Right Ventricular Hypertrophy Index Research Articles

GPS2 ameliorates cigarette smoking-induced pulmonary vascular remodeling by modulating the ras-Raf-ERK axis.

Mitogen-activated protein kinase (MAPK)signaling-mediated smoking-associated pulmonary vascular remodeling (PVR) plays an important role in the pathogenesis of group 3 pulmonary hypertension (PH). And G protein pathway suppressor 2 (GPS2) could suppress G-protein signaling such as Ras and MAPK, but its role in cigarette smoking -induced PVR (CS-PVR) is unclear. An in vivo model of smoke-exposed rats was constructed to assess the role of GPS2 in smoking-induced PH and PVR. In vitro, the effects of GPS2 overexpression and silencing on the function of human pulmonary arterial smooth cells (HPASMCs) and the underlying mechanisms were explored. GPS2 expression was downregulated in rat pulmonary arteries (PAs) and HPASMCs after CS exposure. More importantly, CS-exposed rats with GPS2 overexpression had lower right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness (WT%) than those without. And enhanced proliferation and migration of HPASMCs induced by cigarette smoking extract (CSE) can be evidently inhibited by overexpressed GPS2. Besides, GPS2siRNA significantly enhanced the proliferation, and migration of HPASMCs as well as activated Ras and Raf/ERK signaling, while these effects were inhibited by zoledronic acid (ZOL). In addition, GPS2 promoter methylation level in rat PAs and HPASMCs was increased after CS exposure, and 5-aza-2-deoxycytidine (5-aza) inhibited CSE-induced GPS2 hypermethylation and downregulation in vitro. GPS2 overexpression could improve the CS-PVR, suggesting that GPS2 might serve as a novel therapeutic target for PH-COPD in the future.

Berberine Alleviates Right Heart Dysfunction in MCT-Induced PAH Rats by Targeting Myocardial Mitochondrial Oxidative Phosphorylation

Background: Right ventricular failure (RVF) stands as a leading cause of mortality among pulmonary hypertension (PAH) patients. The underlying mechanisms contributing to Right Ventricular (RV) dysfunction remain enigmatic, with a conspicuous absence of effective treatments. RV functional deterioration is influenced by mitochondria oxidative phosphorylation and fatty acid oxidation. Berberine (BBR), a natural active compound extracted from Berberis vulgaris rhizomes, has shown promise for treating cardiovascular diseases due to its interactions with multiple intracellular targets. This study aimed to investigate the applicability and therapeutic effcacy of BBR in RVF and explore the mechanisms involved in its beneficial effects in monocrotaline (MCT)-induced PAH rats. Methods: Right ventricular hypertrophy (RVH) and RVF were induced via a single injection of MCT (60 mg/kg, ip) in Sprague-Dawley rats. BBR (4 mg/kg body weight per day) was administered through oral gavage. RVH and RVF were confirmed through transthoracic echocardiography (TTE). Transmission electron microscopy (TEM) was utilized to observe changes in cardiomyocyte and mitochondrial ultrastructure within the right ventricle tissue. Comparisons were made among Normal control (NC), RVH, RVH+BBR, RVF, and RVF+ BBR groups. RNA sequencing (RNA-seq) of right ventricle tissue was performed for both the NC (treated with normal saline, ip) and right ventricle dysfunction (RVH and RVF) groups. Gene Ontology and KEGG enrichment analyses were conducted using the R-package “Clusterprofiler.” RV transcriptome results were validated through qPCR and Western blot analysis. Results: Echocardiography and elevated ANP and BNP levels confirmed the presence of RVF following the development of MCT-induced PAH. TEM imaging revealed significant disruptions in mitochondrial ultrastructure, particularly in a subset of interfibrillar mitochondria, which exhibited swelling, disorganization, and reduced cristae density in RVH rats. These disruptions were even more pronounced in RVF rats. Transcriptomic profiles of RV tissue from RVH and RVF identified a total of 175 differentially expressed genes (DEGs). Gene Ontology and KEGG enrichment analyses pinpointed 38 genes associated with oxidative phosphorylation. qPCR demonstrated downregulation of mitochondrial oxidative phosphorylation-related genes (NDUFA2, NDUFA13, COX7B, COX6C, GSTK1, ECH1) in RVF. BBR ameliorated right heart function, leading to decreased RV internal diameter at end-diastole, a reduced RV hypertrophy index, and an increased RV fractional area change. This was accompanied by the recovery of ANP and BNP levels. Furthermore, after BBR application, the mitochondrial structure within RV tissue exhibited marked improvement. Lastly, Western blot analysis revealed that fatty acid oxidation-related proteins (Decr1, Acadm, Acacb, Hadh, Hadhb, Prkag3, Acaa2) were downregulated in right heart dysfunction, and BBR effectively mitigated the decline in oxidation-related protein levels in both RVH and RVF. Conclusion: In summary, our results underscore the critical role of BBR in preserving cardiac function from RVF in MCT-induced PAH rats. This preservation appears to occur through the upregulation of mitochondrial oxidative phosphorylation levels, ultimately enhancing energy metabolism within the mitochondria of RV cardiomyocytes in the right heart of PAH. This work is supported by Natural Science Foundation of Shandong Province(ZR2022MH052), China Postdoctoral Fund (NO.2021M691944) to XMY. 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.

Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

Rosmarinic acid ameliorates the complications of monocrotaline-induced right ventricular hypertrophy on the left ventricle: Investigating the signaling pathway of Wnt/β-catenin in the heart.

Right ventricular hypertrophy (RVH) often results in failure of the right ventricle or even the left ventricle. Rosmarinic acid (RA), a natural polyphenol, is commonly found in Boraginaceae species and some species of ferns and hornworts. This study looked at how RA affects oxidative stress and left ventricular hemodynamic functions as well as RVH in monocrotaline (MCT) induced RVH model rats. To cause RVH, MCT (60 mg/kg) was intraperitoneally (IP) injected. Rats were given saline or RA (10, 15, and 30 mg/kg, gavage, over 21 days). In anesthetized rats, the lead II electrocardiogram was recorded. The hemodynamic functions of the isolated heart were measured using the Langendorff apparatus (at constant pressure). Investigations were made into the right ventricular hypertrophy index (RVHI), the activities of superoxide dismutase, catalase, glutathione, and Wnt and β-catenin gene expressions in the left ventricle. H&E staining was used. A significant decline in electrocardiogram parameters and anti-oxidant enzyme activities, an increase in QTc (Q-T corrected) intervals, MDA (Malondialdehyde), RVHI, and Wnt/β-catenin gene expression, and also significant changes in the hemodynamic parameters were demonstrated in the MCT group. RA improved the above-mentioned factors. According to the findings, RA may act as a cardioprotective agent against cardiovascular complications brought on by RVH due to its capacity to boost the activity of cardiac anti-oxidant enzymes and decrease the expression of genes involved in vascular calcification.

Exosomes derived from mesenchymal stromal cells exert a therapeutic effect on hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway

ObjectiveHypoxic pulmonary hypertension (HPH) is a progressive and life-threatening disease characterized by perivascular inflammation, pulmonary vascular remodeling, and occlusion. Mesenchymal stromal cell-derived exosomes (MSC-exo) have emerged as potential therapeutic agents due to their role in cell communication and the transportation of bioactive molecules. In this study, we aimed to investigate the therapeutic effects of MSC-exo against HPH and elucidate the underlying molecular mechanism. MethodsExosomes were isolated from conditioned media of human bone mesenchymal stromal cells using ultracentrifugation and characterized through western blotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). An HPH animal model was established in male SD rats, and MSC-exo or phosphate-buffered saline (PBS) were administered via the tail vein for three weeks. Subsequently, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. Lung tissues from HPH rats and normal rats underwent high-throughput sequencing and transcriptomic analysis. Gene Ontology (GO) analysis was employed to identify upregulated differentially expressed genes. Additionally, rat pulmonary artery smooth muscle cells (PASMC) exposed to platelet-derived growth factor-BB (PDGF-BB) were used to simulate HPH-related pathological behavior. In vitro cellular models were established to examine the molecular mechanism of MSC-exo in HPH. ResultsMSC-exo administration protected rats from hypoxia-induced increases in RVSP, RVHI, and pulmonary vascular remodeling. Additionally, MSC-exo alleviated PDGF-BB-induced proliferation and migration of PASMC. Transcriptomic analysis revealed 267 upregulated genes in lung tissues of HPH rats compared to control rats. Gene Ontology analysis indicated significant differences in pathways associated with Yes Associated Protein 1 (YAP1), a key regulator of cell proliferation and organ size. RT-qPCR and western blot analysis confirmed significantly increased expression of YAP1 in HPH lung tissues and PASMC, which was inhibited by MSC-exo treatment. Furthermore, analysis of datasets demonstrated that Secreted Phosphoprotein 1 (SPP1), also known as Osteopontin (OPN), is a downstream binding protein of YAP1 and can be upregulated by PDGF-BB. MSC-exo treatment reduced the expression of both YAP1 and SPP1. Lentivirus-mediated knockdown of YAP1 inhibited PDGF-BB-induced PASMC proliferation, migration, and SPP1 protein levels. ConclusionOur findings demonstrate that MSC-exo exert a therapeutic effect against hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway. The inhibition of YAP1 and downstream SPP1 expression by MSC-exo may contribute to the attenuation of pulmonary vascular remodeling and PASMC proliferation and migration. These results suggest that MSC-exo could serve as a potential therapeutic strategy for the treatment of HPH. Further investigations are warranted to explore the clinical applicability of MSC-exo-based therapies in HPH patients.

