Effect of IL-17 on pulmonary artery smooth muscle cells and connective tissue disease-associated pulmonary arterial hypertension.

OBJECTIVE: To explore the role of interleukin (IL)-17 in connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) and to investigate its possible mechanism on pulmonary artery smooth muscle cells (PASMCs). METHODS: Enzyme-linked immunosorbent assay (ELISA) were used to compare levels of serum IL-17 in patients with CTD-PAH and healthy controls (HCs). After treatment for 3 months, the serum IL-17 levels were tested in CTD-PAH. ELISA and immunohistochemistry were used to compare levels of serum IL-17 and numbers of pulmonary artery IL-17+ cells, respectively, in a rat model of monocrotaline-induced PAH and untreated rats. Proliferation, migration, and inflammatory factors expression of PASMCs were assessed after stimulation with different concentrations of IL-17 for various time periods. Proteins in the mitogen-activated protein kinase (MAPK) pathway were examined by western blot. RESULTS: Levels of IL-17 were upregulated in patients with CTD-PAH compared to HCs. After 3 months of treatment, serum IL-17 levels were downregulated with pulmonary artery pressure amelioration. Moreover, serum IL-17 levels and numbers of IL-17+ cells infiltrating lung arterioles were increased in PAH model rats. IL-17 could dose- and time-dependently promote proliferation and migration of PASMCs as well as time-dependently induce IL-6 and intercellular cell adhesion molecule-1 (ICAM-1) expression. The levels of MKK6 increased after IL-17 treatment. Inhibition of MAPK decreased proliferation of PASMCs. CONCLUSION: Levels of IL-17 may increase in CTD-PAH, and IL-17 promotes proliferation, migration, and secretion of IL-6 and ICAM in PASMCs, respectively, which likely involves the p-38 MAPK pathway.

Spermidine attenuates monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting purine metabolism and polyamine synthesis-associated vascular remodeling.

Ensuring the homeostatic integrity of pulmonary artery endothelial cells (PAECs) is essential for combatting pulmonary arterial hypertension (PAH), as it equips the cells to withstand microenvironmental challenges. Spermidine (SPD), a potent facilitator of autophagy, has been identified as a significant contributor to PAECs function and survival. Despite SPD's observed benefits, a comprehensive understanding of its protective mechanisms has remained elusive. Through an integrated approach combining metabolomics and molecular biology, this study uncovers the molecular pathways employed by SPD in mitigating PAH induced by monocrotaline (MCT) in a Sprague-Dawley rat model. The study demonstrates that SPD administration (5 mg/kg/day) significantly corrects right ventricular impairment and pathological changes in pulmonary tissues following MCT exposure (60 mg/kg). Metabolomic profiling identified a purine metabolism disorder in MCT-treated rats, which SPD effectively normalized, conferring a protective effect against PAH progression. Subsequent in vitro analysis showed that SPD (0.8 mM) reduces oxidative stress and apoptosis in PAECs challenged with Dehydromonocrotaline (MCTP, 50 µM), likely by downregulating purine nucleoside phosphorylase (PNP) and modulating polyamine biosynthesis through alterations in S-adenosylmethionine decarboxylase (AMD1) expression and the subsequent production of decarboxylated S-adenosylmethionine (dcSAM). These findings advocate SPD's dual inhibitory effect on PNP and AMD1 as a novel strategy to conserve cellular ATP and alleviate oxidative injuries, thus providing a foundation for SPD's potential therapeutic application in PAH treatment.

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

Purpose: 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. Methods: 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. Results: 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. Conclusion: 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.

The novel roles of YULINK in the migration, proliferation and glycolysis of pulmonary arterial smooth muscle cells: implications for pulmonary arterial hypertension.

