Rat Model Of Pulmonary Arterial Hypertension Research Articles

The TREK-1 potassium channel is a potential pharmacological target for vasorelaxation in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease in which chronic membrane potential (Em) depolarisation of the pulmonary arterial smooth muscle cells (PASMCs) causes calcium overload, a key pathological alteration. Under resting conditions, the negative Em is mainly set by two pore domain potassium (K2P) channels, of which the TASK-1 has been extensively investigated. Ion channel currents and membrane potential of primary cultured human(h) PASMCs were measured using the voltage- and current clamp methods. Intracellular [Ca2+] was monitored using fluorescent microscopy. Pulmonary BP and vascular tone measurements were also performed ex vivo using a rat PAH model. TREK-1 was the most abundantly expressed K2P in hPASMCs of healthy donors and idiopathic(I) PAH patients. Background K+-current was similar in hPASMCs for both groups and significantly enhanced by the TREK activator ML-335. In donor hPASMCs, siRNA silencing or pharmacological inhibition of TREK-1 caused depolarisation, reminiscent of the electrophysiological phenotype of idiopathic PAH. ML-335 hyperpolarised donor hPASMCs and normalised the Em of IPAH hPASMCs. A close link was found between TREK-1 activity and intracellular Ca2+-signalling using a channel activator, ML-335, and an inhibitor, spadin. In the rat, ML-335 relaxed isolated pre-constricted pulmonary arteries and significantly decreased pulmonary arterial pressure in the isolated perfused lung. These data suggest that TREK-1is a key factor in Em setting and Ca2+ homeostasis of hPASMC, and therefore, essential for maintenance of a low resting pulmonary vascular tone. Thus TREK-1 may represent a new therapeutic target for PAH.

Pulmo-protection of long-term swimming exercise via improving insulin sensitivity in monocrotaline-induced pulmonary hypertensive rats

Exercise has been recognized as an effective intervention in the treatment of pulmonary arterial hypertension (PAH), supported by numerous studies. However, the precise effects of exercise on pulmonary function remain to be fully elucidated. In this study, using a rat model of swimming exercise training and monocrotaline-induced PAH, we aimed to explore its impact on pulmonary morphology and function. Our investigations revealed that MCT-treated rats exhibited augmented mean pulmonary arterial pressure (MPAP) and pulmonary vascular remodeling, which can be attenuated by 4 weeks of swimming exercise training (60 min/day, 5 days/week). Notably, MCT-treated rats showed impaired pulmonary function, as manifested by decreased tidal volume and dynamic compliance, which were reversed by exercise training. Assessment of pulmonary substrate in PAH rats indicated a prominent pro-inflammatory substrate, evidenced by macrophage accumulation through quantitative immunohistological analysis of macrophage-like cell expression (CD68), and extracellular matrix remodeling, evaluated by Masson staining. Importantly, both the pro-inflammatory substrate and extracellular matrix remodeling were ameliorated by swimming exercise training. Additionally, serum biochemical analysis demonstrated elevated levels of low-density lipoprotein cholesterol and Apolipoprotein B following MCT treatment, which were reduced with exercise intervention. Moreover, exercise enhanced systemic insulin sensitivity in both MCT-treated and untreated rats. Notably, MCT and exercise treatment both decreased fasting blood glucose (FBG) levels in rats, whereas exercise training reinstated FBG levels to normal in MCT-treated rats. In summary, our study suggests that swimming exercise confers a pulmonary protective effect in MCT-induced PAH rats, highlighting the potential importance of exercise-based rehabilitation in the management of PAH.

C-Src Facilitates ER-to-Mitochondria Ca2+ Transport and Activates Cardiac Fibroblasts under Pulmonary Arterial Hypertension