RGS10 inhibits proliferation and migration of pulmonary arterial smooth muscle cell in pulmonary hypertension via AKT/mTORC1 signaling

ABSTRACT Objective: Excessive proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is a core event of pulmonary hypertension (PH). Regulators of G protein signaling 10 (RGS10) can regulate cellular proliferation and cardiopulmonary diseases. We demonstrate whether RGS10 also serves as a regulator of PH. Methods: PASMC was challenged by hypoxia to induce proliferation and migration. Adenovirus carrying Rgs10 gene (Ad-Rgs10) was used for external expression of Rgs10. Hypoxia/SU5416 or MCT was used to induce PH. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were used to validate the establishment of PH model. Results: RGS10 was downregulated in hypoxia-challenged PASMC. Ad-Rgs10 significantly suppressed proliferation and migration of PASMC after hypoxia stimulus, while silencing RGS10 showed contrary effect. Mechanistically, we observed that phosphorylation of S6 and 4E-Binding Protein 1 (4EBP1), the main downstream effectors of mammalian target of rapamycin complex 1 (mTORC1) as well as phosphorylation of AKT, the canonical upstream of mTORC1 in hypoxia-induced PASMC were negatively modulated by RGS10. Both recovering mTORC1 activity and restoring AKT activity abolished these effects of RGS10 on PASMC. More importantly, AKT activation also abolished the inhibitory role of RGS10 in mTORC1 activity in hypoxia-challenged PASMC. Finally, we also observed that overexpression of RGS10 in vivo ameliorated pulmonary vascular wall thickening and reducing RVSP and RVHI in mouse PH model. Conclusion: Our findings reveal the modulatory role of RGS10 in PASMC and PH via AKT/mTORC1 axis. Therefore, targeting RGS10 may serve as a novel potent method for the prevention against PH.”

Quercetin improves pulmonary arterial hypertension in rats by regulating the HMGB1/RAGE/NF-κB pathway

To explore the mechanism through which quercetin improves pulmonary arterial hypertension (PAH). Rat models of hypoxic pulmonary hypertension were established by exposure to hypoxia for 8-10 h each day (6 days a week for 4 weeks), and before each hypoxic exposure, the rats were given intragastric administration of 100 mg/kg quercetin or saline. After the treatments, the right ventricular systolic pressure (RVSP) and pulmonary artery systolic pressure of the rats were recorded. The right ventricular hypertrophy index (RVHI) was measured to evaluate right ventricular hypertrophy. HE staining was used to observe the remodeling of the pulmonary arterioles. The right cardiac function of the rats was evaluated by ultrasound. The protein levels of HMGB1, RAGE, NF-κB, Bax, Bcl-2 and cleaved caspase-3 in the lung tissue of the rats were detected using Western blotting. Compared with the rats maintained in normoxia, the rats with chronic hypoxic exposure showed significantly increased RVHI and RVSP (P<0.01), which were obviously lowered by quercetin treatment (P<0.01). HE staining showed significant pulmonary artery wall thickening with reduced lumen diameter in hypoxia group, and quercetin treatment effectively improved pulmonary vascular remodeling. Ultrasound examination revealed a significantly increased RVFW and a lowered PAT/PET ratio in hypoxia group (P<0.01), and such changes were ameliorated by quercetin treatment (P<0.01). Chronic hypoxia significantly increased the protein expressions of HMGB1 (P<0.01), RAGE, NF-κB and Bcl-2 (P<0.01) and lowered the protein expressions of Bax and cleaved caspase-3 (P<0.01); Quercetin treatment obviously lowered the protein expressions of HMGB1, NF-κB (P<0.05), RAGE (P<0.01) and (P<0.05) and increased the expressions of Bax and cleaved caspase-3 in the rat models (P<0.01). Quercetin improves pulmonary hypertension in rats possibly by promoting apoptosis through the HMGB1/RAGE/NF-κB pathway.