BACKGROUND: Abnormal remodeling of the pulmonary vasculature, characterized by the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) along with dysregulated glycolysis, is a pathognomonic feature of pulmonary arterial hypertension (PAH). YULINK (MIOS, Entrez Gene: 54468), a newly identified gene, has been recently shown to possess pleiotropic physiologic functions. This study aims to determine novel roles of YULINK in the regulation of PAH-related pathogenesis, including PASMC migration, proliferation and glycolysis. RESULTS: Our results utilized two PAH-related cell models: PASMCs treated with platelet-derived growth factor (PDGF) and PASMCs harvested from monocrotaline (MCT)-induced PAH rats (PAH-PASMCs). YULINK modulation, either by knockdown or overexpression, was found to influence PASMC migration and proliferation in both models. Additionally, YULINK was implicated in glycolytic processes, impacting glucose uptake, glucose transporter 1 (GLUT1) expression, hexokinase II (HK-2) expression, and pyruvate production in PASMCs. Notably, YULINK and GLUT1 were observed to colocalize on PASMC membranes under PAH-related pathogenic conditions. Indeed, increased YULINK expression was also detected in the pulmonary artery of human PAH specimen. Furthermore, YULINK inhibition led to the suppression of platelet-derived growth factor receptor (PDGFR) and the phosphorylation of focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), and protein kinase B (AKT) in both cell models. These findings suggest that the effects of YULINK are potentially mediated through the PI3K-AKT signaling pathway. CONCLUSIONS: Our findings indicate that YULINK appears to play a crucial role in the migration, proliferation, and glycolysis in PASMCs and therefore positioning it as a novel promising therapeutic target for PAH.

Nobiletin attenuates monocrotaline-induced pulmonary arterial hypertension through PI3K/Akt/STAT3 pathway.

OBJECTIVES: Nobiletin is a flavonoid found in the peel of Citrus sinensis (oranges). The purpose of this study is to investigate whether Nobiletin can alleviate the monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and explore the underlying mechanisms. METHODS: The PAH rat model was replicated by subcutaneous injection of MCT. Nobiletin (1, 5 and 10 mg/kg) was administered by gavage from day 1 to day 21. After 21 days of MCT injection, the mean pulmonary artery pressure, pulmonary vascular resistance, Fulton Index, pulmonary artery remodelling, blood routine parameters, liver and kidney functions was measured. The level of inflammatory cytokines and PI3K/Akt/STAT3 were detected by qPCR, ELISA and western blot, the proliferation of pulmonary artery smooth muscle cells (PASMCs) was evaluated by CCK-8. KEY FINDINGS: Nobiletin (10 mg/kg) inhibited the MCT-induced increase in mean pulmonary artery pressure and pulmonary vascular resistance, right ventricular hypertrophy and pulmonary artery remodelling in rats. Nobiletin decreased the levels of inflammatory cytokines and phosphorylation level of PI3K/Akt/STAT3 in lungs of MCT-treated rats. Nobiletin inhibited the proliferation and lowered the inflammatory cytokines level induced by PDGF-BB in PASMCs. CONCLUSION: Nobiletin attenuates MCT-induced PAH, and the potential mechanism is to inhibit inflammation through PI3K/Akt/STAT3 pathway.

Curcumol suppresses endothelial-to-mesenchymal transition via inhibiting the AKT/GSK3ß signaling pathway and alleviates pulmonary arterial hypertension in rats.

Endothelial dysfunction is essential in pulmonary arterial hypertension (PAH) pathogenesis and is considered to be a therapeutic target of PAH. Curcumol is a bioactive sesquiterpenoid with pharmacological properties including restoring endothelial cells damage. This study aimed to evaluate the effect of curcumol on PAH rats and investigate its possible mechanisms. PAH was induced by subcutaneous injection of 60 mg/kg monocrotaline (MCT) in male Sprague Dawley rats. Curcumol (12.5, 25, and 50 mg/kg/day) were administered by intragastric administration for 3 weeks. The results demonstrated that curcumol dose-dependently alleviated MCT-induced right ventricular hypertrophy and pulmonary arterial wall thickness. In addition, endothelial-to-mesenchymal transition (EndMT) in the pulmonary arteries of MCT-challenged rats was inhibited after curcumol treatment, as evidenced by the restored expressions of endothelial and myofibroblast markers. The possible pharmacological mechanisms of curcumol were analyzed using network pharmacology. After screening the common therapeutic targets of PAH and curcumol by searching related databases and comparison, pathway enrichment was performed and AKT/GSK3ß was screened out as a possible signaling pathway which was relevant to the therapeutic mechanism of curcumol on PAH. Western blot analysis verified this in lung tissues. Moreover, combination of TNF-α, TGF-ß1 and IL-1ß-induced EndMT in primary rat pulmonary arterial endothelial cells were blocked by curcumol, and this effect was resembled by PI3K/AKT inhibitor LY294002. Above all, our study suggested that curcumol inhibited EndMT via inhibiting the AKT/GSK3ß signaling pathway, which may contribute to its alleviated effect on PAH. Curcumol may be developed as a therapeutic for PAH in the future.