Introduction: Similar to left ventricular (LV) fibrosis, growing evidence suggests a potential link between right ventricular (RV) fibrosis, poor function of the pressure-overloaded RV, and mortality in patients with pulmonary arterial hypertension (PAH). However, no therapies are currently available that specifically target RV fibrosis. Although the pathological roles of an oxidative stress-sensitive tyrosine kinase c-Src in LV hypertrophy and failure are well characterized, it remains unclear whether c-Src is involved in RV pathology under PAH. Using heterologous expression systems, we previously showed that mitofusin 2 (Mfn2), a key component of the tethering structures between the endoplasmic reticulum (ER) and mitochondria (Mito) is a novel c-Src substrate in mitochondria; and c-Src-dependent tyrosine phosphorylation (P-Tyr) of Mfn2 at the outer mitochondrial membrane (OMM) decreases the ER-Mito distance, possibly through conformational changes of Mfn2, which facilitates ER-to-Mito Ca2+ transfer and increases reactive oxygen species (ROS). Hypothesis: c-Src signaling alters the size of ER-Mito microdomains and enhances oxidative stress in the RV during PAH. Methods: Primary adult cardiac fibroblasts (CFs) from human ventricles and a preclinical rat PAH model induced by Su gen 5416 injection and 3-week h ypo x ia followed by 4-week normoxia (SuHx) were used for in vitro and in vivo assays, respectively. RV tissues from pulmonary hypertension (PH) patients and sex-/aged-matched controls were used for biochemical and cell biological assays. Results: c-Src activation occurred specifically in CFs in the RV, but not in RV myocytes in a rat SuHx PAH model and human PH-RVs as assessed by immunohistochemistry, which is notably different from the reports on LV hypertrophy and failure. P-Tyr of Mfn2 was detectable from human PH-RVs by Western Blots. RV-CFs from PAH rats exhibited increased c-Src activity, decreased ER-Mito distance, increased ROS, and proliferative signaling activation compared to RV-CFs from control rats as assessed by live cell imaging, immunocytochemistry, and transmission electron microscopy. All of these changes in rat CFs from PAH-RVs were recapitulated by overexpressing c-Src in human CFs in vitro. In addition, c-Src significantly increased the speed and amount of mitochondrial Ca2+ uptake without changing cytosolic Ca2+ dynamics in human CFs, suggesting a potential link between c-Src activation, the size of ER-Mito microdomains, and RV fibrosis under PAH. To further dissect the role of c-Src activity in CFs especially in the mitochondria, we specifically inhibited c-Src activity at the OMM, where Mfn2 is located, using an OMM-targeted dominant-negative c-Src (termed mt-c-Src-DN) that was generated by the addition of an OMM-targeting sequence (amino acid 1-33 from human TOM20) to the N-terminus of kinase-dead c-Src-K295R/Y527F. The introduction of mt-c-Src-DN increased the ER-Mito distance and reduced the speed of ER-to-Mito Ca2+ transport in human CFs. Conclusion: c-Src-dependent P-Tyr of Mfn2 alters the ER-Mito tethering structure, facilitates ER-to-Mito Ca2+ transport, and increases mitochondrial ROS generation, which promotes CF activation in PAH. Therefore, CF- and mitochondria-specific inhibition of c-Src may provide an additional therapeutic strategy to attenuate RV fibrosis and failure in response to PAH. NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

A computational study of right ventricular mechanics in a rat model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) presents a significant challenge to right ventricular (RV) function due to progressive pressure overload, necessitating adaptive remodeling in the form of increased wall thickness, enhanced myocardial contractility and stiffness to maintain cardiac performance. However, the impact of these remodeling mechanisms on RV mechanics in not clearly understood. In addition, there is a lack of quantitative understanding of how each mechanism individually influences RV mechanics. Utilizing experimental data from a rat model of PAH at three distinct time points, we developed biventricular finite element models to investigate how RV stress and strain evolved with PAH progression. The finite element models were fitted to hemodynamic and morphological data to represent different disease stages and used to analyze the impact of RV remodeling as well as the altered RV pressure. Furthermore, we performed a number of theoretical simulation studies with different combinations of morphological and physiological remodeling, to assess and quantify their individual impact on overall RV load and function. Our findings revealed a substantial 4-fold increase in RV stiffness and a transient 2-fold rise in contractility, which returned to baseline by week 12. These changes in RV material properties in addition to the 2-fold increase in wall thickness significantly mitigated the increase in wall stress and strain caused by the progressive increase in RV afterload. Despite the PAH-induced cases showing increased wall stress and strain at end-diastole and end-systole compared to the control, our simulations suggest that without the observed remodeling mechanisms, the increase in stress and strain would have been much more pronounced. Our model analysis also indicated that while changes in the RV's material properties-particularly increased RV stiffness - have a notable effect on its mechanics, the primary compensatory factor limiting the stress and strain increase in the early stages of PAH was the significant increase in wall thickness. These findings underscore the importance of RV remodeling in managing the mechanical burden on the right ventricle due to pressure overload.

