recognized in pulmonary hypertension. However, the progression of disease with regard to vascular lesion formation and circulating cytokines/chemokines is unknown. To determine whether vascular lesion formation, changes in hemodynamics, and alterations in circulating chemokines/cytokines differ between males and females, we used a progressive model of pulmonary arterial hypertension (PAH), Sugen/hypoxia, and analyzed cohorts of male and female rats at time points suggested to indicate worsening disease. Our analysis included echocardiography for hemodynamics, morphometry, immunofluoresecence, and chemokine/cytokine analysis of plasma at each time point in both sexes. We found that male rats had significantly increased Fulton index, compared with those for females at each time point, as well as increased medial artery thickening at 8 weeks of PAH. Furthermore, females exhibited fewer obliterative vascular lesions than males at our latest time point. Our data also show increased IL-4, granulocyte-macrophage colony-stimulating factor, IL-10, and macrophage interacting protein-1α that were not observed in females, whereas females were observed to have increased RANTES (whose name derives from Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted) and CXCL-10 that were not found in males. Males also have increased infiltrating macrophages in vascular lesions, compared with females. We found that development of progressive PAH in hemodynamics, morphology, and chemokine/cytokine circulation differs significantly between males and females. These data suggest a macrophage-driven pathology in males, whereas there may be T cell protection from vascular damage in females with PAH.

C-type natriuretic peptide/cGMP/FoxO3 signaling attenuates hyperproliferation of pericytes from patients with pulmonary arterial hypertension.

Pericyte dysfunction, with excessive migration, hyperproliferation, and differentiation into smooth muscle-like cells contributes to vascular remodeling in Pulmonary Arterial Hypertension (PAH). Augmented expression and action of growth factors trigger these pathological changes. Endogenous factors opposing such alterations are barely known. Here, we examine whether and how the endothelial hormone C-type natriuretic peptide (CNP), signaling through the cyclic guanosine monophosphate (cGMP) -producing guanylyl cyclase B (GC-B) receptor, attenuates the pericyte dysfunction observed in PAH. The results demonstrate that CNP/GC-B/cGMP signaling is preserved in lung pericytes from patients with PAH and prevents their growth factor-induced proliferation, migration, and transdifferentiation. The anti-proliferative effect of CNP is mediated by cGMP-dependent protein kinase I and inhibition of the Phosphoinositide 3-kinase (PI3K)/AKT pathway, ultimately leading to the nuclear stabilization and activation of the Forkhead Box O 3 (FoxO3) transcription factor. Augmentation of the CNP/GC-B/cGMP/FoxO3 signaling pathway might be a target for novel therapeutics in the field of PAH.

NOTCH3 and Pulmonary Arterial Hypertension.

NOTCH3 receptor signaling has been linked to the regulation of smooth muscle cell proliferation and the maintenance of smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (World Health Organization Group 1 idiopathic disease: PAH) is a fatal disease characterized clinically by elevated pulmonary vascular resistance caused by extensive vascular smooth muscle cell proliferation, perivascular inflammation, and asymmetric neointimal hyperplasia in precapillary pulmonary arteries. In this review, a detailed overview of the specific role of NOTCH3 signaling in PAH, including its mechanisms of activation by a select ligand, downstream signaling effectors, and physiologic effects within the pulmonary vascular tree, is provided. Animal models showing the importance of the NOTCH3 pathway in clinical PAH will be discussed. New drugs and biologics that inhibit NOTCH3 signaling and reverse this deadly disease are highlighted.

Otud6b induces pulmonary arterial hypertension by mediating the Calpain-1/HIF-1α signaling pathway.

Pulmonary hypertension (PAH) is a cardiopulmonary disease in which pulmonary artery pressure continues to rise, leading to right heart failure and death. Otud6b is a member of the ubiquitin family and is involved in cell proliferation, apoptosis and inflammation. The aim of this study was to understand the role and mechanism of Otud6b in PAH. C57BL/6 and Calpain-1 knockout (KO) mice were exposed to a PAH model induced by 10% oxygen. Human pulmonary artery endothelial cells (HPACEs) and human pulmonary artery smooth muscle cells (HPASMCs) were exposed to 3% oxygen to establish an in vitro model. Proteomics was used to determine the role of Otud6b and its relationship to Calpain-1/HIF-1α signaling. The increased expression of Otud6b is associated with the progression of PAH. ROtud6b activates Otud6b, induces HIF-1α activation, increases the production of ET-1 and VEGF, and further aggravates endothelial injury. Reducing Otud6b expression by tracheal infusion of siOtud6b has the opposite effect, improving hemodynamic and cardiac response to PAH, reducing the release of Calpain-1 and HIF-1α, and eliminating the pro-inflammatory and apoptotic effects of Otud6b. At the same time, we also found that blocking Calpain-1 reduced the effect of Otud6b on HIF-1α, and inhibiting HIF-1α reduced the expression of Calpain-1 and Otud6b. Our study shows that increased Otud6b expression during hypoxia promotes the development of PAH models through a positive feedback loop between HIF-1α and Calpain-1. Therefore, we use Otud6b as a biomarker of PAH severity, and regulating Otud6b expression may be an effective target for the treatment of PAH.