Pulmonary Artery Endothelial Cell Proliferation Research Articles

Investigation into the role of H2-Ab1 in vascular remodeling in pulmonary arterial hypertension via Bioinformatics

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease of vascular remodeling characterized by persistent pulmonary arterial pressure elevation, which can lead to right heart failure and premature death. Given the complex pathogenesis and poor prognosis of PAH, the identification and investigation of biomarkers become increasingly critical for advancing further understanding of the disease.MethodsPAH-related datasets, GSE49114, GSE180169 and GSE154959, were downloaded from the publicly available GEO database. By performing WGCNA on the GSE49114 dataset, a total of 906 PAH-related key module genes were screened out. By carrying out differential analysis on the GSE180169 dataset, a total of 576 differentially expressed genes were identified. Additionally, the GSE154959 single-cell sequencing dataset was also subjected to differential analysis, leading to the identification of 34 DEGs within endothelial cells. By taking intersection of the above three groups of DEGs, five PAH-related hub genes were screened out, namely Plvap, Cyp4b1, Foxf1, H2-Ab1, and H2-Eb1, among which H2-Ab1 was selected for subsequent experiments.ResultsA SuHx mouse model was prepared using the SU5416/hypoxia method, and the successful construction of the model was evaluated through Hematoxylin-Eosin staining, hemodynamic detection, fulton index, and Western Blot (WB). The results of WB and qRT-PCR demonstrated a significant upregulation of H2-Ab1 expression in SuHx mice. Consistent with the results of bioinformatics analysis, a time-dependent increase was observed in H2-Ab1 expression in hypoxia-treated mouse pulmonary artery endothelial cells (PAECs). To investigate whether H2-Ab1 affects the development and progression of PAH, we knocked down H2-Ab1 expression in PAECs, and found that its knockdown inhibited the viability, adhesion, migration, and angiogenesis, while concurrently promoted the apoptosis of PAECs.ConclusionH2-Ab1 could regulate the proliferation, apoptosis, adhesion, migration, and angiogenesis of PAECs.

Ethyl pyruvate alleviates pulmonary arterial hypertension via PI3K-Akt signaling.

Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome that is extremely difficult to manage, and there is currently no effective treatment. We want to elucidate the therapeutic effect of ethyl pyruvate (EP) on PAH and its possible mechanism. Pulmonary artery endothelial cells (PAECs) were cultured in conventional low-oxygen environments, and cellular proliferation was monitored after treatment with EP. Expression of p-PI3K/Akt, LC3-II, and Beclin-1 was detected by Western blot. After hyperkinetic PAH rabbits' models were treated with EP, hemodynamic data were collected. Right ventricular hypertrophy and pulmonary vascular remodeling were evaluated. Expression of p-PI3K/Akt, LC3-II, and Beclin-1 protein was also detected after using autophagy inhibitor and agonists. We found that EP could inhibit PAECs proliferation. After EP treatment, expression of p-PI3K/Akt was upregulated in vitro and in vivo. LC3-II and Beclin-1 were inhibited and their expression was lower after autophagy inhibitor was given, while after administration of autophagy agonists, their expression was higher than that in the EP alone group. Besides, EP attenuated PAH, and right ventricular hypertrophy and pulmonary vascular remodeling were also reversed. EP can reduce PAH and reverse vascular remodeling which is associated with inhibition of autophagy in PAECs based on PI3K-Akt signaling pathway. The results of this study can provide surgical opportunities for patients with severe PAH caused by congenital heart disease in clinical cardiovascular surgery.