SiRNA Targeting ECE-1 Partially Reverses Pulmonary Arterial Hypertensionassociated Damage in a Monocrotaline Model.

The aim of this study was to develop a possible treatment for pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH) is a rare disease characterised by a pulmonary arterial pressure greater than 20 mmHg. One of the factors that contribute to PAH is an increase in the production of endothelin-1, a polypeptide that increases vascular resistance in the pulmonary arteries, leading to increased pulmonary arterial pressure and right ventricular hypertrophy. The objective of this study was to design, synthesize, and evaluate two siRNAs directed against endothelin-1 in a rat model of PAH induced with monocrotaline. Wistar rats were administered monocrotaline (60 mg/kg) to induce a PAH model. Following two weeks of PAH evolution, the siRNAs were administered, and after two weeks, right ventricular hypertrophy was evaluated using the RV/LV+S ratio, blood pressure, weight, and relative expression of ECE-1 (Endothelin-converting enzyme-1) mRNA (messenger RNA) by RT-PCR (real-time PCR). The monocrotaline group showed an increase in the hypertrophy index and in ECE-1 mRNA, as well as a significant decrease in weight compared to the control group, while in the monocrotaline + siRNA group, a significant decrease was observed in the relative expression of ECE-1 mRNA, as well as in right ventricular hypertrophy. Based on the above information, we conclude that the administration of siRNAs directed to ECE-1 decreases the damage associated with PAH.

Transcriptome-Wide N6-Methyladenosine Alternations in Pulmonary Arteries of Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats and Novel Therapeutic Targets.

N6-methyladenosine (m6A) is a post-transcriptional epigenetic change with transcriptional stability and functionality regulated by specific m6A-modifying enzymes. However, the significance of genes modified by m6A and enzymes specific to m6A regulation in the context of pulmonary arterial hypertension (PAH) remains largely unexplored. MeRIP-seq and RNA-seq were applied to explore variances in m6A and RNA expression within the pulmonary artery tissues of control and monocrotaline-induced PAH rats. Functional enrichments were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To screen candidate m6A-related genes, the STRING and Metascape databases were used to construct a protein-protein interaction network followed by a real-time PCR validation of their expression. The expression level of an m6A regulator was further investigated using immunohistochemical staining, immunofluorescence, and Western blot techniques. Additionally, proliferation assays were conducted on primary rat pulmonary artery smooth muscle cells (PASMCs). We identified forty-two differentially expressed genes that exhibited either hypermethylated or hypomethylated m6A. These genes are predominantly related to the extracellular matrix structure, MAPK, and PI3K/AKT pathways. A candidate gene, centromere protein F (CENPF), was detected with increased expression in the PAH group. Additionally, we first identified an m6A reader, leucine rich pentatricopeptide repeat containing (LRPPRC), which was downregulated in the PAH rat model. The in vitro downregulation of Lrpprc mediated by siRNA resulted in the enhanced proliferation and elevated expression of Cenpf mRNA in primary rat PASMCs. Our study revealed a modified transcriptome-wide m6A landscape and associated regulatory mechanisms in the pulmonary arteries of PAH rats, potentially offering a novel target for therapeutic strategies in the future.

Activation of the p62-Keap1-Nrf2 pathway improves pulmonary arterial hypertension in MCT-induced rats by inhibiting autophagy.

Autophagy is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We aimed to investigate whether the p62-Keap1-Nrf2 pathway affects the development of PAH by mediating autophagy. A PAH rat model was established using monocrotaline (MCT). Pulmonary artery smooth muscle cells (PASMCs) were extracted, and the changes in proliferation, migration, autophagy, and oxidative stress were analyzed following overexpression or knockdown of p62. The impact of p62 on the symptoms of PAH rats was assessed by the injection of an adenovirus overexpressing p62. We found that the knockdown of p62 increased the proliferation and migration of PASMCs, elevating the oxidative stress of PASMCs and upregulating gene expression of NADPH oxidases. Co-IP assay results demonstrated that p62 interacted with Keap1. p62 knockdown enhanced Keap1 protein stability and Nrf2 ubiquitination. LC3II/I and ATG5 were expressed more often when p62 was knocked down. Treating with an inhibitor of autophagy reversed the impact of p62 knockdown on PASMCs. Nrf2 inhibitor treatment reduced the expression of Nrf2 and p62, while increasing the expression of Keap1, LC3II/I, and ATG5 in PASMCs. However, overexpressing p62 diminished mRVP, SPAP, and Fulton index in PAH rats and attenuated pulmonary vascular wall thickening. Overexpression of p62 also decreased the expression of Keap1, LC3II/I, and ATG5 and increased the nuclear expression of Nrf2 in PAH rats. Importantly, overexpression of p62 reduced oxidative stress and the NADPH oxidase expression in PAH rats. Overall, activation of the p62-Keap1-Nrf2 positive feedback signaling axis reduces the proliferation and migration of PASMCs and alleviates PAH by inhibiting autophagy and oxidative stress.

