Human Pulmonary Artery Endothelial Cells Research Articles

Persistent Hypoxia with Intermittent Aggravation Causes Imbalance in Smad3/Myocardin-Related Transcription Factor Signaling with Consequent Endothelial Senescence and Pulmonary Arterial Remodeling.

Loss of Smad3 and the consequent activation of myocardin-related transcription factor (MRTF) are associated with vascular pathologies. This study aimed to examine the impact of persistent hypoxia with intermittent aggravation (PI hypoxia) on cellular senescence and pulmonary arterial remodeling mediated by the Smad3/MRTF imbalance. We examined the effects of PI hypoxia on the Smad3/MRTF pathway and cellular senescence using human pulmonary artery endothelial cells (HPAECs) and in vivo studies in rats. The senescent degree was evaluated using β-galactosidase staining, p16 quantitation and the measurement of senescence-associated secretory phenotype. Structural data in the pathological analysis of pulmonary artery remodeling were collected. Compared to the control, HPAECs and pulmonary tissue from rats exposed to PI hypoxia showed a significantly higher senescent degree, lower expression of Smad3, and higher MRTF levels. The overexpression of Smad3 significantly mitigated HPAECs senescence in vitro. Further, treatment with CCG-203971, which inhibits MRTF, increased Smad3 levels and reduced β-galactosidase positive cells in rat lung tissue. This intervention also alleviated PI hypoxia-induced pathological changes, including remodeling indices of pulmonary arterial thickening, muscularization, and collagen formation. In conclusion, imbalanced Smad3/MRTF signaling is linked to PI hypoxia-induced senescence and pulmonary arterial remodeling, making it a potential therapeutic target for patients with sleep apnea and chronic obstructive pulmonary disease.

Polo-like kinase 1 promotes pulmonary hypertension

BackgroundPulmonary hypertension (PH) is a lethal vascular disease with limited therapeutic options. The mechanistic connections between alveolar hypoxia and PH are not well understood. The aim of this study was to investigate the role of mitotic regulator Polo-like kinase 1 (PLK1) in PH development.MethodsMouse lungs along with human pulmonary arterial smooth muscle cells and endothelial cells were used to investigate the effects of hypoxia on PLK1. Hypoxia- or Sugen5416/hypoxia was applied to induce PH in mice. Plk1 heterozygous knockout mice and PLK1 inhibitors (BI 2536 and BI 6727)-treated mice were checked for the significance of PLK1 in the development of PH.ResultsHypoxia stimulated PLK1 expression through induction of HIF1α and RELA. Mice with heterozygous deletion of Plk1 were partially resistant to hypoxia-induced PH. PLK1 inhibitors ameliorated PH in mice.ConclusionsAugmented PLK1 is essential for the development of PH and is a druggable target for PH.

Endothelial FIS1 DeSUMOylation Protects Against Hypoxic Pulmonary Hypertension.

Hypoxia is a major cause and promoter of pulmonary hypertension (PH), a representative vascular remodeling disease with poor prognosis and high mortality. However, the mechanism underlying how pulmonary arterial system responds to hypoxic stress during PH remains unclear. Endothelial mitochondria are considered signaling organelles on oxygen tension. Results from previous clinical research and our studies suggested a potential role of posttranslational SUMOylation in endothelial mitochondria in hypoxia-related vasculopathy. Chronic hypoxia mouse model and Sugen/hypoxia rat model were employed as PH animal models. Mitochondrial morphology and subcellular structure were determined by transmission electron and immunofluorescent microscopies. Mitochondrial metabolism was determined by mitochondrial oxygen consumption rate and extracellular acidification rate. SUMOylation and protein interaction were determined by immunoprecipitation. The involvement of SENP1 (sentrin-specific protease 1)-mediated SUMOylation in mitochondrial remodeling in the pulmonary endothelium was identified in clinical specimens of hypoxia-related PH and was verified in human pulmonary artery endothelial cells under hypoxia. Further analyses in clinical specimens, hypoxic rat and mouse PH models, and human pulmonary artery endothelial cells and human embryonic stem cell-derived endothelial cells revealed that short-term hypoxia-induced SENP1 translocation to endothelial mitochondria to regulate deSUMOylation of mitochondrial fission protein FIS1 (mitochondrial fission 1), which facilitated FIS1 assembling with fusion protein MFN2 (mitofusin 2) and mitochondrial gatekeeper VDAC1 (voltage-dependent anion channel 1), and the membrane tethering activity of MFN2 by enhancing its oligomerization. Consequently, FIS1 deSUMOylation maintained the mitochondrial integrity and endoplasmic reticulum-mitochondria calcium communication across mitochondrial-associated membranes, subsequently preserving pulmonary endothelial function and vascular homeostasis. In contrast, prolonged hypoxia disabled the FIS1 deSUMOylation by diminishing the availability of SENP1 in mitochondria via inducing miR-138 and consequently resulted in mitochondrial dysfunction and metabolic reprogramming in pulmonary endothelium. Functionally, introduction of viral-packaged deSUMOylated FIS1 within pulmonary endothelium in mice improved pulmonary endothelial dysfunction and hypoxic PH development, while knock-in of SUMO (small ubiquitin-like modifier)-conjugated FIS1 in mice exaggerated the diseased cellular and tissue phenotypes. By maintaining endothelial mitochondrial homeostasis, deSUMOylation of FIS1 adaptively preserves pulmonary endothelial function against hypoxic stress and consequently protects against PH. The FIS1 deSUMOylation-SUMOylation transition in pulmonary endothelium is an intrinsic pathogenesis of hypoxic PH.