Compound Tinglizi Decoction intervenes COPD-associated pulmonary hypertension through regulation of HMGB1-mediated pyroptosis and immune imbalance

This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH) in rats and the intervening mechanism of Compound Tinglizi Decoction. Ninety rats were randomly divided into a normal group, a model group, low-dose, medium-dose, and high-dose Compound Tinglizi Decoction groups, and a simvastatin group. The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS) intravascular infusion, which lasted 60 days. Rats in the low, medium, and high-dose Compound Tinglizi Decoction groups were given 4.93, 9.87, and 19.74 g·kg~(-1) Compound Tinglizi Decoction by gavage, respectively. Rats in the simvastatin group were given 1.50 mg·kg~(-1) simvastatin by gavage. After 14 days, the lung function, mean pulmonary artery pressure, and arterial blood gas of rats were analyzed. Lung tissues of rats were collected for hematoxylin-eosin(HE) staining to observe the pathological changes. Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) was used to determine the expression of related mRNA in lung tissues, Western blot(WB) was used to determine the expression of related proteins in lung tissues, and enzyme linked immunosorbent assay(ELISA) was used to determine the levels of inflammatory factors in the lung tissues of rats. The ultrastructure of lung cells was observed by transmission electron microscope. The forced vital capacity(FVC), forced expiratory volume in 0.3 second(FEV_(0.3)), FEV_(0.3)/FVC, peek expiratory flow(PEF), respiratory dynamic compliance(Cdyn), arterial partial pressure of oxygen(PaO_2), and arterial oxygen saturation(SaO_2) were increased, and resistance of expiration(Re), mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index(RVHI), and arterial partial pressure of carbon dioxide(PaCO_2) were decreased by Compound Tinglizi Decoction in rats with COPD-PH. Compound Tinglizi Decoction inhibited the protein expression of HMGB1, receptor for advanced glycation end products(RAGE), pro caspase-8, cleaved caspase-8, and gasdermin D(GSDMD) in lung tissues of rats with COPD-PH, as well as the mRNA expression of HMGB1, RAGE, and caspase-8. Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction. Interferon-γ(IFN-γ) and interleukin-17(IL-17) were reduced, and interleukin-4(IL-4) and interleukin-10(IL-10) were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH. In addition, the lesion degree of trachea, alveoli, and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction. Compound Tinglizi Decoction had dose-dependent effects. The lung function, pulmonary artery pressure, arterial blood gas, inflammation, trachea, alveoli, and pulmonary artery disease have been improved by Compound Tinglizi Decoction, and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2), helper T cell 17(Th17)/regulatory T cell(Treg) imbalance.

Outcomes of pregnancy in mice with pulmonary hypertension induced by Hypoxia/SU5416

BackgroundPulmonary hypertension (PH) seriously affects the health of patients. We have found in clinical studies that PH has adverse effects on both maternal and offspring. ObjectiveTo establish a animal model of PH induced by hypoxia/SU5416 and observe the effects of PH on pregnant mice and their fetuses. MethodsTwenty-four C57 mice aged 7–9 weeks were selected and divided into 4 groups with 6 mice in each group. ① Female mice with normal oxygen; ② Female mice with hypoxia/SU5416; ③ Pregnant mice with normal oxygen; ④ Pregnant mice with hypoxia/SU5416. After 19 days, weight, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were compared in each group. Lung tissue and right ventricular blood were collected. The number and weight of fetal mice were also compared between the two pregnant groups. ResultsThere was no significant difference in RVSP and RVHI between female and pregnant mice under the same condition. Compared with normal oxygen condition, two groups of mice in hypoxia/SU5416 had poor development, RVSP and RVHI were significantly increased, the number of fetal mice was small, hypoplasia, degeneration and even abortion. ConclusionThe model of mice PH was successfully established. PH affects the development and health of female and pregnant mice, and seriously affects the fetuses.

Establishment of a canine model of pulmonary arterial hypertension induced by dehydromonocrotaline and ultrasonographic study of right ventricular remodeling