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

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

Hypusine Signaling Promotes Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a post-translational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) due to excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. To characterize the role of hypusine signaling in PAH. Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Hypusine forming enzymes (deoxyhypusine synthase, DHPS and deoxyhypusine hydroxylase, DOHH) and hypusinated eIF5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using GC7 or short hairpin RNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice and that its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. Using mass spectrometry, we show that hypusine signaling promotes the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. These findings support inhibiting hypusine signaling as a potential treatment for PAH.

Intratracheally Administered Peptide-Modified Lipid Admixture Containing Fasudil and/or DETA NONOate Ameliorates Various Pathologies of Pulmonary Arterial Hypertension.

This study examined the therapeutic potential of a combination therapy using fasudil, a Rho-kinase inhibitor, and DETA NONOate (DN), a nitric oxide donor, delivered as a lipid admixture modified with a cyclic homing peptide known as CAR (CAR-lipid mixture) for the treatment of pulmonary arterial hypertension (PAH). CAR-lipid mixtures were initially prepared via a thin-film hydration method and then combined with fasudil, DN, or a mixture of both. The therapeutic efficacy of this drug-laden lipid mixture was evaluated in a Sugen/Hypoxia (Su/Hx) rat model of PAH by measuring RV systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), Fulton indices, and assessing right ventricular (RV) functions, as well as evaluating pulmonary vascular morphology. Rats that received no treatment exhibited increases in RVSP, mPAP, Fulton indices, and changes in RV functional parameters. However, the treatment with the CAR-lipid mixture containing either fasudil or DN or a combination of both led to a decline in mPAP, RVSP, and Fulton indices compared to saline-treated rats. Similarly, rats that received these treatments showed concurrent improvement in various echocardiographic parameters such as pulmonary acceleration time (PAT), tricuspid annular plane systolic excursion (TAPSE), and ventricular free wall thickness (RVFWT). A significant decrease in the wall thickness of pulmonary arteries larger than 100 µm was observed with the combination therapy. The findings reveal that fasudil, DN, and their combination in a CAR-modified lipid mixture improved pulmonary hemodynamics, RV functions, and pathological alterations in the pulmonary vasculature. This study underscores the potential of combination therapy and targeted drug delivery in PAH treatment, laying the groundwork for future investigations into the optimization of these treatments, their long-term safety and efficacy, and the underlying mechanism of action of the proposed therapy.

Urinary proteomic analysis for biomarker discovery of pulmonary arterial hypertension-induced right ventricular failure

Abstract Background Right ventricular failure (RVF) is one of the independent and strong predictors of mortality in pulmonary arterial hypertension (PAH). Urine is more suitable and sensitive for biomarker discovery with no need for homeostatic control compared to blood. Purpose The purpose of this study is to investigate candidate urinary biomarkers for RV structural and functional monitoring during PAH progression. Methods The urinary proteome of 21 monocrotaline (MCT)-induced PAH rats (8 controls, 8 of compensated RV group, 5 of decompensated RV group) were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). And the differential proteins were verified by parallel reaction monitoring (PRM)-targeted quantitative proteomics. Candidate biomarkers were further validated using ELISA in the urine of PAH patients. Results In total, 1811 urinary proteins were identified with at least 1 unique peptide (FDR≤1%). Compared to the control group of rats, 118 and 184 proteins were altered in the compensated and decompensated groups, respectively, and 62 differential proteins were found between the two stages (2.0-fold change, p< 0.05). The canonical pathways revealed that complement system, glutathione metabolism and iron homeostasis were enriched in the compensated stage, while interferon signaling, fibrosis, Wnt signaling and epithelial-mesenchymal transition were enriched in the failing stage. Four proteins confirmed by PRM, which were also of good discrimination ability of two RV stages were selected for further validation. ELISA results of the urine of PAH patients showed that IL18BP, IFNABR, LGALS3BP and α2-AP were sequentially elevated as disease progressed. Correlation analysis with clinical indicators showed that these proteins were positively correlated with right atrial pressure and right heart dilation, and negatively correlated with cardiac index and tricuspid annular plane systolic excursion. The combination of these four proteins panel could significantly distinguish PAH patients from healthy controls (AUC=0.985, 95%CI 0.955-1) and performs well in distinguishing high-risk PAH patients from low-risk ones (AUC=0.922, 95%CI 0.807-1). Conclusions Novel RVF candidate urinary biomarkers were found through in-depth proteomic analysis of PAH rat model. The panel of these four proteins, which were significantly correlated with RV structural remodeling and systolic function, showed good performance in diagnosis and risk stratification of PAH patients.Figure 1.Study workflowFigure 1.Main results of the study