Abstract P1017: Post-GWAS Functional Analysis Of CBLN2 Locus Reveals The Role Of CUX1 In The Inflammatory Pathogenesis Of Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a deadly pulmonary vascular disease highly relevant to genetic susceptibility and acquired pathogenic triggers, especially inflammation. Chr18q22.3 near cerebellin 2 (CBLN2) gene is the first genetic locus identified by Genome-wide Association Study (GWAS) that is significantly associated with PAH risk in population of European ancestry. This locus includes a group of closely linked non-coding intergenic single nucleotide polymorphisms (SNPs) downstream of CBLN2. Although CBLN2 is upregulated in PAH and causes pulmonary vascular dysfunction, it remains unknown how these non-coding SNPs remotely affect CBLN2 and in turn alter the genetic susceptibility of PAH. Methods and Results: We applied Hi-C analysis, combined with electrophoretic mobility shift assay, to identify rs11151770 as a functional SNP (fSNP) characterized with allele-imbalanced nuclear protein binding and chromatin interaction with CBLN2 promoter, among 19 haplotype SNPs in a linkage disequilibrium (LD r 2 >0.8) in CBLN2 locus. As an intergenic non-coding SNP, rs11151770 is located 110 kbp downstream of CBLN2 promoter. Proteomic analysis revealed nuclear transcription factor CUX1 binds specifically to the risk allele A of rs11151770 but not to the non-risk allele C. Acting as the activator for the expression of target gene CBLN2, CUX1 knockdown prevented, but CUX1 overexpression induced, the pathophenotypes of human pulmonary arterial endothelial cells (PAECs) associated with PAH. CUX1-CBLN2 was upregulated by acquired inflammatory PAH trigger IL-1β and was increased in lung tissues extracted from PAH patients. SNP genotyping in PAH patients and non-PAH controls showed a 2.2-fold risk allele A enrichment only in patients with PAH associated with connective tissue disorders, supporting a pathogenic mechanism of an inflammation-sensitive pathway (CUX1-CBLN2) combined with a PAH susceptible genotype (risk allele A of rs11151770) for the manifestation of clinical PAH. Conclusion: Our post-GWAS functional genomics analysis revealed a CUX1-mediated inflammatory pathway and provided a mechanistic explanation for the GWAS-identified association between the genetic variants in CBLN2 locus and PAH pathogenesis.

