Hypoxia-induced Pulmonary Hypertension In Rats Research Articles

Hydroxycitric Acid Tripotassium Hydrate Attenuates Monocrotaline and Hypoxia-Induced Pulmonary Hypertension in Rats.

Pulmonary hypertension was induced in male Sprague-Dawley rats by a single subcutaneous injection of monocrotaline or hypoxic chamber. In vivo, inflammatory cytokine (including TNF-α, IL-1β, IL-6, and TGF-β, the level of SOD) expression, superoxide dismutase and hydrogen peroxide levels, and p-IκBα and p65 expressions were detected. In vitro, pulmonary artery smooth muscle cell proliferation and migration, ROS production, and hypoxia-inducible factor-1 expression were also studied. Hydroxycitric acid tripotassium hydrate decreased right ventricular systolic pressure and reduced right ventricular fibrosis and pulmonary vascular remodeling in rats with two kinds of pulmonary hypertension. Moreover, the expression of both inflammatory and oxidative stress factors was effectively reduced, and the p65 signaling pathway was found to be inhibited in this study. Additionally, hydroxycitric acid tripotassium hydrate inhibited human pulmonary artery smooth cell proliferation and migration in vitro. This study shows that hydroxycitric acid tripotassium hydrate can alleviate pulmonary hypertension caused by hypoxia and monocycloline in rats, improve remodeling of the right ventricle and pulmonary artery, and inhibit pulmonary artery smooth muscle cell proliferation and migration. The protective effects may be achieved by regulating inflammation and oxidative stress through the p65 signaling pathway.

Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway.

Proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) contribute to hypoxia-induced pulmonary hypertension (HPH). The transcription factor Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) has been implicated in the control of tumor cells and mesenchymal stem cell (MSC) and cardiomyocyte growth or migration. Whether Cited2 is involved in the proliferation and migration of PASMCs and the underlying mechanisms deserve to be explored. Cited2 expression was detected in rat PASMCs under hypoxia conditions and HPH rat models. The effect of Cited2 on the proliferation and migration of PASMC was detected by overexpression or knockdown of the Cited2 gene. After PAMSCs were treated with recombinant TGF-β1 and the lentivirus vector overexpressing Cited2, expression of peroxisome proliferator-activated receptor gamma (PPARγ) was examined by western blotting. We revealed that hypoxia down-regulated the expression of Cited2 in PASMCs and rat pulmonary arteries. Cited2 overexpression inhibited the proliferation and migration of PASMCs under hypoxia, while Cited2 knockdown induced the proliferation and migration of PASMCs. Cited2 inhibits the negative regulation of the TGF-β1 pathway on PPARγ to inhibit the proliferation and migration of PASMCs. These findings suggest that increased Cited2 expression contributes to the inhibition of PASMCs proliferation and migration by regulating TGF-β1-mediated target gene expression in HPH and provides a new target for molecular therapy of HPH.

Tsantan Sumtang, a traditional Tibetan medicine, protects pulmonary vascular endothelial function of hypoxia-induced pulmonary hypertension rats through AKT/eNOS signaling pathway

Ethnopharmacological relevanceTsantan Sumtang (TS), originated from the Four Tantras, is an empirical Tibetan medicine prescription, which has been widely used for treating cardiovascular diseases in the clinic in Qinghai Province of China. Our previous studies found that TS alleviated hypoxia-induced pulmonary hypertension (HPH) in rats. However, the effect and bioactive fractions of TS on hypoxia-injured pulmonary vascular endothelium are unknown. Aim of the studyTo investigate the effect, bioactive fractions and pharmacological mechanism of TS on hypoxia-injured pulmonary vascular endothelium in vivo and in vitro. Materials and methodsIn vivo studies, HPH animal model was established, and TS was administrated for four weeks. Then, hemodynamic indexes, ex vivo pulmonary artery perfusion experiment, morphological characteristics, nitric oxide (NO) production, and the protein expression of protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK)/eNOS signaling were determined. In vitro studies, 1% O2-induced pulmonary artery endothelial cells (PAECs) injury model was applied for screening bioactive fractions of TS by cell proliferation assay and NO production measurement. The associated proteins of AKT/eNOS signaling were further measured to elucidate underlying mechanism of bioactive fraction of TS via using phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. Ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) was used to reveal the chemical profile of bioactive fraction of TS. ResultsTS showed protective effect on the integrity of distal pulmonary arterial endothelium in HPH rats. Tsantan Sumtang dilated pulmonary arterial rings in HPH rats. TS enhanced NO bioavailability in lung tissue via regulating AKT/eNOS signaling. Furthermore, in the cellular level, cell viability as well as NO content of hypoxia-injured PAECs were elevated by fraction 17 of water extract of TS (WTS), through activating the AKT/eNOS signaling. Ellagic acid could be one of compositions in fraction 17 of WTS to produce NO in hypoxia-injured PAECs. ConclusionTS restored pulmonary arterial endothelial function in HPH rats. The bioactive fraction 17 was screened, which protected hypoxia-injured PAECs via upregulating AKT/eNOS signaling.

Hub Genes and Immune Cell Infiltration in Hypoxia-Induced Pulmonary Hypertension: Bioinformatics Analysis and In Vivo Validation.

Hypoxia-induced pulmonary hypertension (HPH) represents a severe pulmonary disorder with high morbidity and mortality, which necessitates identifying the critical molecular mechanisms underlying HPH pathogenesis. The mRNA expression microarray GSE15197 (containing 8 pulmonary tissues from HPH and 13 normal controls) was downloaded from Gene Expression Omnibus (GEO). Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) were executed by RStudio software. The Protein-Protein Interaction (PPI) network was visualized and established using Cytoscape, and the cytoHubba app from Cytoscape was used to pick out the hub modules. The infiltration of immune cells in HPH was analyzed using the CIBERSORTx. To confirm the potential hub genes, real-time quantitative reverse transcription PCR (qRT-PCR) was conducted using lung tissues of rat HPH models and controls. A total of 852 upregulated and 547 downregulated genes were identified. The top terms in biological processes were apoptosis, proliferation, and regulation of the MAPK cascade, including ERK1/2. Cytoplasm, cytosol, and membrane were enriched in cellular component groups. Molecular functions mainly focus on protein binding, protein serine/threonine kinase activity and identical protein binding. KEGG analysis identified pathways in cancer, regulation of actin cytoskeleton and rap1 signaling pathway. There was significantly different immune cell infiltration between HPH and normal control samples. High proportions of the memory subsets of B cells and CD4 cells, Macrophages M2 subtype, and resting Dendritic cells were found in HPH samples, while high proportions of naive CD4 cells and resting mast cells were found in normal control samples. The qRT-PCR results showed that among the ten identified hub modules, FBXL3, FBXL13 and XCL1 mRNA levels were upregulated, while NEDD4L, NPFFR2 and EDN3 were downregulated in HPH rats compared with control rats. Our study revealed the key genes and the involvement of immune cell infiltration in HPH, thus providing new insight into the pathogenesis of HPH and potential treatment targets for patients with HPH.

Restoration of miR-328a-5p function curtails hypoxic pulmonary hypertension through a mechanism involving PIN1/GSK3β/β-catenin axis

Recent evidence has highlighted the involvement of microRNAs (miRs) in hypoxic pulmonary hypertension (PH), which can be induced under hypoxic conditions. We intend to explore whether the miR-328a-5p/PIN1 axis affects hypoxic PH by regulating the GSK3β/β-catenin signaling pathway. The GEO database was retrieved to single out key miRs affecting hypoxic PH. It was observed that downregulation of miR-328a-5p occurred in hypoxia-induced PH samples. The binding affinity between miR-328a-5p to PIN1 was predicted by a bioinformatics tool and verified using a dual luciferase reporter gene assay. Rat primary pulmonary artery smooth muscle cells (PASMCs) were exposed to hypoxia for in vitro cell experiments. miR-328a-5p could target and downregulate PIN1 expression, leading to suppressed GSK3β/β-catenin activation. In addition, GSK3β/β-catenin inactivation curtailed hypoxia-induced vascular inflammatory responses and proliferation and migration in PASMCs in vitro. A hypoxic PH model was established in SD rats to observe the effects of miR-328a-5p on hemodynamic parameters and right heart remodeling. It was demonstrated in vivo that miR-328a-5p downregulated PIN1 expression to suppress GSK3β/β-catenin signaling, thereby reducing the vascular inflammatory response and alleviating disease progression in hypoxia-induced PH rats. The evidence provided by our study highlighted the involvement of miR-328a-5p in the translational suppression of PIN1 and the blockade of the GSK3β/β-catenin signaling pathway, resulting in attenuation of hypoxic PH progression.

4-Terpineol attenuates pulmonary vascular remodeling via suppressing PI3K/Akt signaling pathway in hypoxia-induced pulmonary hypertension rats

The hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) plays a pivotal role in pulmonary arterial remodeling (PAR) of hypoxia-induced pulmonary hypertension (HPH). 4-Terpineol is a constituent of Myristic fragrant volatile oil in Santan Sumtang. Our previous study found that Myristic fragrant volatile oil alleviated PAR in HPH rats. However, the effect and pharmacological mechanism of 4-terpineol in HPH rats remain unexplored. Male Sprague-Dawley rats were exposed to hypobaric hypoxia chamber (simulated altitudes of 4500 m) for 4 weeks to establish an HPH model in this study. During this period, rats were intragastrically administrated with 4-terpineol or sildenafil. After that, hemodynamic indexes and histopathological changes were assessed. Moreover, a hypoxia-induced cellular proliferative model was established by exposing PASMCs to 3% O2. PASMCs were pretreated with 4-terpineol or LY294002 to explore whether 4-terpineol targeted PI3K/Akt signaling pathway. The PI3K/Akt-related proteins expression was also accessed in lung tissues of HPH rats. We found that 4-terpineol attenuated mPAP and PAR in HPH rats. Then, cellular experiments showed 4-terpineol inhibited hypoxia-induced PASMCs proliferation via down-regulating PI3K/Akt expression. Furthermore, 4-terpineol decreased the p-Akt, p-p38, and p-GSK-3β protein expression, as well as reduced the PCNA, CDK4, Bcl-2 and Cyclin D1 protein levels, while increasing levels of cleaved caspase 3, Bax, and p27kip1in lung tissues of HPH rats. Our results suggested that 4-terpineol mitigated PAR in HPH rats by inhibiting the proliferation and inducing apoptosis of PASMCs through suppression of the PI3K/Akt-related signaling pathway.

Kaempferol ameliorates pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension rats via regulating Akt-GSK3β-cyclin axis

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is considered a major contributor to elevated pulmonary vascular resistance and a key mechanism of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Kaempferol is a natural flavonoid compound and can be derived from numerous common medicinal herbs and vegetables, which exhibit antiproliferative and proapoptotic properties, however, the effects of kaempferol on vascular remodeling in HPH remain unexplored. In this study, SD rats were placed in a hypobaric hypoxia chamber for four weeks to establish a pulmonary hypertension model and given either kaempferol or sildenafil (an inhibitor of PDE-5) during days 1–28, after which the hemodynamic parameter and pulmonary vascular morphometry were assessed. Furthermore, primary rat PASMCs were exposed to hypoxic conditions to generate a cell proliferation model, then incubated with either kaempferol or LY294002 (an inhibitor of PI3K). Immunoblotting and real-time quantitative PCR assessed the protein and mRNA expression levels in HPH rat lungs and PASMCs. We found that kaempferol reduced pulmonary artery pressure and pulmonary vascular remodeling, and alleviated right ventricular hypertrophy in HPH rats. The mechanistic analysis demonstrated that kaempferol reduced the protein levels of phosphorylation of Akt and GSK3β, leading to decreased expression of pro-proliferation (CDK2, CDK4, Cyclin D1, and PCNA) and anti-apoptotic related proteins (Bcl-2) and increased expression of pro-apoptosis proteins (Bax and cleaved caspase 3). These results collectively demonstrate that kaempferol ameliorates HPH in rats by inhibiting PASMC proliferation and pro-apoptosis via modulation of the Akt/GSK3β/CyclinD axis.

Comparison of hypoxia-induced pulmonary hypertension rat models caused by different hypoxia protocols

Background and aim: Pulmonary hypertension (PH) is a serious and even fatal disorder with limited treatment strategies. The hypoxia-induced pulmonary hypertension (HPH) rat model is commonly used in this field. While the HPH rat model has strong predictability and repeatability, the model is a chronic model, making it time-consuming, costly, and complicated and limiting the progress of the experiments. Currently, there is no uniform international standard for the HPH model. Our study aimed to find a relatively effective and efficient HPH modeling protocol. Methods: We established HPH rat models with different total hypoxia periods and different daily hypoxia times, and assessed different hypoxia modeling modes in multiple dimensions, such as haemodynamics, right ventricular (RV) hypertrophy, pulmonary arterial remodeling, muscularization, inflammation, and collagen deposition. Results: Longer daily hypoxia time resulted in higher mean pulmonary arterial pressure (mPAP)/right ventricular systolic pressure (RVSP) and more obvious RV hypertrophy, as well as more severe pulmonary arterial remodeling and muscularization, regardless of the total period of hypoxia (3- or 4-week). Moreover, pulmonary perivascular macrophages and collagen deposition showed daily hypoxia time-dependent increases, both in 3- and 4-week hypoxia groups. Conclusion: Our findings showed that the 3-week continuous hypoxia mode was a relatively efficient way to reduce the time needed to induce significant disease phenotypes, which offered methodological evidence for future studies in building HPH models.

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.

Attenuating effect of magnesium on pulmonary arterial calcification in rodent models of pulmonary hypertension.

Vascular calcification has been considered as a potential therapeutic target in pulmonary hypertension. Mg2+ has a protective role against calcification. This study aimed to investigate whether Mg2+ could alleviate pulmonary hypertension by reducing medial calcification of pulmonary arteries. Monocrotaline (MCT)-induced and chronic hypoxia-induced pulmonary hypertension rats were given an oral administration of 10% MgSO4 (10 ml/kg per day). Additionally, we administered Mg2+ in calcified pulmonary artery smooth muscle cells (PASMCs) after incubating with β-glycerophosphate (β-GP, 10 mmol/l). In vivo, MCT-induced and chronic hypoxia-induced pulmonary hypertension indexes, including right ventricular systolic pressure, right ventricular mass index, and arterial wall thickness, as well as Alizarin Red S (ARS) staining-visualized calcium deposition, high calcium levels, and osteochondrogenic differentiation in pulmonary arteries, were mitigated by dietary Mg2+ intake. In vitro, β-GP-induced calcium-rich deposits stained by ARS, calcium content, as well as the detrimental effects of calcification to proliferation, migration, and resistance to apoptosis of PASMCs were alleviated by high Mg2+ but exacerbated by low Mg2+. Expression levels of mRNA and protein of β-GP-induced osteochondrogenic markers, RUNX Family Transcription Factor 2, and Msh Homeobox 2 were decreased by high Mg2+ but increased by low Mg2+; however, Mg2+ did not affect β-GP-induced expression of SRY-Box Transcription Factor 9. Moreover, mRNA expression and protein levels of β-GP-reduced calcification inhibitor, Matrix GLA protein was increased by high Mg2+ but decreased by low Mg2+. Mg2+ supplement is a powerful strategy to treat pulmonary hypertension by mitigating pulmonary arterial calcification as the calcification triggered physiological and pathological changes to PASMCs.

Abstract P2024: Novel Inhibitor Of P38 Mitogen-activated Protein Kinase Reverses Hypoxia-induced Pulmonary Hypertension In Rats

Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular changes with subsequent right ventricular (RV) failure. This work investigated the hypothesis that a new p38-alpha mitogen-activated protein kinase (p38-alpha MAPK) inhibitor, named LASSBio-1824, could interfere on PH model in rats. Methods: HP was induced in male Wistar rats through the exposure to hypoxia (10% O2) during 3 weeks, in combination to i.p. injection of an antagonist of vascular endothelial growth factor receptor (SU5416; 20 mg/kg/weekly). Control rats were kept in normoxia. After 3 weeks, echocardiography images confirmed PH using the pulmonary artery (PA) outflow waveform. After disease onset, animals were randomly divided into three groups: control + vehicle (DMSO), PH + vehicle and PH + LASSBio-1824 (50 mg/kg/day), in which vehicle and LASSBio-1824 were orally administered (gavage) during two weeks. All experiments were in accordance with the Animal Care and Use Committee at Federal University of Rio de Janeiro (039/19). Results: Pulmonary acceleration time (PAT; ms) was reduced from 40.0 ± 2.1 (control) to 24.2 ± 1.8 in PH + vehicle group (p &lt; 0.05) and restored to 36.8 ± 2.9 after treatment with LASSBio-1824 (p &lt; 0.05). RV afterload was detected in PH group because of the increase of systolic pressure (mmHg) from 23.2 ± 1.5 (control) to 46.7 ± 2.8 (p &lt; 0.05) which was reduced to 19.4 ± 2.7 with LASSBio1824 treatment. Medial wall thickness (%) of distal pulmonary arteries (PA, &lt; 50 μm) was evaluated by using immunohistochemistry for alpha smooth muscle actin (alpha-SMA) which was increased from 64.0 ± 4.9 (control) to 80.8 ± 2.7 in PH group (p &lt; 0.05), but it was significantly reduced to 55.0 ± 1.3 after treatment with the inhibitor (p &lt; 0.05). Acetylcholine (ACh)-induced maximal relaxation (%) of PA from normal and PH rats, was reduced from 67.5 ± 1.6 to 51.9 ± 4.9 (p &lt; 0.05). Therefore, the treatment with the p38 inhibitor normalized vascular reactivity to 73.0 ± 3.3 %. The immunohistochemistry for c-fos protein, a product of the p38 pathway related to cell proliferation, showed an increase of stained myocyte nuclei (%) in PH animals of 36.5 ± 1.5 when compared to normal group of 20.2 ± 4.2. The inhibitor significantly reduced ratio to 24.0 ± 2.3% (p &lt; 0.05). PH increased the inflammatory condition because the expression of inducible nitric oxide synthase altered from 10.4 ± 1.9% (control) to 31.9 ± 2.2% and it was recovered by the inhibitor to 15.5 ± 2.2% (p &lt; 0.05). In conclusion, the new p38-alpha MAPK inhibitor represents an important approach for the future treatment of PH, since it improves the underlying remodeling and inflammation processes.

Luteolin ameliorates hypoxia-induced pulmonary hypertension via regulating HIF-2α-Arg-NO axis and PI3K-AKT-eNOS-NO signaling pathway

BackgroundPulmonary hypertension (PH) is a devastating disease with poor prognosis and high mortality. Hypoxia induced pulmonary hypertension (HPH) is a persistent threat to human health, especially to people who live on high altitude plateau. Pulmonary vascular endothelial cell is involved in numerous pathophysiological processes, including in vasoconstriction, oxidative stress, cell growth and differentiation. Endothelial cells (ECs) are the first layer to be exposed to changed oxygen levels and hypoxia could lead to ECs dysfunction. Endothelial-derived nitric oxide (NO) is the most important bioactive molecule, which could regulate endothelial homeostasis. PH pathophysiology has been linked to the disruption of NO pathways. PurposeLuteolin is a kind of plant active ingredient with multiple pharmacological activities. The purpose of this study is to detect the effect of luteolin on HPH with in vivo, ex vivo and in vitro analyses and to further elucidate luteolin's pharmaceutical mechanism with NO related signaling pathway regulation. MethodsHypobaric chamber was used to establish HPH animal model. Rats were intragastrically administrated luteolin for 28 days. Then hemodynamic indexes, histopathological changes, pulmonary artery endothelial function, NO content and arginase activity in lung tissue, NO related pathway proteins expression were measured to evaluate the effect of luteolin on HPH. PAECs were treated with 1% O2 and incubated with or without luteolin. PAECs vitality, NO content in cells supernatant, and NO related pathway proteins expression were tested to reveal the protective mechanism of luteolin. ResultsLuteolin decreased mean pulmonary hypertension of HPH rats, alleviated right ventricular and pulmonary vascular remodeling. Immunofluorescence staining (vWF), isolated perfused/ventilated rat lung experiment indicated that luteolin protected pulmonary vascular endothelial function of HPH rats. Luteolin increased NO content in PAECs supernatant while decreased NO level in lung tissues of HPH rats. Further, it was demonstrated that luteolin inhibited HIF-2α-Arg axis in PAECs and HPH rats. PI3K-AKT-eNOS signaling pathway was upregulated in PAECs, but which was downregulated in lung tissues of HPH rats. Pharmacological effect of luteolin was equivalent or better than sildenafil. ConclusionLuteolin ameliorated HPH in rats by protecting pulmonary vascular endothelial function via regulating HIF-2α-Arg-NO axis and PI3K-AKT-eNOS-NO signaling pathway. This study may provide a novel perspective and approach to alleviate the devastating disease of HPH.

Sodium tanshinone IIA sulfonate enhances the BMP9-BMPR2-Smad1/5/9 signaling pathway in rat pulmonary microvascular endothelial cells and human embryonic stem cell–derived endothelial cells

BackgroundRecent studies have demonstrated the beneficial effects of STS in treating pulmonary hypertension by inhibiting the pulmonary vascular remodeling and suppressing the abnormally elevated proliferation and migration of PASMCs. However, the roles of STS on pulmonary vascular endothelium remain largely known. MethodsIn this study, we investigated the effects and mechanisms of STS on pulmonary vascular endothelial dysfunction by using a chronic hypoxia-induced pulmonary hypertension (HPH) rat model, as well as in primarily cultured rat PMVECs and human ESC-ECs cell models. ResultsFirstly, a 21-day treatment of STS significantly prevents the disease development of HPH by normalizing the right ventricular systolic pressure and right ventricular hypertrophy, improving the cardiac output. Then, STS treatment markedly inhibits the hypoxia-induced medial wall thickening of the distal intrapulmonary arteries. Notably, STS significantly inhibits the hypoxia-induced apoptosis in both the pulmonary endothelium of HPH rats and primarily cultured PMVECs, through the stabilization of BMPR2 protein and protection of the diminished BMP9-BMPR2-Smad1/5/9 signaling pathway. In mechanism, STS treatment retrieves the hypoxic downregulation of BMPR2 by stabilizing the BMPR2 protein, inhibiting the BMPR2 protein degradation via lysosome system, and promoting the plasma membrane localization of BMPR2, all of which together reinforcing the BMP9-induced signaling transduction in both PMVECs and human ESC-ECs. However, these effects are absent in hESC-ECs expressing heterozygous dysfunctional BMPR2 protein (BMPR2+/R899X). ConclusionSTS may exert anti-apoptotic roles, at least partially, via induction of the BMP9-BMPR2-Smad1/5/9 signaling transduction in pulmonary endothelium and PMVECs.

Decoding ceRNA regulatory network in the pulmonary artery of hypoxia-induced pulmonary hypertension (HPH) rat model

BackgroundHypoxia-induced pulmonary hypertension (HPH) is a lethal cardiovascular disease with the characteristic of severe remodeling of pulmonary vascular. Although a large number of dysregulated mRNAs, lncRNAs, circRNAs, and miRNAs related to HPH have been identified from extensive studies, the competitive endogenous RNA (ceRNA) regulatory network in the pulmonary artery that responds to hypoxia remains largely unknown.ResultsTranscriptomic profiles in the pulmonary arteries of HPH rats were characterized through high-throughput RNA sequencing in this study. Through relatively strict screening, a set of differentially expressed RNAs (DERNAs) including 19 DEmRNAs, 8 DElncRNAs, 19 DEcircRNAs, and 23 DEmiRNAs were identified between HPH and normal rats. The DEmRNAs were further found to be involved in cell adhesion, axon guidance, PPAR signaling pathway, and calcium signaling pathway, suggesting their crucial role in HPH. Moreover, a hypoxia-induced ceRNA regulatory network in the pulmonary arteries of HPH rats was constructed according to the ceRNA hypothesis. More specifically, the ceRNA network was composed of 10 miRNAs as hub nodes, which might be sponged by 6 circRNAs and 7 lncRNAs, and directed the expression of 18 downstream target genes that might play important role in the progression of HPH. The expression patterns of selected DERNAs in the ceRNA network were then validated to be consistent with sequencing results in another three independent batches of HPH and normal control rats. The diagnostic effectiveness of several hub mRNAs in ceRNA network was further evaluated through investigating their expression profiles in patients with pulmonary artery hypertension (PAH) recorded in the Gene Expression Omnibus (GEO) dataset GSE117261. Dysregulated POSTN, LTBP2, SPP1, and LSAMP were observed in both the pulmonary arteries of HPH rats and lung tissues of PAH patients.ConclusionsA ceRNA regulatory network in the pulmonary arteries of HPH rats was constructed, 10 hub miRNAs and their corresponding interacting lncRNAs, circRNAs, and mRNAs were identified. The expression patterns of selected DERNAs were further validated to be consistent with the sequencing result. POSTN, LTBP2, SPP1, and LSAMP were suggested to be potential diagnostic biomarkers and therapeutic targets for PAH.

HIF-1α promotes the proliferation and migration of pulmonary arterial smooth muscle cells via activation of Cx43.

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodelling in hypoxia‐induced pulmonary hypertension (HPH). However, its underlying mechanism has not been well elucidated. Connexin 43 (Cx43) plays crucial roles in vascular smooth muscle cell proliferation in various cardiovascular diseases. Here, the male Sprague‐Dawley (SD) rats were exposed to hypoxia (10% O2) for 21 days to induce rat HPH model. PASMCs were treated with CoCl2 (200 µM) for 24 h to establish the HPH cell model. It was found that hypoxia up‐regulated the expression of Cx43 and phosphorylation of Cx43 at Ser 368 in rat pulmonary arteries and PASMCs, and stimulated the proliferation and migration of PASMCs. HIF‐1α inhibitor echinomycin attenuated the CoCl2‐induced Cx43 expression and phosphorylation of Cx43 at Ser 368 in PASMCs. The interaction between HIF‐1α and Cx43 promotor was also identified using chromatin immunoprecipitation assay. Moreover, Cx43 specific blocker (37,43Gap27) or knockdown of Cx43 efficiently alleviated the proliferation and migration of PASMCs under chemically induced hypoxia. Therefore, the results above suggest that HIF‐1α, as an upstream regulator, promotes the expression of Cx43, and the HIF‐1α/Cx43 axis regulates the proliferation and migration of PASMCs in HPH.

C1q/TNF-related protein-9 ameliorates hypoxia-induced pulmonary hypertension by regulating secretion of endothelin-1 and nitric oxide mediated by AMPK in rats

Injury/dysfunction of the endothelium of pulmonary arteries contributes to hypoxia-induced pulmonary hypertension (HPH). We investigated whether C1q/tumor necrosis factor-related protein-9 (CTRP9), a newly identified cardiovascular agent, has protective roles in the development of HPH. HPH was induced in adult male rats by chronic hypobaric hypoxia. CTRP9 overexpression by adeno-associated virus (AAV)-CTRP9 transfection attenuated the increases in right ventricular systolic pressure, right ventricular hypertrophy index, and pulmonary arterial remodeling of rats under hypoxia. Importantly, CTRP9 overexpression improved endothelium-dependent vasodilation in pulmonary arterioles in HPH rats. CTRP9 overexpression enhanced expression of phosphorylated 5′-adenosine monophosphate-activated protein kinase (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS), and reduced phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) expression in pulmonary microvascular endothelial cells (PMVECs) of HPH rats. In cultured PMVECs, CTRP9 not only preserved the decrease of AMPK and eNOS phosphorylation level and nitric oxide (NO) production induced by hypoxia, but also blocked the increase in hypoxia-induced ERK1/2 phosphorylation level and endothelin (ET)-1 production. Furthermore, the effects of CTRP9 were interrupted by inhibitors or knockdown of AMPK. CTRP9 enhances NO production and reduces ET-1 production by regulating AMPK activation. CTRP9 could be a target for HPH.

CENPE contributes to pulmonary vascular remodeling in pulmonary hypertension

Hypoxic pulmonary vascular remodeling is a pathological feature of pulmonary hypertension (PH). Our results showed that centromere-associated protein E (CENPE) expression in PH patients and hypoxia-induced PH rats was significantly higher than that in normal controls. In addition, CENPE deficiency significantly inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy. Moreover, knocking out CENPE effectively inhibited the proliferation and induced the apoptosis of primary pulmonary artery smooth muscle cells (PASMCs) in vivo. Furthermore, CENPE silencing by small interference significantly inhibited abnormal proliferation, apoptosis resistance, migration, and cell cycle arrest in hypoxia-induced PASMCs. Interestingly, we found that CENPE might exert its biological effect by targeting the transcription of CDK1 proteins.

AAV1-Mediated shRNA Knockdown of SASH1 in Rat Bronchus Attenuates Hypoxia-Induced Pulmonary Artery Remodeling.

Pulmonary hypertension (PH) is a proliferative disease characterized by pulmonary arterial remodeling (PAR). SAM and SH3 domain containing 1 (SASH1) is a novel tumor suppressor gene whose biological function in PH is unclear. In this study, a hypoxia-induced pulmonary hypertension (HPH) rat model was constructed to explore the role of SASH1 in PAR. Histopathological changes in the lung tissue and hemodynamic alteration were detected in SASH1-knockdown rats through adeno-associated virus type-1 (AAV1) infection. In vitro, primary human pulmonary arterial smooth muscle cells (HPASMCs) were transfected with SASH1siRNA to investigate the effects of SASH1 on hypoxia-induced proliferation and migration. The molecular mechanisms associated with SASH1 were explored through knockdown and overexpression approaches. We found that SASH1 expression was significantly increased in rat pulmonary arteries and HPASMCs after hypoxia exposure. In vivo, silencing the SASH1 gene expression improved HPH in rats. The SASH1 downregulation inhibited proliferation and migration of hypoxia-induced HPASMCs. The protein expression of phospho-AKT (known as protein kinase B), proliferating cell nuclear antigen, and matrix metalloproteinase 9 (MMP9) in HPASMCs were increased after SASH1 overexpression, whereas these effects were inhibited by SASH1 knockdown. In conclusion, SASH1 downregulation improved hypoxia-induced PAR and PH. SASH1 may be a novel target for PH gene therapy in the era of precision medicine.

Therapeutic Potential of Regorafenib-A Multikinase Inhibitor in Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation®12 platform. The 5-bromo-2′-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH.

Tsantan Sumtang Restored Right Ventricular Function in Chronic Hypoxia-Induced Pulmonary Hypertension Rats.

Background: Tsantan Sumtang originated from Four Tantras, which consisted of Choerospondias axillaris (Roxb.) B. L. Burtt and A. W. Hill, Santalum album L., and Myristica fragrans Houtt. The three herbs are in ratio 1:1:1. This medication is widely used for cardiovascular diseases. Aims: The purpose of this study was to explore the effect of Tsantan Sumtang on right ventricular (RV) function in hypoxia-induced pulmonary hypertension (HPH) rats and investigate the underlying mechanism. Methods: Sixty male Sprague-Dawley (SD) rats were divided into control, hypoxia, and hypoxia + Tsantan Sumtang (1.0, 1.25, and 1.5 g•kg−1•d−1) groups. Chronic hypoxia was induced by putting the rats inside a hypobaric chamber for four weeks and adjusting the inner pressure and oxygen content to match an altitude of 4500 m. Echocardiography was used to assess RV function and right ventricular-pulmonary arterial (RV-PA) coupling. The physiological parameters of the animals were also evaluated. Morphological characteristics of RV were assessed by hematoxylin and eosin (H&E) staining and TEM. Masson’s trichrome staining, immunohistochemical staining, western blotting, and TUNEL assay were used to assess fibrosis and apoptosis levels. The antioxidant and anti-apoptosis properties of Tsantan Sumtang were also evaluated. The effect of Tsantan Sumtang on ROCK signaling pathway was evaluated using real-time quantitative PCR and western blotting. Results: We established an HPH rat model as indicated by the significant increases in the physiological parameters of the rats. Tsantan Sumtang showed a significant cardiac-protective function and an improved effect on RV-PA coupling. Moreover, Tsantan Sumtang treatment inhibited fibrosis and alleviated apoptosis and oxidative stress in RV. In terms of mechanism, Tsantan Sumtang reduced the expression of ROCK (ROCK1, ROCK2) in RV, inhibited cardiac remodeling-related transcription factors (NFATc3, P-STAT3), and regulated apoptosis-related proteins. Conclusion: Tsantan Sumtang was able to restore RV function, improve RV-PA coupling, recover hemodynamic and hematological indexes, and protect RV against structural maladaptive remodeling in the HPH rats. These findings demonstrated that Tsantan Sumtang protects the function of RV in HPH rats. The antioxidant and anti-apoptosis properties of Tsantan Sumtang may be responsible for inhibiting the ROCK signaling pathway.