Abstract 11703: Proteomic Analysis for the Formation of the Plexiform Lesions of a Rat Pulmonary Arterial Hypertension Model

Introduction: Although plexiform lesions (PLs) are the hallmark of the severe pulmonary arterial hypertension, the mechanism of this lesion formation has not been fully elucidated. In this study, to explore the pathways associated PLs formation, we examined the proteome analysis using vascular tissue before and after the formation of PLs obtained by laser capture microdissection (LCM). Methods: 7 weeks old rats were injected Sugen 5416 subcutaneously, followed by exposed to hypoxia for 3 weeks. In addition, the rats were kept in room air for 5 weeks (8-week model) or 10 weeks (13-week model) thereafter. We obtained the tissue of vessels with medial hypertrophy from 8-week models (n=5) and PLs from 13-week models (n=5) by LCM. Those micro-dissected samples were then subject to a qualitative and then quantitative proteomics approach. Differentially expressed proteins (DEPs) were defined by abundance in mass spectrometry (MS) (P-value < 0.05 and |log2 (fold change)| > 0.5 or proteins deteced only in lesions extracted from one model). In addition, we investigated enhanced pathway by gene ontology (GO) analysis of the identified DEPs in each of the 8-week and 13-week models. Results: We identified 718 uniquely expressed proteins including 57 DEPs (31 in 8-week model versus 26 in 13-week model) by using LCM-MS. GO analysis revealed that the actin cytoskeleton organization, actin filament-based process, and actomyosin structure organization processes were enhanced in 8-week model. On the other hand, the innate immune system, G2/M check points, and cellular response to reactive oxygen species were enhanced in 13-week model. Conclusion: Pulmonary vascular lesions of 8-week and PLs of 13-week Sugen hypoxia model evaluated by proteomics using samples obtained by LCM methods showed differential protein expression. Analysis of these DEPs may contribute to elucidate the mechanisms of PLs formation.

Effect of Nicorandil on Endothelial Markers and Tissue Remodeling in Pulmonary Arterial Hypertension Model of Male Rats

Background: Chronic pulmonary arterial hypertension (PAH) is a rare, long-lasting illness that makes pulmonary artery endothelial cells (PAEC) not work properly and leads to heart failure and death. Objective: To evaluate the effect of nicorandil in the treatment of PAH compared to tadalafil in a rat model of monocrotaline-induced PAH. Methods: Monocrotaline injection (60 mg/kg) was used for the induction of PAH in male rats; healthy control and induction groups were not treated. The other 4 groups were treated with either nicorandil or tadalafil with or without treatment blockers (glimepiride and N-Nitroarginine methyl ester (L-NAME)) for 21 days orally. Serum was obtained for assessment of endothelin-1 (ET-1) and tissue harvested for nuclear factor kappa B (NFκB) by ELISA, western blot analysis of endothelial nitric oxide synthase (eNOS), and an apoptosis assay to examine the endothelial function. Results: Nicorandil showed a significant reduction in ET-1 and significant elevation in eNOS compared to the induction group, with comparable efficacy to tadalafil; blocker groups showed significantly elevated levels of ET-1 and reduced levels of eNOS compared to healthy control; NFκB was significantly inhibited in nicorandil and tadalafil groups and significantly elevated in blocker and induction groups; while in the TUNEL apoptosis assay, nicorandil showed the highest level of inhibition to apoptosis with apparently normal endothelium lining. Conclusions: Nicorandil shows anti-inflammatory, antiapoptotic, and enhanced endothelial morphology and function compared to the induction model.