Overexpressed pigment epithelium-derived factor alleviates pulmonary hypertension in two rat models induced by monocrotaline and SU5416/hypoxia

BackgroundPulmonary hypertension (PH) is a progressive and fatal cardiopulmonary disease characterized by vascular remodeling and is associated with endothelial-to-mesenchymal transition (EndoMT). The pigment epithelium-derived factor (PEDF), a secretory protein widely distributed in multiple organs, has been shown to demonstrate anti-EndoMT activity in cardiovascular diseases. In the present study, the role of PEDF in PH was investigated. MethodsFor PEDF overexpression, Sprague Dawley rats were infected with an adeno-associated virus through injection via the internal jugular vein. To establish PH models, the animals were subjected to monocrotaline or Sugen/hypoxia. Four weeks later, pulmonary artery angiography was performed, and hemodynamic parameters, right ventricular function, and vascular remodeling were evaluated. EndoMT and cell proliferation in the pulmonary arteries were assessed via immunofluorescence staining. Moreover, pulmonary artery endothelial cells (PAECs) isolated from experimental PH rats were cultured to investigate the underlying molecular mechanisms involved. ResultsPEDF expression was significantly downregulated in PAECs from PH patients and PH model rats. Overexpressed PEDF alleviated the development of PH by improving pulmonary artery morphology and perfusion, reducing pulmonary artery pressure, improving right ventricular function, and alleviating vascular remodeling. PEDF inhibits EndoMT and reduces excessive PAEC proliferation. Moreover, PEDF overexpression reduced EndoMT in cultured PAECs by competitively inhibiting the binding of wnt to LRP6 and downregulating phosphorylation at the 1490 site of LRP6. ConclusionsOur findings suggest that PEDF may be a potential therapeutic target for PH. We also found that PEDF can inhibit EndoMT in PAECs and may exert these effects by inhibiting the Wnt/LRP6/β-catenin pathway.

CD137 Signaling Mediates Pulmonary Artery Endothelial Cell Proliferation Under Hypoxia By Regulating Mitochondrial Dynamics.

Altered mitochondrial dynamics affect pulmonary artery endothelial cells (PAECs) proliferation, contributing to the development of pulmonary hypertension. CD137 signaling promotes mitochondrial fission. We hypothesize CD137 signaling is involved in the excessive proliferation of PAECs. The levels of CD137 protein were increased in the lung tissue of hypoxic mice and hypoxic-stimulated PAECs. Activation of CD137 signal in hypoxic-PAECs upregulated the levels of hypoxia-inducible factor-2α (HIF-2α), glucose transporters type 4, the lactate transporter monocarboxylate transporter 4, key glycolysis rate-limiting enzymes and promoted mitochondrial division; moreover, increased glucose uptake, lactic acid and ATP production and proliferative cells were observed in these PAECs. Whereas, knockdown HIF-2α reversed CD137 signal-mediated effects in PAECs mentioned above. Compared with wild-type mice, the proliferation of PAECs and the percentage of vascular lateral wall thickness decreased in CD137 knockout mice. Together, CD137 signal participated in pulmonary vascular remodeling through the regulation of mitochondrial dynamics dependent on HIF-2α in PAECs.

FUNDC1/USP15/Drp1 ameliorated TNF-α-induced pulmonary artery endothelial cell proliferation by regulating mitochondrial dynamics.

Mitochondrial dysfunction in pulmonary artery endothelial cells (PAECs) is related to the pathogenesis of pulmonary hypertension (PH). The mitochondrial receptor protein FUN14 domain containing 1 (FUNDC1) was found to be involved in pulmonary artery smooth muscle cell proliferation in PH. However, its role in PAECs remains unclear. We investigated FUNDC1 expression in the pulmonary artery endothelium in both monocrotaline-induced animal models and TNF-α-stimulated cell models. Additionally, the effect of FUNDC1 on PAECs proliferation and its possible mechanism were also investigated. We observed decreased FUNDC1 protein levels in animals and in vitro in PAECs. FUNDC1 deficiency in PAECs upregulated the expression of the deubiquitination enzyme ubiquitin-specific peptidase 15 (USP15), enhanced dynamin-related protein1 (Drp1)-mediated mitochondrial division, and increased mitochondrial ROS levels via the deubiquitination of Drp1. Additionally, FUNDC1 deficiency increased aerobic glycolysis, the production of ATP and lactic acid, and glucose uptake. FUNDC1 overexpression inhibited PAECs proliferation. Moreover, FUNDC1 overexpression in combination with a mitochondrial division or aerobic glycolysis inhibitor enhanced its inhibitory effect on cell proliferation. Our study findings suggest that FUNDC1 deficiency induced by inflammation can promote PAECs proliferation by regulating mitochondrial dynamics and cell energy metabolism via the USP15/Drp1 pathway.