ABSTRACT Objective Pulmonary arterial hypertension (PAH) means high blood pressure in the lungs. We aimed to observe the right ventricular size, wall thickness and characteristic functional changes and their associations with PAH in an established model of beagle dogs, and to explore convenient, reliable and sensitive ultrasound indicators for assessing right ventricular remodeling. Methods Twenty healthy beagle dogs (8–10 kg) were randomly divided into control group (N-dimethylformamide, n = 10) and dehydromonocrotaline (DHMCT) group (DHMCT, n = 10). N-dimethylformamide or DHMCT was injected through a catheter into the right atrium, and then right heart catheterization, routine echocardiography and two-dimensional speckle tracking imaging (2D-STI) were performed before modeling (0 weeks) and 8, 14 weeks after modeling. Hemodynamic parameters and right ventricular function-related ultrasound data were acquired. At the end of the experiment, the animals were killed and the lung tissues were taken for HE staining. Left and right ventricular walls were separated and weighed respectively, and right ventricular hypertrophy index (RVHI) was measured. The associations of the routine ultrasound data and 2D-STI data at each time point with hemodynamic parameters and RVHI were analyzed. Results At 0, 8 and 14 weeks, gradual decreases in the right ventricular global longitudinal strain (RVLS) were found in DHMCT group. RVH occurred in DHMCT group, and DHMCT group had a significantly higher RVHI than that of control group (49.83 ± 4.83% vs. 39.80 ± 1.40%, P < .001) and larger pulmonary artery media thickness. RVLS had significant positive correlations with RVSP (r = 0.74, P < .001), mRVP (r = 0.72, P < .001), PASP (r = 0.75, P < .001), mPAP (r = 0.72, P < .001) and PVR (r = 0.68, P < .001). There was a significant positive correlation between RVLS and RVHI (r = 0.74, P < .001). Conclusion The right ventricular function in PAH can be effectively assessed by echocardiography, and RVLS measured by 2D-STI sensitively reflects right ventricular remodeling following PAH.

The protective role of EP300 in monocrotaline-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a lethal disease characterized by pulmonary vascular remodeling, which is mediated by the abnormal proliferation/migration of pulmonary arterial smooth muscle cells (PASMCs). Recent reports suggest the involvement of histone acetylation in PAH development and that histone deacetylase (HDAC) inhibitors have therapeutic potential for the treatment of PAH. EP300 is an acetyltransferase that plays diverse roles in cell proliferation, differentiation, and apoptosis. However, the functions of EP3000 in PH are rarely studied. In this work, we found that the expression of EP300 was increased in the pulmonary arteries of monocrotaline (MCT)-induced PH rats. Knockdown of EP300 by AAV-mediated shRNA exacerbated the PH, with a higher right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness in the pulmonary artery of MCT-induced PH rat. On the cellular level, the proliferation of PASMCs was promoted by EP300 knockdown. In addition, the expression of EP300 was increased in PASMCs by the overexpression of EGR1, while the deletion of EGR1 binding sites in the EP300 promoter region decreased the activity of EP300 promoter. Moreover, deleting the EP300 promoter region containing EGR1 binding sites using CRISPR/Cas9 abolished the upregulation of EP300 in MCT-induced rats and exacerbated MCT-induced PH. To summarize, our data indicate that EP300 upregulation mediated by EGR1 has a protective effect on MCT-induced PH. These findings showed EP300 expression was increased in the MCT-induced PH model in rats, which could be mediated by EGR1; the EP300 also displayed the potential to provide protection from PH.

Abstract 10753: Phosphodiesterase Type 5 Inhibition and C-c Chemokine Receptor-2 Disruption Synergistically Protect Against Pulmonary Arterial Hypertension in Monocrotaline-Treated Rats: Potential Synergism of Anti-inflammatory Agents and Pulmonary Vasodilator Therapy

BACKGROUND: Whether conventional pulmonary vasodilator therapy and anti-inflammatory agents synergistically protect against pulmonary arterial hypertension (PAH) is unknown. Hypothesis: We tested the hypothesis that a phosphodiesterase type 5 (PDE5) inhibitor and disruption of C-C chemokine receptor (CCR)-2, a receptor for MCP-1, synergistically protect against PAH in monocrotaline (MCT)-treated rats. Methods: Study1:Seven-week-old male Ccr2 (-/-) rats, generated by using the CRISPAR/ Cas9 genome editing technology, or wild-type littermates, treated with MCT (60mg/kg) or vehicle, were subjected to hemodynamic, morphometric and immunohistochemical analyses and mRNA and protein expression studies on day21 (n=40, 4 groups). Study 2: Tadarafil (10mg/kg/d), a PDE5 inhibitor, or vehicle was administered in Ccr2 (-/-) or wild-type MCT rats on days 21-42 (n=71, 4 groups) for evaluating survival and were evaluated hemodynamically on day28. Results: Study1: MCT treatment increased right ventricular systolic pressure (RVSP), an index of right ventricular hypertrophy (Fulton index), indices of pulmonary vascular diseases (%medial wall thickness, %muscularization) and the number of perivascular CD68-positive macrophages in WT rats, all of which were decreased by Ccr2 (-/-) (p&lt;.05). Similarly, an increase in mRNA expression of inflammatory cytokines (IL6 ,TNFa, MCP1, IL1b, TGFb) and a decrease in BMPR2 pathways (BMPR2, pSmad1/5/9 and ID1) as well as an increase in endothelial apoptosis (% cleaved Caspase 3 positive CD31 cells) and a decrease in NO pathways (eNOS and phospho-eNOS/eNOS) in lungs in wild-type MCT rats were reversed by Ccr2 (-/-) on day21 (p&lt;.05) with comparable PDE5 expression. Study2: Tadarafil administration or CCR2(-/-)(p=.03 and p=.02) alone improved the survival in wild type MCT rats. A double intervention synergistically improved the survival in separately treated groups (p=.02 vs CCR2(-/-), p=.04 vs Tadarafil) in MCT rats. Tadarafil treatment on days21-28 decreased RVSP and Fulton index in CCR2(-/-) MCT rats (p&lt;.05). Conclusions PDE5 inhibition and CCR-2 disruption synergistically protect against PAH in MCT-treated rats, suggesting an adjunctive role of a CCR2 inhibitor on top of administering a PDE5 inhibitor for PAH.