Therapeutic potential and protective role of GRK6 overexpression in pulmonary arterial hypertension

Abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key mechanism in the development of pulmonary arterial hypertension (PAH). Signal transducer and activator of transcription 3 (STAT3) signalling plays a critical role in modulating PASMC proliferation, and G-protein-coupled receptor kinase 6 (GRK6) regulates the STAT3 pathway. However, the mechanism underlying the relationship between GRK6 and PAH remains unclear. In this study, we aimed to investigate the role of GRK6 in PAH and determine its potential as a therapeutic target. We utilised hypoxia- and SU5416-induced PAH mouse models and a monocrotaline-induced PAH rat model to analyse the involvement of GRK6. We conducted gain- and loss-of-function experiments using mouse PASMCs. Modulation of GRK6 expression was achieved via a lentiviral vector in vitro and an adeno-associated virus serotype 1 encoding GRK6 in vivo. GRK6 was significantly downregulated in the lung tissues of PAH mice and rats, predominantly in PASMCs. Knockout of GRK6 exacerbated PAH, while both therapeutic and prophylactic overexpression of GRK6 alleviated PAH, as evidenced by a reduction in right ventricular systolic pressure, right ventricular wall to left ventricular wall plus ventricular septum ratio, pulmonary vascular media thickness, and pulmonary vascular muscularisation. Mechanistically, GRK6 overexpression attenuated hypoxia-induced PASMC proliferation and STAT3 phosphorylation. Conversely, knockdown of GRK6 promoted hypoxia-induced proliferation, which was mitigated by a STAT3 inhibitor. Our findings highlight the potential protective and beneficial roles of GRK6 in PAH; we propose a lung-targeted GRK6 gene therapy utilizing adeno-associated virus serotype 1 as a potential treatment approach for patients with PAH.

TNF-α and IL-8 levels are positively correlated with hypobaric hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats.

The expression status of proinflammatory cytokines in high-altitude pulmonary arterial hypertension (PAH) has been well studied. However, the changes in interleukin (IL)-8 and tumor necrosis factor α (TNF-α) during the reversible changes in pulmonary vascular remodeling (PVR) in PAH after detaching from a hypobaric hypoxic environment have not been elucidated. This investigation elucidated a high-altitude PAH rat model. Then, PAH rats in the high-altitude group were maintained in the high-altitude area, and rats in the low-altitude group returned to the low-altitude area. After 0, 10, 20, and 30 days of PAH modeling, right ventricular systolic pressure (RVSP) and the mean pulmonary arterial pressure (mPAP) were assessed. Right ventricular (RV) hypertrophy was reflected by the ratio of RV/[left ventricle + interventricular septum (S)]. Pathological changes in PVR were accessed by hematoxylin-eosin staining, and medial wall thickness (WT%) and medial wall area (WA%) were measured. TNF-α and IL-8 levels in pulmonary artery tissues and blood were measured with Western blot assay and enzyme-linked immunosorbent assay, respectively. Our results showed that PAH rats exhibited a substantial increase in RVSP and mPAP, RV hypertrophy, PVR, and enhanced generation of TNF-α and IL-8. Then, we found that these pathological changes were gradually aggravated and TNF-α and IL-8 levels were increased in rats in the high-altitude group after 10, 20, and 30 days of PAH modeling. In contrast, the mPAP was decreased and PVR was alleviated in rats in the low-altitude group, accompanying with reduced TNF-α and IL-8 production. In conclusion, our study demonstrated that the generation of TNF-α and IL-8 was also reversible during the reversible changes in PVR after detaching from a hypobaric hypoxic environment. Thus, proinflammatory cytokine TNF-α and IL-8 levels are positively correlated with PVR severity.

Development of novel bosentan analogues as endothelin receptor antagonists for pulmonary arterial hypertension