SARS-CoV-2 spike protein receptor-binding domain perturbates intracellular calcium homeostasis and impairs pulmonary vascular endothelial cells

Exposure to the spike protein or receptor-binding domain (S-RBD) of SARS-CoV-2 significantly influences endothelial cells and induces pulmonary vascular endotheliopathy. In this study, angiotensin-converting enzyme 2 humanized inbred (hACE2 Tg) mice and cultured pulmonary vascular endothelial cells were used to investigate how spike protein/S-RBD impacts pulmonary vascular endothelium. Results show that S-RBD leads to acute-to-prolonged induction of the intracellular free calcium concentration ([Ca2+]i) via acute activation of TRPV4, and prolonged upregulation of mechanosensitive channel Piezo1 and store-operated calcium channel (SOCC) key component Orai1 in cultured human pulmonary arterial endothelial cells (PAECs). In mechanism, S-RBD interacts with ACE2 to induce formation of clusters involving Orai1, Piezo1 and TRPC1, facilitate the channel activation of Piezo1 and SOCC, and lead to elevated apoptosis. These effects are blocked by Kobophenol A, which inhibits the binding between S-RBD and ACE2, or intracellular calcium chelator, BAPTA-AM. Blockade of Piezo1 and SOCC by GsMTx4 effectively protects the S-RBD-induced pulmonary microvascular endothelial damage in hACE2 Tg mice via normalizing the elevated [Ca2+]i. Comparing to prototypic strain, Omicron variants (BA.5.2 and XBB) of S-RBD induces significantly less severe cell apoptosis. Transcriptomic analysis indicates that prototypic S-RBD confers more severe acute impacts than Delta or Lambda S-RBD. In summary, this study provides compelling evidence that S-RBD could induce persistent pulmonary vascular endothelial damage by binding to ACE2 and triggering [Ca2+]i through upregulation of Piezo1 and Orai1. Targeted inhibition of ACE2-Piezo1/SOCC-[Ca2+]i axis proves a powerful strategy to treat S-RBD-induced pulmonary vascular diseases.

Hyperoxic exposure alters intracellular bioenergetics distribution in human pulmonary cells

AimsPulmonary oxygen toxicity is caused by exposure to a high fraction of inspired oxygen, which damages multiple cell types within the lung. The cellular basis for pulmonary oxygen toxicity includes mitochondrial dysfunction. The aim of this study was to identify the effects of hyperoxic exposure on mitochondrial bioenergetic and dynamic functions in pulmonary cells. Main methodsMitochondrial respiration, inner membrane potential, dynamics (including motility), and distribution of mitochondrial bioenergetic capacity in two intracellular regions were quantified using cultured human lung microvascular endothelial cells, human pulmonary artery endothelial cells and A549 cells. Hyperoxic (95 % O2) exposures lasted 24, 48 and 72 h, durations relevant to mechanical ventilation in intensive care settings. Key findingsMitochondrial motility was altered following all hyperoxic exposures utilized in experiments. Inhomogeneities in inner membrane potential and respiration parameters were present in each cell type following hyperoxia. The partitioning of ATP-linked respiration was also hyperoxia-duration and cell type dependent. Hyperoxic exposure lasting 48 h or longer provoked the largest alterations in mitochondrial motility and the greatest decreases in ATP-linked respiration, with a suggestion of decreases in respiration complex protein levels. SignificanceHyperoxic exposures of different durations produce intracellular inhomogeneities in mitochondrial dynamics and bioenergetics in pulmonary cells. Oxygen therapy is utilized commonly in clinical care and can induce undesirable decrements in bioenergy function needed to maintain pulmonary cell function and viability. There may be adjunctive or prophylactic measures that can be employed during hyperoxic exposures to prevent the mitochondrial dysfunction that signals the presence of oxygen toxcity.

Decursinol angelate attenuates lipopolysaccharide induced acute lung injury by regulating AKT/NF κB pathway

Purpose: To investigate the effect of decursinol angelate in acute lung injury induced by lipopolysaccharide treatment.
 Methods: Human bronchial epithelial cells (BEAS-2B cells) and human pulmonary artery endothelial cells (HPAECs) were exposed to lipopolysaccharide to induce cell injury and then treated with 20, 40, or 60 μM decursinol angelate. Cell viability was quantified using a cell counting assay, while cell apoptosis was evaluated by western blot and flow cytometry. Colorimetric and enzyme-linked immunosorbent assay (ELISA) was used to measure oxidative stress. Expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1β, and interleukin-6 were evaluated using quantitative real-time-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Phosphorylation levels of p65, IκBα, AKT, and phosphatidylinositol 3-kinase were assessed by western blot, while the entry of p65 into the nucleus was investigated by indirect immunofluorescence assay.
 Results: Treatment with 40 or 60 μM decursinol angelate increased cell viability after lipopolysaccharide-induced damage. B-cell lymphoma 2 (Bcl2) and Bax expression were regulated, and the rate of apoptosis was inhibited. The concentration of superoxide dismutase and glutathione was elevated, and the density of malondialdehyde and myeloperoxidase decreased significantly. Inflammation was also suppressed by decursinol angelate by regulating TNF-α, interleukin-1β, and interleukin-6 expressions. Furthermore, decursinol angelate significantly decreased the phosphorylation of p65, IκBα, AKT, and phosphatidylinositol 3-kinase. Finally, decursinol angelate inhibited p65 entry into the cell nucleus.
 Conclusion: Decursinol angelate alleviates cell damage and apoptosis, reduces oxidative stress, and attenuates inflammation after lipopolysaccharide-induced acute lung injury by inhibiting AKT/NF-κB signaling pathway. Thus, decursinol angelate is a potential candidate for the treatment of lung injury.

Abstract 220: Post-gwas Functional Genomics Analysis Of Abo Locus To Identify Novel Pathogenic Mechanisms For Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and mysterious complication of pulmonary embolism, characterized by chronic pulmonary arterial thrombosis, occlusions and vasculopathy. Emerging evidence has suggested genetic relevance of CTEPH, particularly to ABO blood type. Genome-wide association study (GWAS) has identified ABO gene as the most significant genetic locus associated with CTEPH. This locus, however, consists only intronic SNPs and there is no mechanistic explanation on how these non-coding SNPs control the risk of CTEPH. Methods: GWAS-identified CTEPH-associated ABO locus was analyzed, and haplotype SNPs were identified based on linkage disequilibrium (LD). Electrophoretic mobility shift assay (EMSA) was applied to screen functional SNPs (fSNPs) based on allele-imbalanced nuclear protein binding. SNP-binding nuclear transcription factors (TFs) were identified by proteomics. Hi-C chromatin-interaction analysis was utilized to determine potential target genes affected by fSNPs. Knockdown of fSNP-associated TFs in human pulmonary artery endothelial cells (hPAECs) was applied to validate the regulation of TFs on target genes. Results: Based on GWAS, the minor allele of tag SNP rs2519093 is associated with higher risk of CTEPH (Odds ratio 2.22). It defines 11 haplotype SNPs in a LD with R 2 > 0.8. Screened by EMSA with hPAEC nuclear proteins, intronic SNPs rs579459 and rs550057 were identified functional given their allele-specific protein binding. Proteomics analysis identified 3 candidate TFs that allele-specifically bind to fSNP rs579459: PBX1, CTCF and SAFB2. Candidate target gene ADAMTS13 is chosen based on its prominent interaction with fSNP revealed by Hi-C analysis, and the potential role in thromboembolism through vWF cleavage. TF knockdown by siRNA downregulates ADAMTS13 in hPAECs, suggesting their regulatory function as activators. Conclusion: Leveraging our post-GWAS functional genomics approach, we identified fSNPs in ABO locus that define a genetic architecture controlling the susceptibility of CTEPH. This genetic architecture, along with the newly identified fSNP-associated TFs and target gene ADAMTS13, provides a biological explanation for the pathogenesis of CTEPH.

Hpgd affects the progression of hypoxic pulmonary hypertension by regulating vascular remodeling

BackgroundHypoxic pulmonary hypertension (HPH) is a syndrome of abnormally elevated pulmonary artery pressure, and it is mostly caused by vasoconstriction and remodeling of the pulmonary artery induced by long-term chronic hypoxia. There is a high incidence of HPH, a short survival time of the patients, but currently no effective treatments.MethodsIn this study, HPH-related single cell sequencing (scRNA-seq) and bulk RNA sequencing (RNA-seq) data were downloaded from the public database of Gene Expression Omnibus (GEO) for bioinformatics analysis in order to find out genes with important regulatory roles in the development of HPH. 523 key genes were identified through cell subpopulation identification and trajectory analysis of the downloaded scRNA-seq data, and 41 key genes were identified through weighted correlation network analysis (WGCNA) of the bulk RNA-seq data. Three key genes: Hpgd, Npr3 and Fbln2 were identified by taking intersection of the key genes obtained above, and Hpgd was finally selected for subsequent verification. The human pulmonary artery endothelial cells (hPAECs) were treated with hypoxia for different periods of time, and it was found that the expression of Hpgd decreased in hypoxia-treated hPAECs in a time-dependent manner. In order to further confirm whether Hpgd affects the occurrence and development of HPH, Hpgd was overexpressed in hPAECs.ResultsHpgd was confirmed to regulate the proliferation activity, apoptosis level, adhesiveness and angiogenesis ability of hypoxia-treated hPAECs through multiple experiments.ConclusionsDownregulation of Hpgd can improve the proliferation activity, reduce apoptosis, and enhance adhesion and angiogenesis in endothelial cells (ECs), thus promoting the occurrence and development of HPH.

Impact of l-citrulline on nitric oxide signaling and arginase activity in hypoxic human pulmonary artery endothelial cells.

Impaired nitric oxide (NO) signaling contributes to the development of pulmonary hypertension (PH). The l-arginine precursor, l-citrulline, improves NO signaling and has therapeutic potential in PH. However, there is evidence that l-citrulline might increase arginase activity, which in turn, has been shown to contribute to PH. Our major purpose was to determine if l-citrulline increases arginase activity in hypoxic human pulmonary artery endothelial cells (PAECs). In addition, to avoid potential adverse effects from high dose l-citrulline monotherapy, we evaluated whether the effect on NO signaling is greater using co-treatment with l-citrulline and another agent that improves NO signaling, folic acid, than either alone. Arginase activity was measured in human PAECs cultured under hypoxic conditions in the presence of l-citrulline (0-1 mM). NO production and endothelial nitric oxide synthase (eNOS) coupling, as assessed by eNOS dimer-to-monomer ratios, were measured in PAECs treated with l-citrulline and/or folic acid (0.2 μM). Arginase activity increased in hypoxic PAECs treated with 1 mM but not with either 0.05 or 0.1 mM l-citrulline. Co-treatment with folic acid and 0.1 mM l-citrulline increased NO production and eNOS dimer-to-monomer ratios more than treatment with either alone. The potential to increase arginase activity suggests that there might be plasma l-citrulline concentrations that should not be exceeded when using l-citrulline to treat PH. Rather than progressively increasing the dose of l-citrulline as a monotherapy, co-therapy with l-citrulline and folic acid merits consideration, due to the possibility of achieving efficacy at lower doses and minimizing side effects.

Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4-Human Leukocyte Antigen-DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

Targeting Endothelial ENO1 (Enolase) -PI3K-Akt-mTOR Axis Alleviates Hypoxic Pulmonary Hypertension.

It has been shown that glycolytic protein alpha-ENO1 (enolase) contributes to the pathogenesis of pulmonary hypertension through acting smooth muscle cells; however, the roles of ENO1-caused endothelial and mitochondrial dysfunctions in Group 3 pulmonary hypertension remain unexplored. PCR array and RNA sequencing were used to screen and decipher the differential gene expression by hypoxia-treated ECs. Techniques of small-interfering RNA, specific inhibitor and plasmids carrying gene of ENO1, interventions with specific inhibitor and AAV-ENO1 delivery were employed to explore the role of ENO1 in hypoxic pulmonary hypertension in vitro and in vivo, respectively. Assays for cell proliferation, angiogenesis, and adhesion were employed to analyze cell behaviors, while seahorse analysis was used to measure mitochondrial function of human pulmonary artery endothelial cells. PCR array data showed that ENO1 expression increased in human pulmonary artery endothelial cells exposed to hypoxia, as well as in lung tissues from patients with chronic obstructive lung disease-pulmonary hypertension and murine model of hypoxic pulmonary hypertension. Inhibition of ENO1 restored the hypoxia-induced endothelial dysfunction, including excessive proliferation, angiogenesis, and adhesion, while overexpression of ENO1 promotes these disorders of human pulmonary artery endothelial cells. RNA-seq showed that ENO1 targets mitochondrion-related genes and PI3K-Akt signaling pathway. Mice treated with ENO1 inhibitor exhibited ameliorated pulmonary hypertension and improved right ventricular failure induced by hypoxia. A reversal effect was observed in mice exposed to hypoxia and inhaled adenoassociated virus overexpressing ENO1. These results indicate that hypoxic pulmonary hypertension is associated with an increased level of ENO1 and that targeting ENO1 might reduce experimental hypoxic pulmonary hypertension by improving endothelial and mitochondrial dysfunction via PI3K-Akt-mTOR signaling pathway.

Inhibition of poly (ADP-ribose) Polymerase-1 (PARP-1) improves endothelial function in pulmonary hypertension

Endothelial dysfunction is critical in the pulmonary vasculature during pulmonary hypertension (PH). Moreover, in PH, increased inflammation and oxidative/nitrosative stress cause DNA damage, activating poly (ADP-ribose) polymerase-1 (PARP-1). Meloche et al. (2014) and our previous research have shown that inhibiting PARP-1 is protective in PH and associated RV hypertrophy. However, the role of PARP-1 in pulmonary arterial endothelial dysfunction has not been explored completely. Therefore, the current study aims to investigate the involvement of PARP-1 in endothelial dysfunction associated with PH. Hypoxia (1% O2) was used to induce a PH-like phenotype in human pulmonary artery endothelial cells (HPAECs), and PARP-1 inhibition was achieved via siRNA (60 nM). For the in vivo study, male Sprague Dawley rats were administered monocrotaline (MCT; 60 mg/kg, SC, once) to induce PH, and 1, 5-isoquinolinediol (ISO; 3 mg/kg) was administered daily intraperitoneally to inhibit PARP-1. PARP-1 inhibition decreased proliferation and inflammation, as well as improved mitochondrial dysfunction in hypoxic HPAECs. Furthermore, PARP-1 inhibition also promoted apoptosis by increasing DNA damage in hypoxic HPAECs. In addition, inhibition of PARP-1 reduced cell migration, VEGF expression, and tubule formation in hypoxic HPAECs. In in vivo studies, PARP-1 inhibition by ISO significantly decreased the RVP and RVH as well as improved endothelial function by increasing the pulmonary vascular reactivity and expression of p-eNOS in MCT-treated rats.

Gp120 Envelope Glycoproteins of HIV-1 Group M Subtype A and Subtype B Differentially Affect Gene Expression in Human Vascular Endothelial Cells.

Cardiovascular complications are seen among human immunodeficiency virus (HIV)-positive individuals, who now survive longer due to successful antiretroviral therapies. Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased blood pressure in the lung circulation. The prevalence of PAH in the HIV-positive population is dramatically higher than that in the general population. While HIV-1 Group M Subtype B is the most prevalent subtype in western countries, the majority of HIV-1 infections in eastern Africa and former Soviet Union countries are caused by Subtype A. Research on vascular complications in the HIV-positive population in the context of subtype differences, however, has not been rigorous. Much of the research on HIV has focused on Subtype B, and information on the mechanisms of Subtype A is nonexistent. The lack of such knowledge results in health disparities in the development of therapeutic strategies to prevent/treat HIV complications. The present study examined the effects of HIV-1 gp120 of Subtypes A and B on human pulmonary artery endothelial cells by performing protein arrays. We found that the gene expression changes caused by gp120s of Subtypes A and B are different. Subtype A is a more potent downregulator of perostasin, matrix metalloproteinase-2, and ErbB than Subtype B, while Subtype B is more effective in downregulating monocyte chemotactic protein-2 (MCP-2), MCP-3, and thymus- and activation-regulated chemokine proteins. This is the first report of gp120 proteins affecting host cells in an HIV subtype-specific manner, opening up the possibility that complications occur differently in HIV patients throughout the world.

Astragaloside IV improves pulmonary arterial hypertension by increasing the expression of CCN1 and activating the ERK1/2 pathway.

The aim of the present study was to investigate the underlying mechanism of AS-IV and CCN1 in PAH and to evaluate whether the protective effect of AS-IV against PAH is associated with CCN1 and its related signalling pathway. In vivo, male SD rats were intraperitoneally injected with monocrotaline (MCT, 60 mg/kg) or exposed to hypoxia (10% oxygen) and gavaged with AS-IV (20, 40 and 80 mg/kg/day) to create a PAH model. In vitro, human pulmonary artery endothelial cells (hPAECs) were exposed to hypoxia (3% oxygen) or monocrotaline pyrrole (MCTP, 60 μg/mL) and treated with AS-IV (10, 20 and 40 μM), EGF (10nM, ERK agonist), small interfering CCN1 (CCN1 siRNA) and recombinant CCN1 protein (rCCN1, 100 ng/mL). We identified the differences in the expression of genes in the lung tissues of PAH rats by proteomics. At the same time, we dynamically detected the expression of CCN1 by Western blot both in vivo and in vitro. The Western blot experimental results showed that the expression of CCN1 increased in the early stage of PAH and decreased in the advanced stage of PAH. The results showed that compared with the control group, MCT- and hypoxia-induced increased the hemodynamic parameters and apoptosis. AS-IV can improve PAH, as characterized by decreased hemodynamic parameters, vascular wall area ratio (WA%), vascular wall thickness ratio (WT%) and α-SMA expression and inhibition of cell apoptosis. Moreover, the improvement of PAH by AS-IV was accompanied by increased CCN1 expression, which activated the ERK1/2 signalling pathway. Meanwhile, CCN1 and p-ERK1/2 were inhibited by siCCN1 and promoted by rCCN1. EGF not only activated the ERK1/2 signalling pathway but also induced the expression of CCN1. In conclusion, AS-IV improves PAH by increasing the expression of CCN1 and activating the ERK1/2 signalling pathway. The results of our study provide a theoretical basis for additional study on the protective effect of AS-IV against PAH.

PTPN1 Deficiency Modulates BMPR2 Signaling and Induces Endothelial Dysfunction in Pulmonary Arterial Hypertension.

Bone morphogenic protein receptor 2 (BMPR2) expression and signaling are impaired in pulmonary arterial hypertension (PAH). How BMPR2 signaling is decreased in PAH is poorly understood. Protein tyrosine phosphatases (PTPs) play important roles in vascular remodeling in PAH. To identify whether PTPs modify BMPR2 signaling, we used a siRNA-mediated high-throughput screening of 22,124 murine genes in mouse myoblastoma reporter cells using ID1 expression as readout for BMPR2 signaling. We further experimentally validated the top hit, PTPN1 (PTP1B), in healthy human pulmonary arterial endothelial cells (PAECs) either silenced by siRNA or exposed to hypoxia and confirmed its relevance to PAH by measuring PTPN1 levels in blood and PAECs collected from PAH patients. We identified PTPN1 as a novel regulator of BMPR2 signaling in PAECs, which is downregulated in the blood of PAH patients, and documented that downregulation of PTPN1 is linked to endothelial dysfunction in PAECs. These findings point to a potential involvement for PTPN1 in PAH and will aid in our understanding of the molecular mechanisms involved in the disease.

Increased Thromboxane A2 Levels in Pulmonary Artery Smooth Muscle Cells Isolated from Patients with Chronic Obstructive Pulmonary Disease.

Introduction: Pulmonary hypertension due to chronic obstructive pulmonary disease (COPD) is classified as Group 3 pulmonary hypertension, with no current proven targeted therapies. It has been shown that cigarette smoke, the main risk factor for COPD, can increase thromboxane A2 production in healthy human pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, and that blocking the effect of increased thromboxane A2 using daltroban, a thromboxane A2 receptor antagonist, can inhibit cigarette smoke-induced pulmonary artery cell proliferation. However, it is largely unknown whether thromboxane A2 is increased in smokers with COPD. Therefore, the aim of this study was to assess the level of thromboxane A2 production in patients with COPD who smoke. Methods: Pulmonary artery smooth muscle cells from three smokers with COPD and three healthy donors were cultured in cell culture medium. The culture medium was collected and the thromboxane B2 (a stable metabolite of thromboxane A2) released in the culture medium was quantified using an ELISA kit. The data were normalised with the total protein concentration and then expressed in pg/mg protein. Demographic data were collected and baseline pulmonary function tests of patients with COPD were conducted. Results: The mean age of patients with COPD was 69 ± 7 years. All patients were smokers and had a mean smoking history of 39.66 ± 9.50 packs per year. The mean forced expiratory volume in one second, that is, FEV1%, and the ratio of forced vital capacity (FVC) to FEV1% of COPD patients were 63.33 ± 19.60% and 52.66 ± 14.64%, respectively. The results revealed that thromboxane A2 production was significantly increased in pulmonary artery smooth muscle cells from smokers with COPD (434.56 ± 82.88 pg/mg protein) compared with the thromboxane A2 levels in pulmonary artery smooth muscle cells from healthy donors (160 ± 59.3 pg/mg protein). Conclusions: This is the first report of increased thromboxane A2 production in pulmonary artery smooth muscle cells from smokers with COPD. This observation strongly suggests that thromboxane A2 can be used as a novel therapeutic target for the treatment of patients with COPD-associated pulmonary hypertension.

Involvement of SUR2/Kir6.1 channel in the physiopathology of pulmonary arterial hypertension.

We hypothesized that the ATP-sensitive K+ channels (KATP) regulatory subunit (ABCC9) contributes to PAH pathogenesis. ABCC9 gene encodes for two regulatory subunits of KATP channels: the SUR2A and SUR2B proteins. In the KATP channel, the SUR2 subunits are associated with the K+ channel Kir6.1. We investigated how the SUR2/Kir6.1 channel contributes to PAH pathogenesis and its potential as a therapeutic target in PAH. Using in vitro, ex vivo, and in vivo approaches, we analyzed the localization and expression of SUR2A, SUR2B, and Kir6.1 in the pulmonary vasculature of controls and patients with PAH as in experimental pulmonary hypertension (PH) rat models and its contribution to PAH physiopathology. Finally, we deciphered the consequences of in vivo activation of SUR2/Kir6.1 in the monocrotaline (MCT)-induced PH model. We found that SUR2A, SUR2B, and Kir6.1 were expressed in the lungs of controls and patients with PAH and MCT-induced PH rat models. Organ bath studies showed that SUR2 activation by pinacidil induced relaxation of pulmonary arterial in rats and humans. In vitro experiments on human pulmonary arterial smooth muscle cells and endothelial cells (hPASMCs and hPAECs) in controls and PAH patients showed decreased cell proliferation and migration after SUR2 activation. We demonstrated that SUR2 activation in rat right ventricular (RV) cardiomyocytes reduced RV action potential duration by patch-clamp. Chronic pinacidil administration in control rats increased heart rate without changes in hemodynamic parameters. Finally, in vivo pharmacological activation of SUR2 on MCT and Chronic-hypoxia (CH)-induced-PH rats showed improved PH. We showed that SUR2A, SUR2B, and Kir6.1 are presented in hPASMCs and hPAECs of controls and PAH patients. In vivo SUR2 activation reduced the MCT-induced and CH-induced PH phenotype, suggesting that SUR2 activation should be considered for treating PAH.

Second International Pulmonary Hypertension/Heart Failure Symposium—Structural heart disease, right ventricular dysfunction, and stem cell therapy: The European Pediatric Pulmonary Vascular Disease Network

Second International Pulmonary Hypertension/Heart Failure Symposium—Structural heart disease, right ventricular dysfunction, and stem cell therapy: The European Pediatric Pulmonary Vascular Disease Network

Decursin alleviates LPS-induced lung epithelial cell injury by inhibiting NF-κB pathway activation.

To reveal the possible effects of decursin on viability, oxidative stress, and inflammatory response in lipopolysaccharide (LPS)-treated human bronchial epithelial cells-2B (BEAS-2B) and human pulmonary artery endothelial cells (HPAEC) cells, and revealed the potential mechanisms. LPS was used to induce acute lung injury (ALI) in normal human lung epithelial cells, including BEAS-2B and HPAEC cells. Cell viability and apoptosis in response to LPS and decursin in BEAS-2B and HPAEC cells were, respectively, evaluated by MTT colorimetric and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The oxidative stress and inflammatory response in LPS-treated BEAS-2B and HPAEC cells were detected by enzyme-linked-immunosorbent serologic assay. In addition, the role of decursin in nuclear -factor-kappa B (NF-κB) activation was analyzed by immunoblot and immunofluorescence assays. Our data revealed that decursin could alleviate the viability of LPS-induced BEAS-2B and HPAEC cells. Decursin could also reduce LPS-induced oxidative stress in BEAS-2B and HPAEC cells. In addition, it could reduce LPS-induced inflammation in BEAS-2B and HPAEC cells. Mechanically, decursin suppressed the activation of NF-κB pathway. Decursin suppressed NF-κB pathway, and therefore alleviated ALI.