In Vitro Experimental Approach for Studying Human Pulmonary Artery Smooth Muscle Cells and Endothelial Cells Proliferation and Migration.

Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

Membrane stretch due to hypoosmolality leads to the development of various pulmonary and cardiovascular diseases

High blood pressures over a long period of time can lead to the development of diseases such as congestive heart failure(CHF). Congestive heart failure also commonly leads to the development of conditions such as hyponatremia, a common electrolyte disorder which increases the body’s water levels, creating hypoosmotic conditions for cells; these hypoosmotic conditions cause cell swelling, which subsequently causes mechanical stress on the cells. In these hypoosmotic environments, specific mechanosensitive ion channels which are activated by these mechanical stresses on cells may play a critical role in the progression of conditions resulting from these hypoosmotic environments, such as pulmonary arterial hypertension (PAH) and CHF. This study aims to look at the connection between the effects of hypoosmotic conditions in cells on the pathogenesis of the development of chronic diseases such as PAH and CHF. Mechanical actions on specific mechanosensitive ion channels such as Piezo1 have already been shown to play a critical role in regulating intracellular Ca2+homeostasis, which play an important part in the regulation of the vascular tone in places such as the pulmonary arteries; mechanical stimulation from hypotonic environments such as those seen in hyponatremia lead to Piezo1-mediated increases of intracellular Ca2+, which are subsequently linked to the increased contraction and proliferation of pulmonary arterial endothelial cells(PAECs). The proliferation of PAECs contribute to the pathogenesis of diseases, such as pulmonary arterial hypertension(PAH) or CHF, which there is currently no cure for. Patch-clamp experiments were conducted to examine the activity of mechanosensitive cation currents; graphed results showed that the activity of mechanosensitive cation currents were significantly increased (p<0.05) in PAECs treated with a hypoosmotic solution when compared with PAECs treated in an isosmotic control solution. Digital fluorescence microscopy using fura-2 AM, a membrane-permeable Ca2+ sensitive fluorescent indicator, and a Nikon digital imaging fluorescent microscopy system was also used to measure intracellular Ca2+ concentration in PAECs; graphed data indicated that the change in intracellular calcium concentration in PAECs was significantly increased(p<0.05) when placed in a hypoosmotic solution, than when placed in an isosmotic solution. The collective data indicates that hypoosmotic conditions increase the activity of mechanosensitive ion channels, as well as intracellular Ca2+ levels, which may prove the role of mechanosensitive ion channels like Piezo1 and TRPC6 in the development of diseases such as PAH and CHF. This work is supported in part by the National Heart, Lung, and Blood Institute of the National Institutes of Health Grants HL135807 and HL146764. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mice lacking 1,4,5-triphosphate inositol type III receptor demonstrate inhibition of hypoxic pulmonary hypertension

Hypoxic pulmonary hypertension (HPH) is a respiratory disease characterized by increased pulmonary vascular resistance and pulmonary arterial pressure. Persistent hypoxia alters the metabolic and transport functions of endothelial cells and promotes thrombosis and inflammation. Type 3 inositol-1,4,5-trisphosphate receptor (IP3R3) controls the release of calcium ions from the endoplasmic reticulum to the cytoplasm and mitochondria and is involved in cell proliferation, migration, and protein synthesis. In this study, we investigated the role and function of IP3R3 in HPH. The results showed that the expression level of IP3R3 was increased in pulmonary artery endothelial cells (PAECs) in a rat HPH model. The pulmonary artery pressure indices of IP3R3(−/−) mice with persistent hypoxia were significantly lower than those of HPH mice. The expression level of IP3R3 was significantly increased in hypoxia-treated PAECs. Knockdown of IP3R3 significantly inhibited the proliferation, migration and mesenchymal transition of PAECs induced by hypoxia. In conclusion, knockdown of IP3R3 can inhibit hypoxia-induced dysfunctions in PAECs, thus enabling IP3R3(−/−) mice to avoid HPH development. IP3R3 plays a key role in HPH and can be used as a potential target for the prevention and treatment of HPH.

MicroRNA-146-5p Promotes Pulmonary Artery Endothelial Cell Proliferation under Hypoxic Conditions through Regulating USP3

Objective MicroRNAs play a pivotal role in the progression of pulmonary hypertension (PAH). Although microRNA-146-5p is specifically expressed in many diseases, but in PAH, its role remains elusive. Patients and Methods. 30 patients with PAH and 20 healthy volunteers in our hospital were enrolled, and their serum samples were extracted for the detection of microRNA-146-5p and ubiquitin specific protease 3 (USP3) expression. In addition, the interaction between microRNA-146-5p and USP3 was examined by luciferase reporting assay. Furthermore, the potential mechanism was explored by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), and Western blotting experiments. Results It was found that microRNA-146-5p was higher in PAH patients than in healthy volunteers. Meanwhile, in hypoxia-induced human pulmonary artery endothelial cell lines (HPAECs), microRNA-146-5p expression was dramatically downregulated while USP3 protein expression was conversely upregulated. Under hypoxic conditions, microRNA-146-5p mimics was able to prompt the growth of HPAECs. In addition, after overexpression of microRNA-146-5p, luciferase reporting assay revealed a reduced luciferase activity of the reporter gene containing the USP3 3′-untranslated region, and a reduction of USP3 protein expression was also confirmed. However, USP3 overexpression partially attenuated the impact of upregulated microRNA-146-5p on the proliferation capacity of HPAECs. Conclusions MicroRNA-146-5p was able to enhance the proliferation ability of HPAEC cells under hypoxic conditions through targeting USP3, suggesting the microRNA-146-5p/USP3 axis may act as a target for PAH treatment.

Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension.

Background: The hypoxia-induced pro-proliferative and anti-apoptotic characteristics of pulmonary arterial endothelial cells (PAECs) play critical roles in pulmonary vascular remodeling and contribute to hypoxic pulmonary arterial hypertension (PAH) pathogenesis. However, the mechanism underlying this hypoxic disease has not been fully elucidated.Methods: Bioinformatics was adopted to screen out the key hypoxia-related genes in PAH. Gain- and loss-function assays were then performed to test the identified hypoxic pathways in vitro. Human PAECs were cultured under hypoxic (3% O2) or normoxic (21% O2) conditions. Hypoxia-induced changes in apoptosis and proliferation were determined by flow cytometry and Ki-67 immunofluorescence staining, respectively. Survival of the hypoxic cells was estimated by cell counting kit-8 assay. Expression alterations of the target hypoxia-related genes, cell cycle regulators, and apoptosis factors were investigated by Western blot.Results: According to the Gene Expression Omnibus dataset (GSE84538), differentiated embryo chondrocyte expressed gene 1-peroxisome proliferative-activated receptor-γ (Dec1-PPARγ) axis was defined as a key hypoxia-related signaling in PAH. A negative correlation was observed between Dec1 and PPARγ expression in patients with hypoxic PAH. In vitro observations revealed an increased proliferation and a decreased apoptosis in PAECs under hypoxia. Furthermore, hypoxic PAECs exhibited remarkable upregulation of Dec1 and downregulation of PPARγ. Dec1 was confirmed to be crucial for the imbalance of proliferation and apoptosis in hypoxic PAECs. Furthermore, the pro-surviving effect of hypoxic Dec1 was mediated through PPARγ inhibition.Conclusion: For the first time, Dec1-PPARγ axis was identified as a key determinant hypoxia-modifying signaling that is necessary for the imbalance between proliferation and apoptosis of PAECs. These novel endothelial signal transduction events may offer new diagnostic and therapeutic options for patients with hypoxic PAH.

WITHDRAWN: Tanshinone-IIA inhibits the inflammatory response and proliferation of PAECs under hypoxic conditions by repressing HMGB1 via the TLR4/NF-κB signalling pathway

To explore the potential molecular mechanism of Tanshinone-IIA (Tan-IIA) in pulmonary artery endothelial cells (PAECs) under hypoxic conditions. Rat PAECs were observed under an inverted microscope, and the biomarkers CD31 and factor VIII related antigen (FVIII-Rag) were identified by immunofluorescence (IF). The PAECs were divided into a 20% normoxia group and a 4% hypoxia group, and the cells were subjected to Tan-IIA treatment, high mobility group box 1 (HMGB1) overexpression, or the relevant controls. ELISA and MTT were performed to evaluate proinflammatory factor production and cell proliferation, respectively. RT-qPCR was used to measure HMGB1 mRNA expression, and western blotting was performed to evaluate the protein levels of HMGB1, Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB)-p65. Immunocytochemistry (ICC) and western blotting indicated the subcellular distribution of NF-κB-p65. Rat PAECs were round or short spindle-shaped and abundantly expressed CD31 and FVIII-Rag. Compared with the normoxia group, the hypoxia group produced significantly increased levels of proinflammatory factors, which decreased with increasing concentrations of Tan-IIA (p<0.05). The expression of HMGB1 and TLR4 and the phosphorylation of NF-κB-p65 presented gradually decreasing trends in the 4% hypoxia group with increasing concentrations of Tan-IIA. Moreover, ICC indicated that in the 4% hypoxia group, NF-κB-p65 translocated to the nucleus (p<0.05), and western blotting analysis showed that the higher the Tan-IIA concentration was, the lower the NF-κBp65 level in the nucleus was. Compared with the Hypoxia + Vector group, the Hypoxia+ pHMGB1 group exhibited significantly enhanced effects of the hypoxia-induced inflammatory response (p<0.05), and the inhibitory effects of Tan-IIA on the hypoxia-induced inflammatory response were reversed in the Hypoxia+Tan-IIA+pHMGB1 group due to the overexpression of HMGB1 (p<0.05); HMGB1 exerted similar effects on cell proliferation, as illustrated by MTT assay. Tan-IIA inhibits the proliferation and inflammatory response of PAECs by downregulating the expression of HMGB1 via the TLR4/NF-κB signalling pathway under hypoxic conditions.

SIRT1 promotes pulmonary artery endothelial cell proliferation by targeting the Akt signaling pathway.

Pulmonary arterial hypertension (PAH) is a disease characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodeling; however, the pathogenesis of the disease is not completely understood. Sirtuin 1 (SIRT1) is a histone deacetylase involved in cell survival and metabolism. The present study explored the potential role of SIRT1 in human pulmonary arterial endothelial cells (HPAECs) under hypoxic conditions. In vitro HPAECs were cultured and exposed to hypoxic conditions. Subsequently, SIRT1 expression levels were measured via western blotting, the generation of reactive oxygen species (ROS) was evaluated, and the interaction between SIRT1 and Akt was assessed via reverse transcription-quantitative PCR and western blotting. In addition, the effects of SIRT1 on cell proliferation and apoptosis were also investigated. The results indicated that hypoxia induced SIRT1 expression in pulmonary arterial endothelial cells, which may be associated with ROS generation. SIRT1 expression activated the Akt signaling pathway, which increased the expression levels of Bcl-2 and hypoxia-inducible factor-1 in HPAECs. Moreover, SIRT1 promoted HPAEC proliferation and inhibited HPAEC apoptosis. ROS generation enhanced the SIRT1/Akt axis, which was essential for epithelial cell injury under hypoxic conditions. Therefore, blocking SIRT1 may reduce hypoxia-induced pathological damage in HPAECs.

Sex differences in the proliferation of pulmonary artery endothelial cells: implications for plexiform arteriopathy.

The sex-biased disease pulmonary arterial hypertension (PAH) is characterized by the proliferation and overgrowth of dysfunctional pulmonary artery endothelial cells (PAECs). During inflammation associated with PAH, granzyme B cleaves intersectin-1 to produce N-terminal (EHITSN) and C-terminal (SH3A-EITSN) protein fragments. In a murine model of PAH, EHITSN triggers plexiform arteriopathy via p38-ELK1-c-Fos signaling. The SH3A-EITSN fragment also influences signaling, having dominant-negative effects on ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively). Using PAECs engineered to express tagged versions of EHITSN and SH3A-EITSN, we demonstrate that the two ITSN fragments increase both p38-ELK1 activation and the ratio of p38 to ERK1 and ERK2 activity, leading to PAEC proliferation, with female cells being more responsive than male cells. Furthermore, expression of EHITSN substantially upregulates the expression and activity of the long non-coding RNA Xist in female PAECs, which in turn upregulates the X-linked gene ELK1 and represses expression of krüppel-like factor 2 (KLF2). These events are recapitulated by the PAECs of female idiopathic PAH patients, and may account for their proliferative phenotype. Thus, upregulation of Xist could be an important factor in explaining sexual dimorphism in the proliferative response of PAECs and the imbalanced sex ratio of PAH.

Molecular Mechanism and Functional Role of Hypoxia‐induced Reactive Oxygen Species in Pulmonary Arterial Endothelial Cells

Hypoxia‐induced (Group 3) pulmonary hypertension (PH) is a common devastating lung disease. Pulmonary artery endothelial cells (PAECs) are a major contributor in hypoxic PH due to possible roles of reactive oxygen species (ROS). However, the production up‐and‐down, molecular mechanisms and functional roles of hypoxia‐induced ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed‐derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2−) production. Our assays showed that hypoxia largely increased O2− production. Hypoxia also enhanced H2O2 production in isolated mitochondria from PAECs. In support, using the genetically encoded H2O2 sensor HyPer, we revealed that hypoxia caused a large increase in HyPer‐derived fluorescence (ROS production) in PAECs, which was blocked by the mitochondrial inhibitor rotenone or myxothiazol. Hypoxia largely increased proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. The hypoxic cell proliferation was blocked by rotenone or myxothiazol. Like rotenone and myxothiazol, lentiviral shRNA‐mediated knockdown of Rieske iron‐sulfur protein nicotine (RISP), known to be a primary molecule in mitochondrial ROS generation, inhibited the hypoxic proliferation as well. Hypoxic exposure evoked a significant increase in migration of PAECs, evaluated by wound healing assay. Pharmacological and genetic inhibition of mitochondrial ROS generation with myxothiazol and RISP knockdown both diminished the hypoxic cell migration. Taken together, we conclude that hypoxia significantly increases intracellular ROS generation in human PAECs, the hypoxia‐induced intracellular ROS generation primarily occur in mitochondria, and RISP serves as an essential molecule in controlling the hypoxic mitochondrial ROS generation. Equally Importantly, and RISP‐dependent mitochondrial proliferation that is likely to be ROS‐dependent. Conceivably, hypoxia remarkably causes proliferation and migration of human PAECs, which is predominantly mediated by RISP‐dependent mitochondrial ROS, thereby making an essential contribution to hypoxic pulmonary hypertension.

MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a dangerous disease, featured by pulmonary vascular remodeling. Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs) plays crucial roles in this process. MicroRNA-150 (miR-150) level has been found to be reduced in patients with PH, and correlated with the poor survival. This study aimed to investigate the beneficial effect of miR-150 on PH in hypoxia-induced rats, PASMCs and PAECs. The results showed that miR-150 level was reduced in the lung tissue and plasma of hypoxia-treated rats. Lentivirus-mediated overexpression of miR-150 restrained hypoxia-induced increase in right ventricular systolic pressure and decrease in cardiac output. Moreover, as assessed by HE staining, hypoxia-induced thickening of vessel wall was relieved by miR-150 up-regulation. Overexpression of miR-150 also suppressed hypoxia-induced formation of collagen fiber, expressions of α-SMA, TGF-β1, and collagen I in lung tissues and PASMCs. In addition, the excessive proliferation of PASMCs induced by hypoxia was repressed by miR-150 overexpression via AKT/mTOR signaling pathway. Increased NFATc3 expression in response to hypoxia was restrained by miR-150 overexpression in lung tissue and PAMSCs. Finally, miR-150 overexpression inhibited hypoxia-induced proliferation and apoptosis resistance in PAECs. In conclusion, these results indicate that miR-150 protects against hypoxia-induced pulmonary vascular remodeling, fibrosis, abnormal proliferation of PASMCs and PAECs, which suggests miR-150 as a promising therapeutic target for PH.

Exosomal 15-LO2 mediates hypoxia-induced pulmonary artery hypertension in vivo and in vitro

Our previous studies have shown that 15-LO2/15-HETE induced by hypoxia played an important role in pulmonary arterial hypertension (PH). However, the transportations of 15-LO2/15-HETE among the cells remain elusive. In this study, we investigated the specific involvement of 15-LO2-containing exosomes in the overproliferation of pulmonary artery endothelial cells (PAECs) induced by hypoxia and the underlying mechanism. In vitro, 15-LO2 was abundantly expressed and enriched in exosomes secreted from hypoxic PAECs, which subsequently activated the STAT3 signaling pathway, resulting in a robust increase in PAECs proliferation. In vivo treatment with the exosomes inhibitor GW4869 protected the pulmonary vascular homeostasis from dysfunctional and abnormal remodeling. Moreover, 15-LO2 was ubiquitinated under hypoxia, and further inhibition of the ubiquitin-proteasome system significantly suppressed PAECs proliferation, suggesting that ubiquitination of 15-LO2 may contribute to its sorting into exosomes. Overall, these findings indicate a previously unrecognized effect of exosomes and the cargo 15-LO2 in pulmonary vascular homeostasis on the pathogenesis of PH.

Growth Differentiation Factor 11 Promotes Abnormal Proliferation and Angiogenesis of Pulmonary Artery Endothelial Cells.

Disordered proliferation and angiogenesis of pulmonary artery endothelial cells is an important stage in the development of pulmonary arterial hypertension. Recent studies revealed that GDF11 (growth differentiation factor 11) induces endothelial cells proliferation and migration; however, whether GDF11 is directly involved in the pathogenesis of pulmonary arterial hypertension remains unknown. Here, we found that GDF11 was significantly upregulated and activated in 2 experimental pulmonary arterial hypertension models and cultured pulmonary artery endothelial cells. Genetic ablation of gdf11 in endothelial cells rescued pulmonary arterial hypertension features, as demonstrated by right ventricle hypertrophy, right ventricular systolic pressure, hemodynamics, cardiac function, and vascular remodeling. Moreover, we found that hypoxia significantly increased cell cycle progression, proliferation, migration, adhesion, and tube formation, which were significantly inhibited by GDF11 small interfering RNA. These events could be reproduced using cultured pulmonary artery endothelial cells and were dependent on Smad signaling. Moreover, hypoxia-induced GDF11 expression was regulated by the transcription factor zinc finger protein 740, which assisted RNA polymerase in recognizing and binding to the GDF11 promoter sequence located at a site (-753/-744; CCCCCCCCAC) upstream of the gene. This study identified a novel growth and differentiation factor signaling pathway involved in the zinc finger protein 740/GDF11/transforming growth factor-β receptor I/Smad signaling axis and involved in pulmonary artery endothelial cells proliferation and angiogenesis. These results provide critical insights for the development of novel therapeutic strategies for pulmonary arterial hypertension involving components of the GDF11 signaling system.

1,25(OH)2D3 attenuates pulmonary arterial hypertension via microRNA-204 mediated Tgfbr2/Smad signaling

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by high pulmonary artery pressure. It is reported that microRNA-204 (miR-204) plays an important role in the development of PAH. Calcitriol [1,25-dihydroxyvitamin D3, 1,25(OH)2D3] mediates multiple pathophysiological processes. The aim of the current study was to explore the role of 1,25(OH)2D3 in PAH. PAH was induced in rats and rat pulmonary arterial endothelial cells (PAECs) were isolated as in vitro PAH model. The mean pulmonary artery pressure, morphologic changes, and expressions of transforming growth factor-beta1 (Tgfbr2), Smad2/7, alpha smooth muscle actin (α-SMA), and p21 were then measured. Furthermore, the effect of 1,25(OH)2D3 on rat PAECs with or without hypoxia treatment was also assessed by measuring the proliferation, migration, and cell cycle distribution of PAECs. The potential targets of miR-204 were also predicted and validated with a dual-luciferase reporter system. Then the role of miR-204 and Tgfbr2 in the anti-PAH effect of 1,25(OH)2D3 was further explored by modulating the expression of the two genes. The overall pulmonary hypertension and hypoxia-induced proliferation and migration of PAECs were attenuated by administration of 1,25(OH)2D3, which was associated with the suppressed expressions of Tgfbr2, α-SMA, and Smad7 and induced expressions of miR-204, p21 and Smad2 both in vitro and in vivo. Moreover, the luciferase reporter assay identified Tgfbr2 as a novel direct target of miR-204. Both overexpression of miR-204 and inhibition of Tgfbr2 would strengthen the effect of 1,25(OH)2D3 administration. Findings outlined in the current study demonstrated that 1,25(OH)2D3 was a promising therapeutic modality for treatment of PAH, function of which was exerted through miR-204 mediated Tgfbr2 signaling.

Fatty acid synthase modulates proliferation, metabolic functions and angiogenesis in hypoxic pulmonary artery endothelial cells

Endothelial dysfunction plays an important role in structural remodeling occurring in the pulmonary vasculature during pulmonary hypertension (PH). Endothelial injury causes apoptosis and activation of endothelial cells. However, some endothelial cells show apoptosis-resistance and later proliferate extensively leading to vascular oculopathy and formation of plexiform lesions in PH. Studies have shown that rapidly proliferating cells exhibit increased expression of Fatty acid synthase (FAS), a regulatory enzyme responsible for the production of fatty acids. Our previous study has shown that FAS inhibition prevented smooth muscle cell proliferation, reversed pulmonary vascular remodeling and improved pulmonary vasoreactivity in monocrotaline induced PH model. However, the role of FAS in pulmonary artery endothelial cell proliferation and angiogenesis has not been explored. The present study was designed to explore the role of FAS in proliferation, metabolic dysfunctions, and angiogenesis in endothelial dysfunction associated with PH. The human pulmonary artery endothelial cells (HPAECs) were exposed to hypoxia and FAS siRNA (60nM) was used for the FAS inhibition. Increased expression and activity of FAS were observed in hypoxic HPAECs. Inhibition of FAS increased apoptosis and glucose oxidation, but decreased cellular proliferation, markers of autophagy and glycolysis in hypoxic HPAECs. FAS inhibition decreased the angiogenesis as evident by decreased tubule length and VEGF expression in hypoxic HPAECs. Inhibition of FAS also increased expression of endothelial NOS in hypoxic HPAECs, a marker of endothelial function. Our results proved, and further supported previous findings, that inhibition of FAS is beneficial for endothelial function in pulmonary hypertension.