Development Of Hypoxia-induced Pulmonary Hypertension Research Articles

PD-1/PD-L1 Provides Protective Role in Hypoxia-Induced Pulmonary Vascular Remodeling.

Hypoxia-induced pulmonary hypertension (HPH) is a T helper 17 cell response-driven disease, and PD-1 (programmed cell death 1)/PD-L1 (programmed cell death-ligand 1) inhibitor-associated pulmonary hypertension has been reported recently. This study is designed to explore whether the PD-1/PD-L1 pathway participates in HPH via regulating endothelial dysfunction and T helper 17 cell response. Lung tissue samples were obtained from eligible patients. Western blotting, immunohistochemistry, and immunofluorescence techniques were used to assess protein expression, while immunoprecipitation was utilized to detect ubiquitination. HPH models were established in C57BL/6 WT (wild-type) and PD-1-/- mice, followed by treatment with PD-L1 recombinant protein. Adeno-associated virus vector delivery was used to upregulate PD-L1 in the endothelial cells. Endothelial cell function was assessed through assays for cell angiogenesis and adhesion. Expression of the PD-1/PD-L1 pathway was downregulated in patients with HPH and mouse models, with a notable decrease in PD-L1 expression in endothelial cells compared with the normoxia group. In comparison to WT mice, PD-1-/- mice exhibited a more severe HPH phenotype following exposure to hypoxia, However, administration of PD-L1 recombinant protein and overexpression of PD-L1 in lung endothelial cells mitigated HPH. In vitro, blockade of PD-L1 with a neutralizing antibody promoted endothelial cell angiogenesis, adhesion, and pyroptosis. Mechanistically, hypoxia downregulated PD-L1 protein expression through ubiquitination. Additionally, both in vivo and in vitro, PD-L1 inhibited T helper 17 cell response through the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in HPH. PD-1/PD-L1 plays a role in ameliorating HPH development by inhibiting T helper 17 cell response through the PI3K/AKT/mTOR pathway and improving endothelial dysfunction, suggesting a novel therapeutic indication for PD-1/PD-L1-based immunomodulatory therapies in the treatment of HPH.

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.

Dan-Shen-Yin Granules Prevent Hypoxia-Induced Pulmonary Hypertension via STAT3/HIF-1α/VEGF and FAK/AKT Signaling Pathways.

Traditional Chinese medicine (TCM) plays an important role in the treatment of complex diseases, especially cardiovascular diseases. However, it is hard to identify their modes of action on account of their multiple components. The present study aims to evaluate the effects of Dan-Shen-Yin (DSY) granules on hypoxia-induced pulmonary hypertension (HPH), and then to decipher the molecular mechanisms of DSY. Systematic pharmacology was employed to identify the targets of DSY on HPH. Furthermore, core genes were identified by constructing a protein-protein interaction (PPI) network and analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analysis. Related genes and pathways were verified using a hypoxia-induced mouse model and hypoxia-treated pulmonary artery cells. Based on network pharmacology, 147 potential targets of DSY on HPH were found, constructing a PPI network, and 13 hub genes were predicted. The results showed that the effect of DSY may be closely associated with AKT serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription 3 (STAT3), and HIF-1 signaling pathways, as well as biological processes such as cell proliferation. Consistent with network pharmacology analysis, experiments in vivo demonstrated that DSY could prevent the development of HPH in a hypoxia-induced mouse model and alleviate pulmonary vascular remodeling. In addition, inhibition of STAT3/HIF-1α/VEGF and FAK/AKT signaling pathways might serve as mechanisms. Taken together, the network pharmacology analysis suggested that DSY exhibited therapeutic effects through multiple targets in the treatment of HPH. The inferences were initially confirmed by subsequent in vivo and in vitro studies. This study provides a novel perspective for studying the relevance of TCM and disease processes and illustrates the advantage of this approach and the multitargeted anti-HPH effect of DSY.

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.

SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach. C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-β1 (transforming growth factor β1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

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.

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.

NDUFA4L2 in smooth muscle promotes vascular remodeling in hypoxic pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodelling (PVR). The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important cause of PVR leading to PAH. Mitochondria play a key role in the production of hypoxia‐induced pulmonary hypertension (HPH). However, there are still many issues worth studying in depth. In this study, we demonstrated that NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 like 2 (NDUFA4L2) was a proliferation factor and increased in vivo and in vitro through various molecular biology experiments. HIF‐1α was an upstream target of NDUFA4L2. The plasma levels of 4‐hydroxynonene (4‐HNE) were increased both in PAH patients and hypoxic PAH model rats. Knockdown of NDUFA4L2 decreased the levels of malondialdehyde (MDA) and 4‐HNE in human PASMCs in hypoxia. Elevated MDA and 4‐HNE levels might be associated with excessive ROS generation and increased expression of 5‐lipoxygenase (5‐LO) in hypoxia, but this effect was blocked by siNDUFA4L2. Further research found that p38‐5‐LO was a downstream signalling pathway of PASMCs proliferation induced by NDUFA4L2. Up‐regulated NDUFA4L2 plays a critical role in the development of HPH, which mediates ROS production and proliferation of PASMCs, suggesting NDUFA4L2 as a potential new therapeutic target for PAH.

Tribbles Homolog 3-Mediated Vascular Insulin Resistance Contributes to Hypoxic Pulmonary Hypertension in Intermittent Hypoxia Rat Model.

This study aimed to investigate the role of vascular insulin resistance (VIR) and Tribbles homolog 3 (TRIB3) in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). Rats were subjected to low air pressure and low oxygen intermittently for 4 weeks to induce HPH. The mean right ventricular pressure (mRVP), mean pulmonary arterial pressure (mPAP), and right ventricular index (RVI) were significantly increased in HPH rats. Pulmonary arteries from HPH rats showed VIR with reduced vasodilating effect of insulin. The protein levels of peroxisome proliferator-activated receptor gamma (PPARγ), phosphoinositide 3-kinase (PI3K), phosphorylations of Akt, and endothelial nitric oxide (NO) synthase (eNOS) were decreased, and TRIB3 and phosphorylated extracellular signal-regulated protein kinases (ERK1/2) were increased in pulmonary arteries of HPH rats. Early treatment of pioglitazone (PIO) partially reversed the development of HPH, improved insulin-induced vasodilation, and alleviated the imbalance of the insulin signaling. The overexpression of TRIB3 in rat pulmonary arterial endothelial cells (PAECs) reduced the levels of PPARγ, PI3K, phosphorylated Akt (p-Akt), and phosphorylated eNOS (p-eNOS) and increased p-ERK1/2 and the synthesis of endothelin-1 (ET-1), which were further intensified under hypoxic conditions. Moreover, TRIB3 knockdown caused significant improvement in Akt and eNOS phosphorylations and, otherwise, a reduction of ERK1/2 activation in PAECs after hypoxia. In conclusion, impaired insulin-induced pulmonary vasodilation and the imbalance of insulin-induced signaling mediated by TRIB3 upregulation in the endothelium contribute to the development of HPH. Early PIO treatment improves vascular insulin sensitivity that may help to limit the progression of hypoxic pulmonary hypertension.

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.

Overexpression of ACE2 prevents hypoxia-induced pulmonary hypertension in rats by inhibiting proliferation and immigration of PASMCs.

Imbalance of the ratio of angiotensin converting enzyme (ACE) and ACE2 may lead to pathological conditions in lung. However, its effect on hypoxia-induced pulmonary hypertension (HPH) remains unclear. Therefore, the aim of this study was to investigate the effects of ACE2 overexpression on rat primary pulmonary arterial smooth muscle cells (PASMCs) and HPH rat model. ACE and ACE2 expression in rat PASMCs under hypoxia condition, as well as in HPH rat model, was detected. The overexpressed ACE2 gene was transfected into PASMCs by Lentiviral. Later, the proliferation and migration of PASMCs were evaluated. Meanwhile, the overexpressed ACE2 gene was transfected into rats and exposed to hypoxia for four weeks. Finally, the right ventricular systolic pressure, the right ventricular hypertrophy, and the percentage of the medial wall thickness were measured to evaluate the development of HPH. Imbalance of the expression of ACE/ACE2 was indicated in rat PASMCs under hypoxia condition and in the HPH rat model, respectively. The overexpression of ACE2 significantly inhibited PASMCs proliferation and migration. Moreover, the overexpressed ACE2 could significantly attenuate pulmonary hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy in HPH rat model. ACE2 is related to the formation of pulmonary vascular remodeling and pulmonary hypertension. Furthermore, it may prevent hypoxia-induced pulmonary hypertension by inhibiting the proliferation of PASMCs.

Senescence Marker Protein 30 Deficiency Exacerbates Pulmonary Hypertension in Hypoxia-Exposed Mice.

Pulmonary arterial hypertension is a fatal disease caused by pulmonary arterial vasoconstriction and organic stenosis due to the proliferation of pulmonary smooth muscle cells and endothelial cells. Endothelial dysfunction, including impaired nitric oxide (NO) bioavailability, plays a crucial role in the pathogenesis of pulmonary hypertension, and endothelial nitric oxide synthase (eNOS) is an important modulator of pulmonary vasodilatation. Although senescence marker protein (SMP) 30 is known as an anti-aging protein, the role of SMP30 in pulmonary vessels is still unclear. In this study, we examined the role of SMP30 in pulmonary vasculature using SMP30-deficient mice.We used female SMP30-deficient mice and wild-type littermate (WT) mice at the age of 12 to 18 weeks. The WT and SMP30-deficient mice were exposed to normoxia or hypoxia (10% oxygen for 4 weeks). In normoxia, the right ventricular systolic pressure (RVSP) was not different between the WT and SMP30-deficient mice, but in hypoxia, the RVSP was significantly higher in the SMP30-deficient mice compared to the WT mice (P < 0.05). The hypoxia-induced increases in right ventricular hypertrophy and medial smooth muscle area of the pulmonary artery were comparable between the WT and the SMP30-deficient mice. Western blotting showed that eNOS phosphorylation in lung tissue was reduced in the SMP30-deficient mice compared to the WT mice in normoxia. However, in hypoxic conditions, eNOS phosphorylation was reduced in both the WT and SMP30-deficient mice with no differences in Akt phosphorylation.Our study demonstrated that SMP30 is involved in the development of hypoxia-induced pulmonary hypertension by impairment of eNOS activity.

Reciprocal actions of constrictor prostanoids and superoxide in chronic hypoxia-induced pulmonary hypertension: roles of EETs.

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by CYP/epoxygenase and metabolized by soluble epoxide hydrolase (sEH). Roles of EETs in hypoxia-induced pulmonary hypertension (HPH) remain elusive. The present study aimed to investigate the underlying mechanisms, by which EETs potentiate HPH. Experiments were conducted on sEH knockout (sEH-KO) and wild type (WT) mice after exposure to hypoxia (10% oxygen) for three weeks. In normal/normoxic conditions, WT and sEH-KO mice exhibited comparable pulmonary artery acceleration time (PAAT), ejection time (ET), PAAT/ET ratio, and velocity time integral (VTI), along with similar right ventricular systolic pressure (RVSP). Chronic hypoxia significantly reduced PAAT, ET, and VTI, coincided with an increase in RVSP; these impairments were more severe in sEH-KO than WT mice. Hypoxia elicited downregulation of sEH and upregulation of CYP2C9 accompanied with elevation of CYP-sourced superoxide, leading to enhanced pulmonary EETs in hypoxic mice with significantly higher levels in sEH-KO mice. Isometric tension of isolated pulmonary arteries was recorded. In addition to downregulation of eNOS-induced impairment of vasorelaxation to ACh, HPH mice displayed upregulation of thromboxane A2 (TXA2) receptor, paralleled with enhanced pulmonary vasocontraction to a TXA2 analog (U46619) in an sEH-KO predominant manner. Inhibition of COX-1 or COX-2 significantly prevented the enhancement by ∼50% in both groups of vessels, and the remaining incremental components were eliminated by scavenging of superoxide with Tiron. In conclusion, hypoxia-driven increases in EETs, intensified COXs/TXA2 signaling, great superoxide sourced from activated CYP2C9, and impaired NO bioavailability work in concert, to potentiate HPH development.

ALDH2 (Aldehyde Dehydrogenase 2) Protects Against Hypoxia-Induced Pulmonary Hypertension.

Hypoxia-induced pulmonary hypertension (HPH) increases lipid peroxidation with generation of toxic aldehydes that are metabolized by detoxifying enzymes, including ALDH2 (aldehyde dehydrogenase 2). However, the role of lipid peroxidation and ALDH2 in HPH pathogenesis remain undefined. Approach and Results: To determine the role of lipid peroxidation and ALDH2 in HPH, C57BL/6 mice, ALDH2 transgenic mice, and ALDH2 knockout (ALDH2-/-) mice were exposed to chronic hypoxia, and recombinant tissue-specific ALDH2 overexpression adeno-associated viruses were introduced into pulmonary arteries via tail vein injection for ALDH2 overexpression. Human pulmonary artery smooth muscle cells were used to elucidate underlying mechanisms in vitro. Chronic hypoxia promoted lipid peroxidation due to the excessive production of reactive oxygen species and increased expression of lipoxygenases in lung tissues. 4-hydroxynonenal but not malondialdehyde level was increased in hypoxic lung tissues which might reflect differences in detoxifying enzymes. ALDH2 overexpression attenuated the development of HPH, whereas ALDH2 knockout aggravated it. Specific overexpression of ALDH2 using AAV1 (adeno-associated virus)-ICAM (intercellular adhesion molecule) 2p-ALDH2 and AAV2-SM22αp (smooth muscle 22 alpha)-ALDH2 viral vectors in pulmonary artery smooth muscle cells, but not endothelial cells, prevented the development of HPH. Hypoxia or 4-hydroxynonenal increased stabilization of HIF (hypoxia-inducible factor)-1α, phosphorylation of Drp1 (dynamin-related protein 1) at serine 616, mitochondrial fission, and pulmonary artery smooth muscle cells proliferation, whereas ALDH2 activation suppressed the latter 3. Increased 4-hydroxynonenal level plays a critical role in the development of HPH. ALDH2 attenuates the development of HPH by regulating mitochondrial fission and smooth muscle cell proliferation suggesting ALDH2 as a potential new therapeutic target for pulmonary hypertension.

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.

MicroRNA410 Inhibits Pulmonary Vascular Remodeling via Regulation of Nicotinamide Phosphoribosyltransferase

Nicotinamide phosphoribosyltransferase (NAMPT) upregulation in human pulmonary artery endothelial cells (hPAECs) is associated with pulmonary arterial hypertension (PAH) progression and pulmonary vascular remodeling. The underlying mechanisms regulating NAMPT expression are still not clear. In this study, we aimed to study the regulation of NAMPT expression by microRNA410 (miR410) in hPAECs and explore the role of miR410 in the pathogenesis of experimental pulmonary hypertension. We show that miR410 targets the 3′ UTR of NAMPT and that, concomitant with NAMPT upregulation, miR410 is downregulated in lungs of mice exposed to hypoxia-induced pulmonary hypertension (HPH). Our results also demonstrate that miR410 directly inhibits NAMPT expression. Overexpression of miR410 in hPAECs inhibits basal and VEGF-induced proliferation, migration and promotes apoptosis of hPAECs, while miR410 inhibition via antagomirs has the opposite effect. Finally, administration of miR410 mimics in vivo attenuated induction of NAMPT in PAECs and prevented the development of HPH in mice. Our results highlight the role of miR410 in the regulation of NAMPT expression in hPAECs and show that miR410 plays a potential role in PAH pathobiology by targeting a modulator of pulmonary vascular remodeling.

Beraprost Upregulates KV Channel Expression and Function via EP4 Receptor in Pulmonary Artery Smooth Muscle Cells Obtained from Rats with Hypoxia-Induced Pulmonary Hypertension

The reduced expression and function of voltage-dependent potassium (K_V) channels have been involved in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH), leading to pulmonary vasoconstriction and vascular remodeling, while the upregulation of K_V channels is of therapeutic significance for pulmonary hypertension. Beraprost sodium (BPS) has been shown to be effective in patients with pulmonary hypertension. However, the effect of BPS on O₂-sensitive K_V channels in pulmonary artery smooth muscle cells (PASMCs) remains unclear. In the present study, the effect of BPS on rats with HPH was observed, and the influence of BPS on the expression and function of O₂-sensitive K_V channels in PASMCs was investigated. The results revealed that BPS reduced mean pulmonary artery pressure, suppressed right ventricular hypertrophy, and attenuated the remodeling of pulmonary arteries in rats exposed to discontinuous hypoxia for 4 weeks (8 h/day). This was accompanied with the significantly upregulated expression of K_V channel α-subunits (K_V1.2, K_V1.5 and K_V2.1) and O₂-sensitive voltage-gated K⁺ (K_V) channel current (I_K(V)) in small pulmonary arteries in HPH model rats, as well as in hypoxia-induced PASMCs. Furthermore, in vitrostudies have revealed that the upregulation of BPS on O₂-sensitive K_V channels was significantly inhibited after treatment with prostaglandin E₂ receptor subtype EP₄ antagonist GW627368X. Taken together, these results suggest that BPS attenuates the development of HPH through the upregulation of O₂-sensitive K_V channels, which was probably via the EP₄ receptor-related pathway.

Recent Advances in the Understanding of Thrombosis.

Recent Advances in the Understanding of Thrombosis.

MiR-593-5p promotes the development of hypoxic-induced pulmonary hypertension via targeting PLK1.

The aim of this study was to investigate the role of microRNA-593-5p (miR-593-5p) in hypoxia-induced pulmonary hypertension (HPH), and to explore its underlying mechanism. Sprague-Dawley (SD) rats were housed in a hypoxia environment 8 hours per day for consecutive 4 weeks. After the establishment of the HPH rat model, we detected the right ventricular systolic pressure (RVSP) and right heart hypertrophy index (RVHI) in HPH rats and controls. The expression levels of miR-593-5p and polo-like kinase 1 (PLK1) in rat lungs were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Subsequently, miR-593-5p mimics and inhibitor were constructed and transfected into cells. The proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) were accessed by cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The protein level of PLK1 in PASMCs after transfection with miR-593-5p mimics or inhibitor was detected by Western blot. Dual-luciferase reporter gene assay was conducted to verify the binding condition of miR-593-5p and PLK1. Finally, rescue experiments were performed to explore whether the regulatory effect of miR-593-5p on HPH development was associated with PLK1. RVSP and RVHI in rats of the hypoxic group were significantly higher than those of controls. MiR-593-5p was significantly downregulated while PLK1 was remarkably upregulated in lung tissues of HPH rats than those of controls. Similarly, miR-593-5p expression in PASMCs decreased gradually with the prolongation of hypoxia induction. Overexpression of miR-593-5p remarkably inhibited the proliferation and migration of PASMCs. Subsequently, dual-luciferase reporter gene verified the binding condition of miR-593-5p and PLK1. Both the mRNA and protein levels of PLK1 were negatively regulated by miR-593-5p. Also, rescue experiments demonstrated that the inhibitory effects of miR-593-5p on the proliferation and migration of PASMCs could be reversed by PLK1 overexpression. MiR-593-5p is lowly expressed in lung tissues of HPH rats. Meanwhile, it stimulates the proliferation and migration of PASMCs via targeting PLK1, thereby promoting HPH development.

MiR-143 and miR-145 promote hypoxia-induced proliferation and migration of pulmonary arterial smooth muscle cells through regulating ABCA1 expression.

Excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) play an important role in the occurrence and development of pulmonary arterial hypertension (PAH). miR-143/145 was reported to be up-regulated in the animal models and PAH patients, and deletion of miR-143/145 cluster prevented the development of hypoxia-induced pulmonary hypertension, but its underlying mechanism has not been elucidated. qRT-PCR and Western blot were performed to detect the expressions of miR-143/145 and ATP-binding cassette transporter A1 (ABCA1) in PAH patients and PASMCs under hypoxic conditions. Cell proliferation and migration were assessed by Cell Counting Kit-8 and wound-healing assay, respectively. Luciferase reporter assay and RNA immunoprecipitation were conducted to confirm the interaction between miR-143/145 and ABCA1. Hypoxia-induced PAH rat model was established to confirm the functions of miR-143/145 in the pathogenesis of PAH and its underlying mechanism in vivo. miR-143 and miR-145 were up-regulated and ABCA1 was down-regulated in PAH patients and PASMCs under hypoxic conditions for different time, as well as hypoxia-induced PAH rats. miR-143/145 inhibition and ABCA1 overexpression suppressed hypoxia-induced proliferation and migration in PASMCs. ABCA1 was identified as a direct target of miR-143/145 in PASMCs. Moreover, ABCA1 partially reversed miR-143/145-mediated promotion of hypoxia-induced cell proliferation and migration in PASMCs. Furthermore, in vivo experiments confirmed that inhibition of miR-143/145 prevents hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling by targeting ABCA1. miR-143/145 promoted hypoxia-induced proliferation and migration of PASMCs by targeting ABCA1, contributing to better understanding of the mechanism underlying the pathogenesis of PAH.