A study of the efficacy of sacubitril/valsartan plus dapagliflozin combination treatment in pulmonary arterial hypertension due to left heart disease.

OBJECTIVE: To determine the efficacy of sacubitril/valsartan plus dapagliflozin in the treatment of patients with pulmonary arterial hypertension (PAH) due to left heart disease and to explore new treatment regimen for PAH due to left heart disease. METHODS: This study is a randomized controlled trial (RCT) study of 120 patients with PAH due to left heart disease admitted to the cardiovascular department of our hospital from Dec. 2019 to Dec. 2021. The patients were randomized 1:1 to the study group and control group. All patients were given baseline treatments targeting left heart disease and symptoms of PAH. In addition to the baseline treatments, patients in the control group were given sacubitril/valsartan tablets, while patients in the study group were given sacubitril/valsartan tablets plus dapagliflozin tablets. After 6 months of treatment, parameters including left heart function and exercise tolerance, Hemodynamics (left ventricular end systolic diameter [LVSED], left ventricular end diastolic diameter [LVEDD], left ventricular ejection fraction [LVEF], 6 min walk distance (6MWD), mean pulmonary artery pressure (mPAP) and pulmonary artery systolic pressure (PASP)), vascular endothelial function (plasma endothelin (ET) -1 and nitric oxide [NO]), heart failure markers (plasma N-terminal pro-brain natriuretic peptide (NT-proBNP)], inflammatory factors (serum C reactive protein [CRP], interleukin (IL)-6, and tumor necrosis factor (TNF)-α], and adverse drug reactions (ADRs) were assessed in both groups. RESULTS: Both groups had reduced LVESD and LVEDD, increased LVEF, and extended 6MWD after 6 months of treatment. The improvements in these parameters were significantly greater in the study group than in the control group (all P < 0.05). In addition, both the mPAP and PASP showed a decrease, and the mPAP and PASP in the study group were lower than those in the control group (p<0.05). Furthermore, both groups had decreased plasma ET-1 and NT-proBNP but increased plasma NO after 6 months of treatment. The improvements in these parameters were significantly greater in the study group than in the control group (all P < 0.05). Serum CRP, IL-6 and TNF-α levels were decreased in both groups after 6 months of treatment, and were significantly lower in the study group than in the control group (all P < 0.05). There was no significant difference in the overall incidence of ADRs between the two groups (P > 0.05). CONCLUSION: Sacubitril/valsartan plus dapagliflozin in the treatment with PAH due to left heart disease can improve left heart function of patients by improving vascular endothelial functions and alleviating inflammation, which helps to reduce the PAH process. Therefore, this combination treatment is safe and effective in PAH due to left heart disease.

[Pulmonary Vascular Remodeling Characteristics of Pulmonary Arterial Hypertension Mouse Model Induced by Left Pneumonectomy and Jugular Vein Injection of Monocrotaline Pyrrole].

Objective: To compare and investigate the differences and characteristics of pulmonary vascular remodeling in three mouse models of pulmonary arterial hypertension (PAH) constructed by left pneumonectomy, jugular vein injection of monocrotaline pyrrole, and left pneumonectomy combined with jugular vein injection of monocrotaline pyrrole, to explore for a PAH animal model that approximates the clinical pathogenesis of PAH, and to create a model that will provide sound basis for thorough investigation into the pathogenesis of severe PAH. Methods: 59 male C57/BL mice (10-12 weeks, 24-30 g) were randomized into four groups, a control group ( n=9), a group that had left pneumonectomy (PE, n=15), a group that had jugular vein injection of monocrotaline pyrrole (MCTP, n=15), and the last group that had left pneumonectomy combined with jugular injection of monocrotaline pyrrole (P+M, n=20). To evaluate the effect of modeling and the characteristics of pulmonary vascular remodeling, hemodynamic and morphological parameters, including right ventricular systolic pressure (RVSP), right ventricle/(left ventricle plus septum) (RV/LV+S), percent of wall thickness in the pulmonary artery (WT%), muscularization of non-muscular arteries, neointima formation, and vascular obstruction score (VOS), were measured in each group. Results: 1) Compared with those of the control group, the RVSP, RV/LV+S, WT%, and the degree of small pulmonary arteries muscularization in the P+M group were significantly increased ( P<0.01). The MCTP group had just slightly higher findings for these indicators ( P<0.05), while no significant change in these indicators was observed in the PE group ( P>0.05). 2) Neointima formation in the acinus pulmonary arteries, which caused obvious stenosis of the lumen, was observed in the P+M group, the VOS being 1.25±0.80 points ( P<0.001). In contrast, neointima formation was not observed in the MCTP group or the PE groups, the VOS being 0 point ( P>0.05). Conclusion: Left pneumonectomy combined with jugular intravenous injection of MCTP could induce severe PAH formation in mouse. The model provides a good simulation of neointima formation, the characteristic pathological change of clinical severe PAH.

Inhibition of Bruton's Tyrosine Kinase Alleviates Monocrotaline-Induced Pulmonary Arterial Hypertension by Modulating Macrophage Polarization.

Macrophage accumulation and activation contribute to the development of pulmonary arterial hypertension (PAH), while Bruton's tyrosine kinase (BTK) is an important regulator for the activation and polarization of macrophage. However, the role of BTK in PAH remains unknown. In the present study, a selective BTK inhibitor (BTKi) BGB-3111 was applied to investigate the role of BTK in monocrotaline- (MCT-) induced PAH rat and phorbol myristate acetate- (PMA-) differentiated U937 macrophages. Our results showed that BTK was mainly distributed and upregulated in CD68+ macrophages in the lungs of PAH rats. Daily treated with BTKi BGB-3111 alleviated MCT-induced PAH, as indicated by the decrease in right ventricular systolic pressure (RVSP), attenuation in right ventricle hypertrophy and pulmonary vascular remodeling, reduction in perivascular collagen deposition, as well as inhibition of inflammation and endothelial-to-mesenchymal transition (EndMT) in the lung. Moreover, BTK inhibition suppressed MCT-induced recruitment of macrophages, especially the classical activated macrophages (M1) in the lung. In vitro, BGB-3111 significantly suppressed lipopolysaccharide- (LPS-) induced M1 polarization and proinflammatory cytokine production in U937-derived macrophages. The underlying mechanism is associated with the inhibition of NF-κB/MAPK pathways and nucleotide-binding oligomerization domain-like receptor with pyrin domain 3 (NLRP3) inflammasome activation. Furthermore, macrophage conditioned medium (CM) from LPS-induced M1 macrophages promoted migration and EndMT of HPAECs, while CM from BGB-3111-pretreated LPS-induced M1 macrophages failed to induce this response. These findings suggest that BTK inhibition alleviates PAH by regulating macrophage recruitment and polarization and may be a potential therapeutic strategy for the treatment of PAH.

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

PURPOSE: It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. METHOD: PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. RESULTS: FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. CONCLUSION: FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

Dapagliflozin reduces the vulnerability of rats with pulmonary arterial hypertension-induced right heart failure to ventricular arrhythmia by restoring calcium handling.

BACKGROUND: Malignant ventricular arrhythmia (VA) is a major contributor to sudden cardiac death (SCD) in patients with pulmonary arterial hypertension (PAH)-induced right heart failure (RHF). Recently, dapagliflozin (DAPA), a sodium/glucose cotransporter-2 inhibitor (SGLT2i), has been found to exhibit cardioprotective effects in patients with left ventricular systolic dysfunction. In this study, we examined the effects of DAPA on VA vulnerability in a rat model of PAH-induced RHF. METHODS: Rats randomly received monocrotaline (MCT, 60 mg/kg) or vehicle via a single intraperitoneal injection. A day later, MCT-injected rats were randomly treated with placebo, low-dose DAPA (1 mg/kg/day), or high-dose (3 mg/kg/day) DAPA orally for 35 days. Echocardiographic analysis, haemodynamic experiments, and histological assessments were subsequently performed to confirm the presence of PAH-induced RHF. Right ventricle (RV) expression of calcium (Ca2+) handling proteins were detected via Western blotting. RV expression of connexin 43 (Cx43) was determined via immunohistochemical staining. An optical mapping study was performed to assess the electrophysiological characteristics in isolated hearts. Cellular Ca2+ imaging from RV cardiomyocytes (RVCMs) was recorded using Fura-2 AM or Fluo-4 AM. RESULTS: High-dose DAPA treatment attenuated RV structural remodelling, improved RV function, alleviated Cx43 remodelling, increased the conduction velocity, restored the expression of key Ca2+ handling proteins, increased the threshold for Ca2+ and action potential duration (APD) alternans, decreased susceptibility to spatially discordant APD alternans and spontaneous Ca2+ events, promoted cellular Ca2+ handling, and reduced VA vulnerability in PAH-induced RHF rats. Low-dose DAPA treatment also showed antiarrhythmic effects in hearts with PAH-induced RHF, although with a lower level of efficacy. CONCLUSION: DAPA administration reduced VA vulnerability in rats with PAH-induced RHF by improving RVCM Ca2+ handling.

Silencing TUFM Inhibits Development of Monocrotaline-Induced Pulmonary Hypertension by Regulating Mitochondrial Autophagy via AMPK/mTOR Signal Pathway.

Pulmonary arterial hypertension (PAH) is an extremely malignant cardiovascular disease which mainly involves the uncontrollable proliferation of the pulmonary arterial smooth muscular cells (PASMCs). Recent studies have confirmed that mitochondria play an important role in the pathogenesis of pulmonary hypertension through sensing cell hypoxia, energy metabolism conversion, and apoptosis. As a mitochondrial membrane protein, TUFM has been regarded to be related to mitochondrial autophagy (mitophagy), apoptosis, and oxidative stress. Considering these factors are closely associated with the pathogenesis of PAH, we hypothesize that TUFM might play a role in the development of PAH. Our preliminary examination has showed TUFM mainly expressed in the PASMCs, and the subsequent test indicated an increased TUFM expression in the SMCs of pulmonary arteriole in monocrotaline- (MCT-) induced PAH rat model compared with the normal rat. The TUFM knockdown (Sh-TUFM) or overexpressed (OE-TUFM) rats were used to establish PAH by treating with MCT. A notable lower pulmonary arterial systolic pressure together with slightly morphological changes of pulmonary arteriole was observed in the Sh-TUFM group compared with the single MCT-induced PAH group. Increased levels of P62 and Bax and reduced LC3II/I, BECN1, and Bcl2 were detected in the Sh-TUFM group, while the expressions of these proteins in the OE-TUFM group were contrast to the results of the Sh-TUFM group. To elucidate the possible mechanism underlying biological effect of TUFM in PAH, PASMCs were treated with silence or overexpression of TUFM and then exposed to hypoxia condition. An obviously high levels of P62 and Bax along with a decreased LC3 II/I, BECN1, ULK1, Atg12, Atg13, and Bcl2 levels were noticed in cells with silence of TUFM. Moreover, the phosphorylated AMPK and mTOR which was well known in mitophagy modulating vary by the alternation of TUFM. These observations suggested that TUFM silence inhibits the development of MCT-induced PAH via AMPK/mTOR pathway.

Paeoniflorin attenuates monocrotaline-induced pulmonary arterial hypertension in rats by suppressing TAK1-MAPK/NF-κB pathways.

Pulmonary arterial hypertension (PAH) characterized by pulmonary vascular remodeling is a lethal disease. Paeoniflorin (PF) is a monoterpene glycoside with numerous beneficial functions, such as vasodilation, anti-inflammation and immunomodulation. This study aims to investigate the effects of PF on monocrotaline (MCT)-induced PAH rats. Our data showed that both prophylactic or therapeutic administration of PF alleviated MCT-induced increasing of right ventricular systolic pressure (RVSP), prevented right ventricle hypertrophy and pulmonary arterial remodeling, as well as inhibited inflammatory cell infiltration around pulmonary arteries. Meanwhile, PF blocked MCT-induced endothelial-mesenchymal transition (EndMT) as indicated by the restored expression of endothelial markers in lung. Moreover, PF inhibited MCT-induced down-regulation of bone morphogenetic protein receptor 2 (BMPR2) and suppressed MCT-induced phosphorylation of transforming growth factor-ß (TGFß) activated kinase 1 (TAK1) in vivo. In vitro studies indicated that PF prevented human pulmonary arterial smooth muscle cells (PASMCs) from platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferation and migration. PF also partially reversed TGFß1, interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-α) co-stimulated endothelial-to-mesenchymal transition (EndMT) in cultured human pulmonary artery endothelial cells (HPAECs). Signaling pathway analysis demonstrated that the underlying mechanism might be associated with the inhibition of TAK1-MAPK/NF-κB pathways. Taken together, our results suggested that PF could be a potential drug for the treatment of PAH.

MicroRNA-663 prevents monocrotaline-induced pulmonary arterial hypertension by targeting TGF-ß1/smad2/3 signaling.

OBJECTIVE: Pulmonary vascular remodeling due to excessive growth factor production and pulmonary artery smooth muscle cells (PASMCs) proliferation is the hallmark feature of pulmonary arterial hypertension (PAH). Recent studies suggest that miR-663 is a potent modulator for tumorigenesis and atherosclerosis. However, whether miR-663 involves in pulmonary vascular remodeling is still unclear. METHODS AND RESULTS: By using quantitative RT-PCR, we found that miR-663 was highly expressed in normal human PASMCs. In contrast, circulating level of miR-663 dramatically reduced in PAH patients. In addition, in situ hybridization showed that expression of miR-663 was decreased in pulmonary vasculature of PAH patients. Furthermore, MTT and cell scratch-wound assay showed that transfection of miR-663 mimics significantly inhibited platelet derived growth factor (PDGF)-induced PASMCs proliferation and migration, while knockdown of miR-663 expression enhanced these effects. Mechanistically, dual-luciferase reporter assay revealed that miR-663 directly targets the 3'UTR of TGF-ß1. Moreover, western blots and ELISA results showed that miR-663 decreased PDGF-induced TGF-ß1 expression and secretion, which in turn suppressed the downstream smad2/3 phosphorylation and collagen I expression. Finally, intratracheal instillation of adeno-miR-663 efficiently inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy in monocrotaline (MCT)-induced PAH rat models. CONCLUSION: These results indicate that miR-663 is a potential biomarker for PAH. MiR-663 decreases PDGF-BB-induced PASMCs proliferation and prevents pulmonary vascular remodeling and right ventricular hypertrophy in MCT-PAH by targeting TGF-ß1/smad2/3 signaling. These findings suggest that miR-663 may represent as an attractive approach for the diagnosis and treatment for PAH.

Betaine alleviates right ventricular failure via regulation of Rho A/ROCK signaling pathway in rats with pulmonary arterial hypertension.

Pulmonary vascular remodeling was shown to lead to pulmonary arterial hypertension (PAH), further trigger excessive apoptosis of cardiomyocytes, and ultimately cause right ventricular failure (RVF), which involves the activation of Rho A/ROCK signaling pathway. Betaine has been found efficacious for attenuating PAH through its anti-inflammatory effects in our previous research while its effects on RVF due to PAH remains inconclusive. Thus, we attempted to elucidate the protective effects of betaine on PAH, RVF due to PAH as well as the potential mechanisms. To this end, male Sprague Dawley rats received a single subcutaneous injection of monocrotaline (50 mg/kg) to imitate PAH and RVF, and subsequently oral administration of betaine (100, 200, and 400 mg/kg/day). Betaine treatment improved the hemodynamics and histomorphological parameters and echocardiographic changes. Moreover, betaine also alleviated the pulmonary vascular remodeling and cardiomyocyte apoptosis. The mechanisms study revealed that administration of betaine significantly increased the expression of Rho A, ROCK1, and ROCK2. Furthermore, betaine alleviated the changes of its downstream molecules P53, Bcl-2, Bax, phosphorylated MYPT1 (p-MYPT1), total MYPT1 (t-MYPT1), p27kip1, and Cleaved Caspase-3. According to what we observed, this study indicated that betaine treatment could protect RVF due to PAH, which may be achieved through an altered Rho A/ROCK signaling pathway.

Penetrance of Severe Pulmonary Arterial Hypertension in Response to Vascular Endothelial Growth Factor Receptor 2 Blockade in a Genetically Prone Rat Model Is Reduced by Female Sex.

Background We have previously reported important strain differences in response to SU5416 (SU, a vascular endothelial growth factor receptor 2 inhibitor) in rats and have identified a specific colony of Sprague-Dawley rats that are hyperresponsive (SDHR) to SU alone and develop severe pulmonary arterial hypertension (PAH) with a single injection of SU, even in the absence of hypoxia. Interestingly, SDHR rats exhibit incomplete penetrance of the severe PAH phenotype with an "all-or-none" response to SU alone, which provides a unique opportunity to assess the influence of female sex and sex hormones on susceptibility to PAH after endothelial injury in a genetically prone model. Methods and Results SDHR rats were injected with SU (20 mg/kg SC) and, in the absence of hypoxia, 72% of male but only 27% of female rats developed severe PAH at 7 weeks, which was associated with persistent endothelial cell apoptosis. This sex difference in susceptibility for severe PAH was abolished by ovariectomy. Estradiol replacement, beginning 2 days before SU (prevention), inhibited lung endothelial cell apoptosis and completely abrogated severe PAH phenotype in both male and ovariectomized female rats, while progesterone was only protective in ovariectomized female rats. In contrast, delayed treatment of SDHR rats with established PAH with estradiol or progesterone (initiated at 4 weeks post-SU) failed to reduce lung endothelial cell apoptosis or improve PAH phenotype. Conclusions Female sex hormones markedly reduced susceptibility for the severe PAH phenotype in response to SU alone in a hyperresponsive rat strain by abolishing SU-induced endothelial cell apoptosis, but did not reverse severe PAH in established disease.

Integrated analysis of m6A mRNA methylation in rats with monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: N6-methyladenosine (m6A) modification is one of the most common chemical modifications of eukaryotic mRNAs, which play an important role in tumors and cardiovascular disease through regulating mRNA stability, splicing and translation. However, the changes of m6A mRNA and m6A-related enzymes in pulmonary arterial hypertension (PAH) remain largely unexplored. METHODS: MeRIP-seq was used to identify m6A methylation in lung tissues from control and MCT-PAH rats. Western blot and immunofluorescence were used to evaluate expression of m6A-related enzymes. RESULTS: Compared with control group, m6A methylation was mainly increased in lung tissues from MCT-PAH rats. The up-methylated coding genes in MCT-PAH rats were primarily enriched in processes associated with inflammation, glycolysis, ECM-receptor interaction and PDGF signal pathway, while genes with down-methylation were enriched in processes associated with TGF-ß family receptor members. The expression of FTO and ALKBH5 downregulated, METTL3 and YTHDF1 increased and other methylation modification-related proteins was not significantly changed in MCT-PAH rats lung tissues. Immunofluorescence indicated that expression of FTO decreased and YTHDF1 increased in small pulmonary arteries of MCT-PAH rats. CONCLUSION: m6A levels and the expression of methylation-related enzymes were altered in PAH rats, in which FTO and YTHDF1 may play a crucial role in m6A modification.