Effects of panax notoginseng saponins on pulmonary vascular remodeling in rats with pulmonary hypertension and its mechanisms

Objective: To investigate the effects of panax notoginseng saponins (PNS) on pulmonary vascular remodeling and SIRT1/FOXO3a/p27 pathway in pulmonary arterial hypertension (PAH) rats. Methods: Male SD rats weighing 200~250g were randomly divided into control group, monocrotaline group (MCT) and monocrotaline + panax notoginseng saponins group (MCT+PNS), with 10 rats in each group. The rats in control group were injected intraperitoneally with normal saline 3 ml/kg on the first day, then injected intraperitoneally with normal saline 2.5 ml/kg every day. The rats in MCT group were injected intraperitoneally with MCT 60 mg/kg on the first day, followed by daily injection of normal saline 2.5 ml/kg. In MCT+PNS group, 60 mg/kg MCT was injected intraperitoneally on the first day, and 50 mg/kg PNS was injected intraperitoneally every day. The above models were fed conventionally for 4 weeks. After the modeling was completed, the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) of rats in each group were detected by right heart catheter method, weighed and calculated right ventricular hypertrophy index (RVHI), and the pulmonary vascular structure and morphological changes were observed by HE and Masson staining. The protein and gene expressions of SIRT1, FOXO3a, p27, PCNA and Caspase-3 were detected by qPCR and Western blot. Results: Compared with control group, mPAP, RVSP and RVHI in MCT group were increased significantly (P＜0.01), pulmonary vessels were thickened significantly and collagen fibers were increased, protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were decreased (P＜0.05 or P＜0.01). The protein and gene expressions of PCNA were increased (P＜0.05). Compared with MCT group, the levels of mPAP, RVSP and RVHI in MCT+PNS group were decreased significantly (P＜0.05 or P＜0.01), pulmonary vascular thickening was alleviated and collagen fibers were reduced. The protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were increased (P＜0.05 or P＜0.01), while the protein and gene expressions of PCNA were decreased (P＜0.05 or P＜0.01). Conclusion: Panax notoginseng saponins can relieve pulmonary vascular remodeling in rats with pulmonary hypertension by activating SIRT1/FOXO3a/p27 pathway.

Human umbilical cord mesenchymal stem cells attenuate experimental pulmonary arterial hypertension

Abstract Background Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. Until now, there is still a lack of effective treatment applied. Experimental research has reported beneficial effects of mesenchymal stem cells (MSCs) therapy in PAH. However, there are few studies on human umbilical cord mesenchymal stem cells (hUC-MSCs) transplantation in the treatment of PAH. Purpose The purpose of the present study was to observe the protective effect of hUC-MSCs against experimental PAH and the colonization of hUC-MSCs in lung tissue. Methods Sprague Dawley rats were randomly divided into three groups: control group, monocrotaline (MCT)-induced PAH group, and stem cell therapy group (PAH+ hUC-MSCs). Rats with PAH were induced by a one-time intraperitoneal injection of 60mg/kg monocrotaline (PAH and PAH+hUC-MSCs groups) at the beginning of the experiment, while a control group received saline (SAL) instead. Huc-Mscs were treated by tail vein injection twice 1 and 2 weeks later (PAH+huc-MSC group dose: 3x106/each time), Ctr and PAH groups were given the same volume of SAL in tail vein. On day 28, Right ventricular systolic pressures (RVSP) were measured by right cardiac catheterization. WT% and WA% of pulmonary arterioles were evaluated by ipp6.0 Image Analysis Software. Right ventricular hypertrophy index (RVHI) were measured by weighing method. The colonization of hUC-MSCs in lung tissue was detected by immunohistochemistry. Results The RVHI and RVSP in MCT-induced PAH group were significantly higher than those in the control group (P&amp;lt;0.05). Compared with the control group, the WT% and the WA% were significantly increased in MCT-induced PAH group (P&amp;lt;0.05). The RVHI, RVSP, WT%, and the WA% in stem cell therapy group were significantly lower than those in the MCT-induced PAH group (P&amp;lt;0.05). Immunohistochemical examination of lung tissue showed that the nuclear staining of the stem cell therapy group was brown, while no staining was observed in the control group and PAH group. Conclusions In MCT-induced PAH, hUC-MSCs can transplanted into the lung tissues by tail vein injection. hUC-MSCs therapy can reduced lung vascular remodeling, improved hemodynamics. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Natural Science Fundation of China (82060018) and Natural Science Fundation of Yunnan (202101AS070043, 202102AA31003-7).

Saluisn-β contributes to endothelial dysfunction in monocrotaline-induced pulmonary arterial hypertensive rats

BackgroundThe endothelial dysfunction and the consequent attenuated pulmonary vasodilatation are the major causes of elevated pulmonary arterial resistance and pressure in pulmonary arterial hypertension (PAH). Current study aimed to explore the effects of a TOR2A gene product, salusin-β, on endothelium-dependent vascular relaxation and the progression of PAH in monocrotaline (MCT)-induced PAH rats as well as the relevant signaling pathway. MethodsAcetylcholine (ACh)-induced dose-dependent relaxation was used to evaluate the endothelium-dependent vasodilatation function. ResultsThe salusin-β level in plasma and pulmonary artery (PA) in MCT-PAH rats were significantly increased, while the ACh-induced endothelium-dependent vasodilatation was attenuated. After salusin-β incubation or overexpression of salusin-β gene, the endothelium-dependent relaxation was further deteriorated, while anti-salusin-β IgG incubation or knockdown of salusin-β improved it in PAH rats. The superoxide anions scavenger NAC or the antioxidant apocynin inhibited the effect of salusin-β, while the SOD inhibitor DETC further enhanced it. The nitric oxide (NO) synthase inhibitor L-NAME almost blocked the effect of anti-salusin-β IgG. Silencing of salusin-β in PAH rats decreased right ventricular (RV) systolic pressure, RV hypertrophy index, NAD(P)H oxidase activity and ROS level, and increased the eNOS activity and NO level of PA. Overexpression of salusin-β played opposite roles. ConclusionsThe elevated saluisn-β level in PAH rats plays important roles in the reduction of endothelium-dependent vasodilatation and participates in the progression of PAH through stimulating NAD(P)H oxidase-ROS production and inhibiting eNOS-NO release.

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

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. 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. 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. FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

The Mechanism of Volatile Oil of Rhodiola tangutica against Hypoxia-Induced Pulmonary Hypertension in Rats Based on RAS Pathway.

Materials and Methods Seventy-five male Sprague-Dawley (SD) rats were separated into control (Ctr), hypoxia (Hyp), and Hyp+VORA treatment (100 mg/kg/d, 80 mg/kg/d, and 40 mg/kg/d) groups in random. To achieve the chronic hypoxia condition, rats were kept inside the hypobaric chamber with automatically adjusted inner pressure as well as oxygen content equal to those of 4500 m in altitude for 4 continuous weeks. After 4 weeks, the rats' physiological parameters were determined (mean pulmonary artery pressure (mPAP); right ventricular hypertrophy index (RVHI)). Based on hematoxylin and eosin (HE) staining and transmission electron microscope (TEM), morphological features of their lung tissues were also analyzed. Proliferation of pulmonary arterial smooth muscle cells (PASMCs) was detected by MTS Cell Proliferation Colorimetric assay. The levels of glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in PASMCs were detected through corresponding kits, respectively. The protein levels in PASMCs and HPH rats were evaluated by Western blot (WB). Chemical components of VORA were detected through gas chromatography-mass spectrometer (GC-MS). Results After induced by hypoxia for 4 weeks, the mPAP and RVHI levels were increased significantly in hypoxia group in contrast to the Ctr group, indicating the establishment of HPH rat model. The subsequent administration of VORA decreased the mPAP and RVHI level. The vascular wall thickness and lumen size were also decreased after treated by VORA compared with Hyp group. Meanwhile, VORA suppressed the proliferation and oxidant stress in PASMCs. Therefore, the effect of VORA on decreasing vascular wall thickening and lumen size could be related to its antiproliferation effect on PASMCs. In addition, compared to the Hyp group, VORA downregulated the ACE, AngII, and AT1R protein expressions but increased ACE2 and MAS protein expressions (P < 0.05). A total of 48 constituents in VORA were identified by GC-MS in comparison with reference standards as well as the reference pieces of literatures. Conclusions HPH rat model as established based on the significant increased mPAP and RVHI. VORA presented a significant antihypoxia function plus an inhibiting effect on PASMC proliferation induced by hypoxia. Moreover, VORA treatment inhibited oxidative stress among PASMCs. With regard to the mechanism, VORA reduced ACE, AngII, and AT1R protein expressions but increased ACE2 and MAS protein expressions. There were 48 constituents in VORA identified by GC-MS.

Effects of YM155 on the proliferation and apoptosis of pulmonary artery smooth muscle cells in a rat model of high pulmonary blood flow-induced pulmonary arterial hypertension

ABSTRACT Introduction Proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) play an important role in the occurrence and development of pulmonary arterial hypertension (PAH). The purpose of this study was to investigate the effects of survivin inhibitor YM155 on the proliferation and apoptosis of PASMCs in rats with PAH induced by high pulmonary blood flow. Methods Thirty male Sprague-Dawley (SD) rats were randomly divided into control, model, and YM155 intervention groups. A rat model of PAH induced by high pulmonary blood flow was established, and it was confirmed by assessments of right-ventricular pressure (RVP) and right ventricular hypertrophy index (RVHI). Immunohistochemical staining and western blot analysis were used to detect the expression of survivin, and the proliferation and apoptosis of PASMCs. Lastly, the effects of in vivo treatment of YM155 were tested. Results The increased expression of survivin mRNA and protein were observed in the model group, accompanied by pulmonary arteriolar wall thickening, lumen stenosis, and perivascular inflammatory cell infiltration. Elevated expression of survivin and pulmonary vascular remodeling were significantly mitigated after YM155 treatment. Specifically, the YM155 intervention group had a significantly lower PASMC proliferation rate and a higher PASMC apoptotic rate. Conclusion YM155 suppressed PASMC proliferation and promoted PASMC apoptosis by inhibiting survivin expression and thereby reducing pulmonary vascular remodeling in high pulmonary blood flow-induced PAH in vivo.

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

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

Differential Expression Profile of microRNAs and Tight Junction in the Lung Tissues of Rat With Mitomycin-C-Induced Pulmonary Veno-Occlusive Disease.

BackgroundPulmonary veno-occlusive disease (PVOD) is characterized by increased pulmonary vascular resistance. Currently, there is a lack of effective treatment. It is of great significance to explore molecular targets for treatment. This study investigated the differential expression profile of miRNAs and tight junction in the lung tissues of rats with mitomycin-C (MMC)-induced PVOD.MethodsA total of 14 rats were divided into the control group and he PVOD group. We measured mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index (RVHI). Pathological changes including those in lung tissues, pulmonary venules, and capillary were detected by H&E and orcein staining. Western blot was used to detect GCN2, ZO-1, occludin, and claudin-5 expression. We analyzed the miRNAs profile in the rat lung tissues by high-throughput sequencing. The top differentially expressed miRNAs were validated by using real-time polymerase chain reaction (RT-PCR).ResultsThere were severe pulmonary artery hypertrophy/hyperplasia, thickening, and occlusion in the small pulmonary veins, pulmonary edema, and dilated capillaries in MMC-induced rats with PVOD. In addition, mPAP and RVHI were significantly increased (P < 0.05). The expression of GCN2 was significantly decreased (P < 0.05). A total of 106 differentially expressed miRNAs were identified. According to the fold changes, the top ten upregulated miRNAs were miRNA-543-3p, miRNA-802-5p, miRNA-493-3p, miRNA-539-3p, miRNA-495, miRNA-380-5p, miRNA-214-5p, miRNA-539-5p, miRNA-190a-3p, and miRNA-431. The top 10 downregulated miRNAs were miRNA-201-3p, miRNA-141-3p, miRNA-1912-3p, miRNA-500-5p, miRNA-3585-5p, miRNA-448-3p, miRNA-509-5p, miRNA-3585-3p, miRNA-449c-5p, and miRNA-509-3p. RT-PCR confirmed that miRNA-214-5p was upregulated, while miRNA-141-3p was downregulated (P < 0.05). Functional analysis showed various signaling pathways and metabolic processes, such as fatty acid biosynthesis, tight junction, and the mTOR signaling pathway. In addition, the expression of the tight junction-related protein of ZO-1, occludin, and claudin-5 was significantly decreased in rats with PVOD (P < 0.05).ConclusionmiRNAs may be involved in the pathogenesis of PVOD. Furthermore, ZO-1, occludin, and claudin-5 verification confirmed that the tight junction may be involved in the development of the disease.