Since decades, bosentan has been in use for the treatment of pulmonary arterial hypertension (PAH). However, chronic exposure to bosentan leads to the development of resistance, tolerance, and serious adverse effects that have restricted its usage in clinical practices. To surmount these limitations, some new bosentan derivatives have been synthesized and evaluated for their therapeutic efficacy in PAH. Molecular docking analyses of all the synthesized derivatives were carried out using the endothelin (ET) receptor. In addition, the inhibitory ability of synthesized derivatives was determined in in vitro assay employing an ET-1 human ELISA kit. Among the synthesized derivatives, three derivatives namely 17d, 16j, and 16h with higher docking scores and lower IC50 values were selected for determination of the magnitude of the binding force between the derivative and ET receptor using molecular dynamics (MD) simulations study. Further, these derivatives were subjected to in vivo studies using monocrotaline (MCT) induced PAH in rat model. Results of in vivo studies inferred that the derivatives exhibit impressive ability to reduce PAH. Besides, its protective role was also evidenced in hemodynamic and right ventricular hypertrophy analyses, histological analysis, cardiac biomarkers, hypoxia-inducible factor 1 alpha (HIF1α) levels, and biochemical studies. Furthermore, gene quantification by quantitative RT-PCR and Western blot analysis was also performed to examine its effect on the expression of key proteins in PAH.Notably, amongst three, derivative 16h exhibited the most encouraging results in molecular docking analysis, in vitro, in vivo, histopathological, biochemical, protein expression, and MD studies. Besides, derivative 16h also showed impressive pharmacokinetic features in ADMET analysis.In conclusion, derivative 16 h could act as a reliable ET receptor antagonist and requires further exploration to attain its therapeutic utility in PAH management.

T18/S19 diphosphorylation of myosin regulatory light chain impairs pulmonary artery relaxation in monocrotaline-induced pulmonary hypertensive rats.

Phosphorylation of Ser19 (S19-p) on the myosin regulatory light chain (MLC2) is critical for arterial contraction. It has been shown that elevated RhoA-dependent kinase (ROCK) activity or decreased MLC phosphatase (MLCP) activity leads to further phosphorylation of Thr18 (T18/S19-pp), which has been linked to vasospastic diseases. However, this phenomenon has not yet been studied in the context of pulmonary arterial hypertension (PAH). In the monocrotaline-induced PAH (PAH-MCT) rat model, we observed a significant delay in pulmonary artery (PA) relaxation following high potassium-induced contraction, which persisted even with the use of an L-type calcium channel blocker or in a calcium-free solution. Immunoblot analysis showed increased levels of both S19-p and T18/S19-pp in unstimulated PAs from PAH-MCT rats. Proteomics analysis revealed a reduction in soluble guanylate cyclase (sGC) and protein kinase G (PKG) levels, and immunoblotting confirmed decreased levels of MYPT1 (a component of MLCP) and increased ROCK in PAH-MCT. In the control PAs, the pharmacological inhibition of sGC with ODQ resulted in a prominent delay of relaxation and increased T18/S19-pp as in PAH-MCT. The delayed relaxation and the T18/S19-pp in PAH-MCT were reversed by ROCK inhibitor, Y27632, while not by membrane permeable 8-Br-cGMP. The delayed relaxation and T18/S19-diP in the ODQ-treated control PA were also reversed by Y27632. Taken together, the decreased sGC and MLCP, and increased ROCK increased T18/S19-pp, which leads to the decreased ability of PA to relax in PAH-MCT rats. PA specific inhibition of ROCK or activation of MLCP are expected to serve as potential drugs in the treatment of PAH.

Metabolic Deregulation in Pulmonary Hypertension

The high morbidity and mortality rate of pulmonary arterial hypertension (PAH) is partially explained by metabolic deregulation. The present study complements our previous publication in “Genes” by identifying significant increases of the glucose transporter solute carrier family 2 (Slc2a1), beta nerve growth factor (Ngf), and nuclear factor erythroid-derived 2-like 2 (Nfe2l2) in three standard PAH rat models. PAH was induced by subjecting the animals to hypoxia (HO), or by injecting with monocrotaline in either normal (CM) or hypoxic (HM) atmospheric conditions. The Western blot and double immunofluorescent experiments were complemented with novel analyses of previously published transcriptomic datasets of the animal lungs from the perspective of the Genomic Fabric Paradigm. We found substantial remodeling of the citrate cycle, pyruvate metabolism, glycolysis/gluconeogenesis, and fructose and mannose pathways. According to the transcriptomic distance, glycolysis/gluconeogenesis was the most affected functional pathway in all three PAH models. PAH decoupled the coordinated expression of many metabolic genes, and replaced phosphomannomutase 2 (Pmm2) with phosphomannomutase 1 (Pmm1) in the center of the fructose and mannose metabolism. We also found significant regulation of key genes involved in PAH channelopathies. In conclusion, our data show that metabolic dysregulation is a major PAH pathogenic factor.

Quantitative Proteomic and Phosphoproteomic Profiling of Lung Tissues from Pulmonary Arterial Hypertension Rat Model.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH.