Hypoxia-induced Pulmonary Hypertension Model Research Articles

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

SOCS5, targeted by miR-155-5p, plays a negative regulatory role in pulmonary hypertension through inhibiting JAK2/STAT3 signaling pathway

Pulmonary hypertension (PH) is a chronic pulmonary vascular disease and causes massive deaths. Here, we intended to investigate the function and mechanism of SOCS5 in PH. We engineered a hypoxia-induced PH model in mice. HE staining were implemented to evaluate pathological alterations in the lung tissues. The potential mechanism of SOCS5 in regulating hypoxia-induced pulmonary artery smooth muscle cell (PASMC) function was explored in vitro. RT-qPCR and western blot revealed that the level of SOCS5 was decreased both in PH mice and hypoxia-induced HPASMCs. Functional assays were performed for confirming the role of SOCS5 in modulating the cell phenotype and JAK2/STAT3 pathway in HPASMCs. Results revealed that overexpression of SOCS5 suppressed proliferation, migration and contraction of HPASMCs and negatively regulated the JAK2/STAT3 signaling pathway in HPASMCs under hypoxia in vitro, while knockdown of SOCS5 accelerated it. As evidenced by mechanism studies, SOCS5 was targeted and regulated by miR-155-5p, hence affecting on HPASMC proliferation, migration and contraction. These outcomes indicated that the decreased level of SOCS5 in hypoxia-induced HPASMCs promoted the cell proliferation, cell migration, and cell contraction through activating JAK2/STAT3 signaling pathway. Moreover, SOCS5 was targeted by miR-155-5p. All in all, our work hinted that miR-155-5p/SOCS5/JAK2/STAT3 axis played a crucial part in PH.

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.

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.

Airway delivery of Streptococcus salivarius is sufficient to induce experimental pulmonary hypertension in rats.

The causal relationship between altered host microbiome composition, especially the respiratory tract microbiome, and the occurrence of pulmonary hypertension (PH) has not yet been studied. An increased abundance of airway streptococci is seen in patients with PH compared with healthy individuals. This study aimed to determine the causal link between elevated airway exposure to Streptococcus and PH. The dose-, time- and bacterium-specific effects of Streptococcus salivarius (S. salivarius), a selective streptococci, on PH pathogenesis were investigated in a rat model established by intratracheal instillation. Exposure to S. salivarius successfully induced typical PH characteristics, such as elevated right ventricular systolic pressure (RVSP), right ventricular hypertrophy (Fulton's index) and pulmonary vascular remodelling, in a dose- and time-dependent manner. Moreover, the S. salivarius-induced characteristics were absent in either the inactivated S. salivarius (inactivated bacteria control) treatment group or the Bacillus subtilis (active bacteria control) treatment group. Notably, S. salivarius-induced PH is characterized by elevated inflammatory infiltration in the lungs, in a pattern different from the classic hypoxia-induced PH model. Moreover, in comparison with the SU5416/hypoxia-induced PH model (SuHx-PH), S. salivarius-induced PH causes similar histological changes (pulmonary vascular remodelling) but less severe haemodynamic changes (RVSP, Fulton's index). S. salivarius-induced PH is also associated with altered gut microbiome composition, suggesting potential communication of the lung-gut axis. This study provides the first evidence that the delivery of S. salivarius in the respiratory tract could cause experimental PH in rats.

Important Role of Endogenous Nerve Growth Factor Receptor in the Pathogenesis of Hypoxia-Induced Pulmonary Hypertension in Mice.

Pulmonary arterial hypertension (PAH) remains a disease with poor prognosis; thus, a new mechanism for PAH treatment is necessary. Circulating nerve growth factor receptor (Ngfr)-positive cells in peripheral blood mononuclear cells are associated with disease severity and the prognosis of PAH patients; however, the role of Ngfr in PAH is unknown. In this study, we evaluated the function of Ngfr using Ngfr gene-deletion (Ngfr-/-) mice. To elucidate the role of Ngfr in pulmonary hypertension (PH), we used Ngfr-/- mice that were exposed to chronic hypoxic conditions (10% O2) for 3 weeks. The development of hypoxia-induced PH was accelerated in Ngfr-/- mice compared to littermate controls. In contrast, the reconstitution of bone marrow (BM) in Ngfr-/- mice transplanted with wild-type BM cells improved PH. Notably, the exacerbation of PH in Ngfr-/- mice was accompanied by the upregulation of pulmonary vascular remodeling-related genes in lung tissue. In a hypoxia-induced PH model, Ngfr gene deletion resulted in PH exacerbation. This suggests that Ngfr may be a key molecule involved in the pathogenesis of PAH.

PS-BPB09-7: ENDOTHELIAL CNP-GC-B SYSTEM PLAYS A PROTECTIVE ROLE IN THE DEVELOPMENT OF PULMONARY HYPERTENSION

Background: Pulmonary hypertension (PH) is a disease with a poor prognosis despite the development of various treatments, and a further therapeutic agent is required. C-type natriuretic peptide (CNP), one of the natriuretic peptide family, affects vasorelaxation and inhibition of vascular remodeling through its receptor guanylyl cyclase-B (GC-B). However, the beneficial effect of CNP on PH is still controversial. In this study, we examined the role of the CNP-GC-B pathway in the pathogenesis of PH. Methods and Results: To clarify the effect of the endogenous CNP-GC-B system on PH, we evaluated two types of PH models (monocrotaline pyrrole-induced and hypoxia-induced PH models) by using endothelial cell-specific CNP knockout mice (ecCNP-cKO), endothelial cell-specific GC-B knockout mice (ecGC-B-cKO), and smooth muscle cell-specific CG-B knockout mice (smGC-B-cKO). In both PH models, ecGC-B-cKO and ecCNP-cKO mice exacerbated PH compared to control cre-mice. Besides, mRNA levels of Edn1, IL6 and Ccl2 of the lung were significantly increased in the PH models of these mice. However, PH models of smGC-B-cKO mice showed no significant difference compared to those of control cre-mice. In cultured pulmonary arterial endothelial cells, synthesised CNP significantly attenuated PDGF- or hypoxia-induced increase in mRNA expression of Edn1, IL-6, and Ccl2. Moreover, continuous subcutaneous administration of synthesised CNP by osmotic mini-pump significantly ameliorated PH in PH model mice. Conclusion: CNP plays a protective role in the development of PH via endothelial GC-B through inhibiting endothelin-1 and inflammatory signals involvement. These data suggest CNP could be a novel therapeutic agent for PH.

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.

Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension.

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease characterised by pro-proliferative and anti-apoptotic phenotype in vascular cells, leading to pulmonary vascular remodelling and right heart failure. Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1), a highly conserved enzyme, which binds to and catalyses the isomerisation of specific phosphorylated Ser/Thr-Pro motifs, acts as a molecular switch in multiple coordinated cellular processes. We hypothesised that Pin1 plays a substantial role in PAH, and its inhibition with a natural organic compound, Juglone, would reverse experimental pulmonary hypertension.ResultsWe demonstrated that the expression of Pin1 was markedly elevated in experimental pulmonary hypertension (i.e. hypoxia-induced mouse and Sugen/hypoxia-induced rat models) and pulmonary arterial smooth muscle cells of patients with clinical PAH. In vitro Pin1 inhibition by either Juglone treatment or short interfering RNA knockdown resulted in an induction of apoptosis and decrease in proliferation of human pulmonary vascular cells. Stimulation with growth factors induced Pin1 expression, while its inhibition reduced the activity of numerous PAH-related transcription factors, such as hypoxia-inducible factor (HIF)-α and signal transducer and activator of transcription (STAT). Juglone administration lowered pulmonary vascular resistance, enhanced right ventribular function, improved pulmonary vascular and cardiac remodelling in the Sugen/hypoxia rat model of PAH and the chronic hypoxia-induced pulmonary hypertension model in mice.ConclusionOur study demonstrates that targeting of Pin1 with small molecule inhibitor, Juglone, might be an attractive future therapeutic strategy for PAH and right heart disease secondary to 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.

Halofuginone, a promising drug for treatment of pulmonary hypertension.

Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH). Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments. Halofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 μM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice. Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

Resistin-like molecule \u03b2 acts as a mitogenic factor in hypoxic pulmonary hypertension via the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK signaling pathways

BackgroundPulmonary arterial smooth muscle cell (PASMC) proliferation plays a crucial role in hypoxia-induced pulmonary hypertension (HPH). Previous studies have found that resistin-like molecule β (RELM-β) is upregulated de novo in response to hypoxia in cultured human PASMCs (hPASMCs). RELM-β has been reported to promote hPASMC proliferation and is involved in pulmonary vascular remodeling in patients with PAH. However, the expression pattern, effects, and mechanisms of action of RELM-β in HPH remain unclear.MethodsWe assessed the expression pattern, mitogenetic effect, and mechanism of action of RELM-β in a rat HPH model and in hPASMCs.ResultsOverexpression of RELM-β caused hemodynamic changes in a rat model of HPH similar to those induced by chronic hypoxia, including increased mean right ventricular systolic pressure (mRVSP), right ventricular hypertrophy index (RVHI) and thickening of small pulmonary arterioles. Knockdown of RELM-β partially blocked the increases in mRVSP, RVHI, and vascular remodeling induced by hypoxia. The phosphorylation levels of the PI3K, Akt, mTOR, PKC, and MAPK proteins were significantly up- or downregulated by RELM-β gene overexpression or silencing, respectively. Recombinant RELM-β protein increased the intracellular Ca2+ concentration in primary cultured hPASMCs and promoted hPASMC proliferation. The mitogenic effects of RELM-β on hPASMCs and the phosphorylation of PI3K, Akt, mTOR, PKC, and MAPK were suppressed by a Ca2+ inhibitor.ConclusionsOur findings suggest that RELM-β acts as a cytokine-like growth factor in the development of HPH and that the effects of RELM-β are likely to be mediated by the Ca2+-dependent PI3K/Akt/mTOR and PKC/MAPK pathways.

Abstract 12990: High Fat Diet Delays Reverse Remodeling of Pulmonary Artery in Hypoxia-induced Pulmonary Hypertension Mice

Background: Previous studies suggest the association of metabolic disorder with pulmonary hypertension. Elevated pulmonary artery pressure induced by hypoxia in mice returns to normal when they are placed back in normoxia. In this study, we investigated the effect of high-fat diet (HFD) on pulmonary artery pressure and reverse remodeling of pulmonary artery in mouse model of hypoxia-induced pulmonary hypertension. Methods: We used the female C57/Bl6 mice at the age of 8 weeks. After exposed to hypoxia (10% oxygen for 4 weeks) for induction of pulmonary hypertension, mice were returned to normoxic condition and fed HFD or low-fat diet (LFD) for 12 weeks. Results: The body weight (BW), glucose, low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C), but not triglyceride (TG) were significantly increased in HFD group (36.9±5.1 vs. 24.0±1.4 g [BW], P<0.05; 404.2±55.1 vs. 326.5±8.7 mg/dl [glucose], P<0.05; 10.2±3.6 vs. 7.5±0.9 mg/dl [LDL-C], P<0.05; 47.2±10.0 vs. 30.9±8.7 mg/dl [HDL-C], P<0.05 and 40.0±21.1 vs. 53.4±12.9 mg/dl [TG], P=0.09). The right ventricular systolic pressure (RVSP) measured by a micro-manometer catheter and the Fulton index in HFD group were significantly higher than those in LFD group (23.8±2.3 vs. 19.3±2.9 mmHg [RVSP], P<0.05; 0.30±0.06 vs. 0.25±0.04 [Fulton index], P<0.05, respectively). The medial smooth muscle area (expressed as a percentage of the external area of the vessel) was larger in HFD group (40.1±5.3 vs. 34.5±3.1%, P<0.05). In addition, the levels of active caspase-3 detected by Western blotting was lower in HFD group than in LFD group. TUNEL staining revealed that the apoptosis of pulmonary artery smooth muscle cells was suppressed in HFD group. Conclusion: Our results suggest that metabolic disorder attenuates pulmonary artery reverse remodeling, at least in part, by suppressing apoptosis of pulmonary artery smooth muscle cells when hypoxia-induced pulmonary hypertension mice were returned to normoxia.

TIGAR reduces smooth muscle cell autophagy to prevent pulmonary hypertension.

Yamanaka R, Hoshino A, Fukai K, Urata R, Minami Y, Honda S, Fushimura Y, Hato D, Iwai-Kanai E, Matoba S. TIGAR reduces smooth muscle cell autophagy to prevent pulmonary hypertension. Am J Physiol Heart Circ Physiol 319: H1087-H1096, 2020. First published September 18, 2020; doi:10.1152/ajpheart.00314.2020.-Pulmonary arterial hypertension (PAH) is a refractory disease. Its prognosis remains poor; hence, establishment of novel therapeutic targets is urgent. TP53-induced glycolysis and apoptosis regulator (TIGAR) is a downstream target of p53 and exhibits functions inhibiting autophagy and reactive oxygen species (ROS). Recently, p53 was shown to suppress PAH progression. Because inhibition of autophagy and ROS is known to improve PAH, we examined the effect of TIGAR on PAH progression. We compared pulmonary hypertension (PH) development between TIGAR-deficient knockout (KO) and wild-type (WT) mice using a hypoxia-induced PH model. Human pulmonary artery smooth muscle cells (PASMCs) were used for in vitro experiments with small interfering RNA (siRNA) to investigate the possible molecular mechanisms. From the analysis of right ventricular pressure, right ventricular weight, and mortality rate, we concluded that the hypoxia-induced PH development was remarkably higher in TIGAR KO than in WT mice. Pathological investigation revealed that medial thickening of the pulmonary arterioles and cell proliferation were increased in TIGAR KO mice. Autophagy and ROS activity were also increased in TIGAR KO mice. TIGAR knockdown by siRNA increased cell proliferation and migration, exacerbated autophagy, and increased ROS generation during hypoxia. Autophagy inhibition by chloroquine and ROS inhibition by N-acetylcysteine attenuated the proliferation and migration of PASMCs caused by TIGAR knockdown and hypoxia exposure. TIGAR suppressed the proliferation and migration of PASMCs via inhibiting autophagy and ROS and, therefore, improved hypoxia-induced PH. Thus, TIGAR might be a promising therapeutic target for PAH.NEW & NOTEWORTHY Pulmonary arterial hypertension is a refractory disease. TP53-induced glycolysis and apoptosis regulator (TIGAR) is a downstream target of p53 and exhibits functions inhibiting autophagy and reactive oxygen species (ROS). By using TIGAR-deficient knockout mice and human pulmonary artery smooth muscle cells, we found that TIGAR suppressed the proliferation and migration of PASMCs via inhibiting autophagy and ROS and, therefore, improved hypoxia-induced PH. TIGAR will be a promising therapeutic target for PAH.

AMPKα2 deficiency exacerbates hypoxia-induced pulmonary hypertension by promoting pulmonary arterial smooth muscle cell proliferation.

Increased evidence indicates that adenosine monophosphate-activated protein kinase (AMPK) plays a vital role in vascular homeostasis, especially under hypoxia, and protects against the progression of pulmonary hypertension (PH). However, the role of AMPK in the pathogenesis of PH remains to be clarified. In the present study, we confirmed that a loss of AMPKα2 exacerbated the development of PH by using hypoxia-induced PH model in AMPKα2 -/- mice. After a 4-week period of hypoxic exposure, AMPKα2 -/- mice exhibited more severe pulmonary vascular remodeling and pulmonary vascular smooth muscle cell (SMC) proliferation when compared with wild type (WT) mice. In vitro, AMPKα2 knockdown promoted the proliferation of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. This phenomenon was accompanied by upregulated Skp2 and downregulated p27kip1 expression and was abolished by rapamycin, an inhibitor of mTOR. These results indicate that AMPKα2 deficiency exacerbates hypoxia-induced PH by promoting PASMC proliferation via the mTOR/Skp2/p27kip1 signaling axis. Therefore, enhanced AMPKα2 activity might underlie a novel therapeutic strategy for the management of PH.

Drag-reducing polymers attenuates pulmonary vascular remodeling and right ventricular dysfunction in a rat model of chronic hypoxia-induced pulmonary hypertension.

Drag-reducing polymers (DRPs) was previously demonstrated to increase blood flow, tissue perfusion, and reduce vascular resistance. The purpose of this study was to investigate the effect of DRPs on pulmonary vascular remodeling and right ventricular dysfunction in a rat model of chronic hypoxia-induced pulmonary hypertension (HPH). A total of forty male Wistar rats were randomly and equally assigned into four experimental groups (Group I: normoxia + saline, Group II: normoxia + PEO, Group III: hypoxia + saline, Group IV: hypoxia + PEO) and maintained in normoxia (21% O2) or hypobaric hypoxia (10% O2). After four weeks, comparisons were made of the following aspects: the mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), right ventricular hypertrophy, wall thickness of pulmonary trunk and arteries, internal diameter of pulmonary arteries, cardiomyocyte cross-sectional area (CM CSA), and ultrastructure of right ventricular. Treatment with PEO in Group IV attenuated the increases in RVSP and mPAP (40.5±7.2 and 34.7±7.0 mmHg, respectively, both P < 0.05), compared with Group III. Distal vascular remodeling was visible as a significant increase in medial wall thickness (64.2±12.3% vs. 43.95±7.0%, P < 0.01) and a remarkable decrease in internal diameter of small pulmonary arteries (35.2±9.7μ m vs. 50.4±14.7μ m, P < 0.01) in Group III, to a greater extent than that detected in Group IV. Nevertheless, no significant histopathological differences in medial wall thickness was observed in pulmonary trunk between Group III and Group IV (P > 0.05), denoting that PEO chiefly attenuated the remodeling of small pulmonary arteries rather than main arteries in hypoxic environment. Infusion of DRPs (intravenous injection twice weekly) also attenuated the index of right ventricular hypertrophy, protected against the increase of cardiomyocyte cross-sectional area, and provided protection for cardiac ultrastructure. DRP treatment with intravenous injection elicited a protective effect against pulmonary vascular remodeling and right ventricular dysfunction in the rat model of HPH. DRPs may offer a new potential approach for the treatment of HPH, which may have theoretical significance and application value to society.

Transplantation of Mesenchymal Stem Cells Attenuates Pulmonary Hypertension by Normalizing the Endothelial-to-Mesenchymal Transition.

For decades, stem cell therapies for pulmonary hypertension (PH) have progressed from laboratory hypothesis to clinical practice. Promising preclinical investigations have laid both a theoretical and practical foundation for clinical application of mesenchymal stem cells (MSCs) for PH therapy. However, the underlying mechanisms are still poorly understood. We sought to study the effects and mechanisms of MSCs on the treatment of PH. For in vivo experiments, the transplanted GFP+ MSCs were traced at different time points in the lung tissue of a chronic hypoxia-induced PH (CHPH) rat model. The effects of MSCs on PH pathogenesis were evaluated in both CHPH and sugen hypoxia-induced PH models. For in vitro experiments, primary pulmonary microvascular endothelial cells were cultured and treated with the MSC conditioned medium. The specific markers of endothelial-to-mesenchymal transition (EndMT) and cell migration properties were measured. MSCs decreased pulmonary arterial pressure and ameliorated the collagen deposition, and reduced the thickening and muscularization in both CHPH and sugen hypoxia-induced PH rat models. Then, MSCs significantly attenuated the hypoxia-induced EndMT in both the lungs of PH models and primary cultured rat pulmonary microvascular endothelial cells, as reflected by increased mesenchymal cell markers (fibronectin 1 and vimentin) and decreased endothelial cell markers (vascular endothelial cadherin and platelet endothelial cell adhesion molecule-1). Moreover, MSCs also markedly inhibited the protein expression and degradation of hypoxia-inducible factor-2α, which is known to trigger EndMT progression. Our data suggest that MSCs successfully prevent PH by ameliorating pulmonary vascular remodeling, inflammation, and EndMT. Transplantation of MSCs could potentially be a powerful therapeutic approach against PH.

EIF2α promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats.

PurposeEukaryotic initiation factor 2α (eIF2α) plays important roles in the proliferation and survival of pulmonary artery smooth muscle cells (PASMCs) in animal hypoxia-induced pulmonary hypertension models. However, the underlying mechanism remains unknown at large. Autophagy has been reported to play a key role in the vascular remodeling in pulmonary arterial hypertension (PAH). The purposes of this study are to determine the functions of eIF2α and autophagy in the vascular remodeling of the monocrotaline-induced PAH rats and to clarify the correlation between eIF2α and autophagy.MethodsWe established a rat model of monocrotaline-induced PAH, and we established a cell model of platelet derived growth factor (PDGF)-induced PASMCs proliferation. The vascular morphology and the expression of eIF2α, LC3B, and p62 were assessed in the pulmonary arterial tissue of Sprague-Dawleyrats and PDGF-induced PASMCs.ResultsAutophagy was significantly active in monocrotaline model group (MCT)-induced PAH rats, which obviously promotes vascular remodeling in MCT-induced PAH rats. Furthermore, the proliferation of PASMCs was induced by PDGF in vitro. The expression of LC3B, eIF2α was increased in the PDGF-induced PASMCs proliferation, and the expression of p62 was reduced in the PDGF-induced PASMCs proliferation. Moreover, eIF2α siRNA downregulated the expression of eIF2α and LC3B, and upregulated the expression of p62 in PDGF-induced PASMCs proliferation. eIF2α siRNA inhibited the PDGF-induced PASMCs proliferation. Finally, chloroquine can upregulate the protein expression of LC3B and p62, it also can inhibit proliferation in PDGF-induced PASMCs.ConclusionBased on these observations, we conclude that eIF2α promotes the proliferation of PASMCs and vascular remodeling in monocrotaline-induced PAH rats through accelerating autophagy pathway.

Crosstalk between the Akt/mTORC1 and NF-κB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs.

Abnormal wound healing by pulmonary artery smooth muscle cells (PASMCs) promotes vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Increasing evidence shows that both the mammalian target of rapamycin complex 1 (mTORC1) and nuclear factor-kappa B (NF-κB) are involved in the development of HPH. In this study, we explored the crosstalk between mTORC1 and NF-κB in PASMCs cultured under hypoxic condition and in a rat model of hypoxia-induced pulmonary hypertension (HPH). We showed that hypoxia promoted wound healing of PASMCs, which was dose-dependently blocked by the mTORC1 inhibitor rapamycin (5-20 nM). In PASMCs, hypoxia activated mTORC1, which in turn promoted the phosphorylation of NF-κB. Molecular docking revealed that mTOR interacted with IκB kinases (IKKs) and that was validated by immunoprecipitation. In vitro kinase assays and mass spectrometry demonstrated that mTOR phosphorylated IKKα and IKKβ separately. Inhibition of mTORC1 decreased the level of phosphorylated IKKα/β, thus reducing the phosphorylation and transcriptional activity of NF-κB. Bioinformatics study revealed that dipeptidyl peptidase-4 (DPP4) was a target gene of NF-κB; DPP4 inhibitor, sitagliptin (10-500μM) effectively inhibited the abnormal wound healing of PASMCs under hypoxic condition. In the rat model of HPH, we showed that NF-κB activation (at 3 weeks) was preceded by mTOR signaling activation (after 1 or 2 weeks) in lungs, and administration of sitagliptin (1-5 mg/kg every day, ig) produced preventive effects against the development of HPH. In conclusion, hypoxia activates the crosstalk between mTORC1 and NF-κB, and increased DPP4 expression in PASMCs that leads to vascular remodeling. Sitagliptin, a DPP4 inhibitor, exerts preventive effect against HPH.

Inhaled nebulized nitrite and nitrate therapy in a canine model of hypoxia-induced pulmonary hypertension

Dysfunction in the nitric oxide (NO) signaling pathway can lead to the development of pulmonary hypertension (PH) in mammals. Discovery of an alternative pathway to NO generation involving reduction from nitrate to nitrite and to NO has motivated the evaluation of nitrite as an alternative to inhaled NO for PH. In contrast, inhaled nitrate has not been evaluated to date, and potential benefits include a prolonged half-life and decreased risk of methemoglobinemia. In a canine model of acute hypoxia-induced PH we evaluated the effects of inhaled nitrate to reduce pulmonary arterial pressure (PAP). In a randomized controlled trial, inhaled nitrate was compared to inhaled nitrite and inhaled saline. Exhaled NO, PAP and systemic blood pressures were continuously monitored. Inhaled nitrite significantly decreased PAP and increased exhaled NO. In contrast, inhaled nitrate and inhaled saline did not decrease PAP or increase exhaled NO. Unexpectedly, we found that inhaled nitrite resulted in prolonged (>5 h) exhaled NO release, increase in nitrate venous/arterial levels and a late surge in venous nitrite levels. These findings do not support a therapeutic role for inhaled nitrate in PH but may have therapeutic implications for inhaled nitrite in various disease states.