Aryl hydrocarbon receptor is essential for the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by arteriopathy in the small to medium-sized distal pulmonary arteries, often accompanied by infiltration of inflammatory cells. Aryl hydrocarbon receptor (AHR), a nuclear receptor/transcription factor, detoxifies xenobiotics and regulates the differentiation and function of various immune cells. However, the role of AHR in the pathogenesis of PAH is largely unknown. Here, we explore the role of AHR in the pathogenesis of PAH. AHR agonistic activity in serum was significantly higher in PAH patients than in healthy volunteers and was associated with poor prognosis of PAH. Sprague-Dawley rats treated with the potent endogenous AHR agonist, 6-formylindolo[3,2-b]carbazole, in combination with hypoxia develop severe pulmonary hypertension (PH) with plexiform-like lesions, whereas Sprague-Dawley rats treated with the potent vascular endothelial growth factor receptor 2 inhibitors did not. Ahr-knockout (Ahr-/- ) rats generated using the CRISPR/Cas9 system did not develop PH in the SU5416/hypoxia model. A diet containing Qing-Dai, a Chinese herbal drug, in combination with hypoxia led to development of PH in Ahr+/+ rats, but not in Ahr-/- rats. RNA-seq analysis, chromatin immunoprecipitation (ChIP)-seq analysis, immunohistochemical analysis, and bone marrow transplantation experiments show that activation of several inflammatory signaling pathways was up-regulated in endothelial cells and peripheral blood mononuclear cells, which led to infiltration of CD4+ IL-21+ T cells and MRC1+ macrophages into vascular lesions in an AHR-dependent manner. Taken together, AHR plays crucial roles in the development and progression of PAH, and the AHR-signaling pathway represents a promising therapeutic target for PAH.

Phenotype and function of macrophage polarization in monocrotaline-induced pulmonary arterial hypertension rat model.

Pulmonary arterial hypertension (PAH) isa fatal disease characterized by vascular remodeling and chronic inflammation. Macrophages are the key orchestrators of inflammatory and repair responses, and have been demonstrated to be vital in the pathogenesis of PAH. However, specific phenotype of macrophage polarization (M1 & M2 macrophage) in the development of PAH and the underlying mechanisms how they work are still largely unclear. A rat model of monocrotaline (MCT) induced PAH was used. Hemodynamic analysis and histopathological experiments were conducted at day 3, 7, 14, 21 and 28, respectively. In PAH rat lung tissue, confocal microscopic images showed that CD68+NOS2+ M1-like macrophages were remarkably infiltrated on early stage, but dramatically decreased in mid-late stage. Meanwhile, CD68+CD206+ M2-like macrophages in lung tissue accumulated gradually since day 7 to day 28, and the relative ratio of M2/M1 macrophage increased over time. Results detected by western blot and immunohistochemistry were consistent. Further vitro functional studies revealed the possible mechanism involved in this pathophysiological process. By using Transwell co-culture system, it was found that M1 macrophages inducedendothelial cellapoptosis, while M2 macrophages significantly promoted proliferation of both endothelial cell and smooth muscle cell.These data preliminarily demonstrated a temporal dynamic change of macrophage M1/M2 polarization status in the development of experimental PAH. M1 macrophages participated in the initial stage of inflammation by accelerating apoptosis of endothelial cell, while M2 macrophages predominated in the reparative stage of inflammation and the followed stage of aberrant tissue remodeling.

Selective deletion of connective tissue growth factor attenuates experimentally-induced pulmonary fibrosis and pulmonary arterial hypertension.

Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-ß-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia/Sugen model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease.