The neo-potential therapeutic strategy in preeclampsia: Downregulated miR-26a-2-3p motivates endothelial cell injury by targeting 15-LOX-1.

Preeclampsia (PE) poses a life-threatening risk for both mothers and babies, and its onset and progression are linked to endothelial injury. The enzyme 15-lipoxygenase-1 (15-LOX-1), critical in arachidonic acid metabolism, is implicated in various diseases, yet its specific role and precise mechanisms in PE remain largely unknown. In this study, we found that 15-LOX-1 and its main metabolite, 15-HETE, were significantly increased in both the placenta and serum of PE patients. This increase was accompanied by elevated levels of endothelial injury markers, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). A positive correlation between 15-LOX-1 and those markers in the placenta. In Alox15-/- mice, Alox15 deficiency reduced endothelial cell injury in PE-like mice induced by L-NAME. In vitro studies showed that hypoxia-induced upregulation of 15-LOX-1 reduced the cell viability, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs), while increasing apoptosis and inflammatory cell adhesion. Mechanistically, the p38 MAPK pathway was identified as a downstream target of 15-LOX-1. Knocking down 15-LOX-1 or inhibiting p38 MAPK activation improved endothelial cell injury in hypoxia-treated HUVECs. Furthermore, downregulation of miR-26a-2-3p was found to correlate negatively and colocalize with 15-LOX-1 upregulation in the placenta of PE patients. Luciferase reporter assays further confirmed that miR-26a-2-3p directly bind to the 3'UTR of 15-LOX-1, targeting its expression. Moreover, miR-26a-2-3p agomir ameliorated the PE-like phenotype in mice through the 15-LOX-1/p38 MAPK axis, improving endothelial dysfunction. Therefore, our study provides novel insights into the pathogenesis of PE and highlight modulating the miR-26a-2-3p/15-LOX-1/p38 MAPK axis as a potential therapeutic target for PE.

Risk Factors for Length of Hospital Stay in Acute Exacerbation Chronic Obstructive Pulmonary Disease: A Multicenter Cross-Sectional Study.

Background/Purpose: A patient's length of hospital stay (LHS) is associated with the severity and outcome of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Therefore, identification of patients with prolonged LHS at an early stage can potentially reduce the risk of adverse events and treatment costs in patients with AECOPD. Therefore, this study aimed to explore the independent predictors of prolonged LHS in AECOPD patients. Patients and Methods: This multicenter cross-sectional study was conducted at two tertiary hospitals between January 2019 and August 2020. Demographic data, underlying diseases, symptoms, and laboratory findings were collected. Univariate analysis was used to identify variables with significant differences. A collinearity diagnostic was applied to the selected variables before the establishment of the regression model. Ordinal logistic regression was performed to explore the independent risk factors for prolonged LHS in patients with AECOPD. Results: In total, 598 patients with AECOPD were screened. Finally, the LHS of 111, 218, and 100 patients was <7, 7-10, and ≥11 days, respectively. Significant differences in the 12 variables were found in the univariate analysis. Because collinearities among white blood cells (WBC), neutrophils (NS), and NS% were observed, WBC and NS% were excluded. Subsequently, an ordinal logistic regression model identified that rates of hypertension and chronic cor pulmonale (CCP), neutrophil-lymphocyte ratio (NLR), and erythrocyte sedimentation rate (ESR) were independent predictors of prolonged LHS in AECOPD patients. Conclusion: Collectively, our results showed that inflammatory status, hypertension, and CCP were independently associated with LHS in patients with AECOPD. These data indicate that early and appropriate administration of antibiotics and anti-inflammatory drugs is essential for reducing LHS. Hypertension and CCP were independent predictors of worse outcomes in patients with AECOPD. Therefore, advanced management and care should be provided to AECOPD patients with hypertension and/or CCP on admission.

Circular RNA hsa_circ_101555 promotes hepatocellular carcinoma cell proliferation and migration by sponging miR-145-5p and regulating CDCA3 expression.

Circular RNAs have been reported to play significant roles in regulating pathophysiological processes while also guiding clinical diagnosis and treatment of hepatocellular carcinoma (HCC). However, only a few circRNAs have been identified thus far. Herein, we investigated the role of a specific closed-loop structure of hsa_circ_101555 that was generated by back-splicing of the host gene casein kinase 1 gamma 1 (CSNK1G1) in the development and proliferation of HCC. We investigated the expression of Hsa_circ_101555 in HCC and normal tissues using bioinformatics. The expression level of hsa_circ_101555 was further detected by fluorescence in situ hybridization and qRT-PCR in ten HCC patients. Transwell, migration, WST-1 assays, and colony formation assays were used to evaluate the role of hsa_circ_101555 in HCC development and proliferation. The regulatory mechanisms of hsa_circ_101555 in miR-145-5p and CDCA3 were determined by dual luciferase reporter assay. A mouse xenograft model was also used to determine the effect of hsa_circ_101555 on HCC growth in vivo. hsa_circ_101555 showed greater stability than the linear RNA; while in vitro and in vivo results demonstrated that hsa_circ_101555 silencing significantly suppressed cell proliferation, migration, and invasion of HCC cells. Rescue experiments further demonstrated that suppression of miR-145-5p significantly attenuated the biological effects of hsa_circ_101555 knockdown in HCC cells. We also identified a putative oncogene CDCA3 as a potential miR-145-5p target. Thus, our results demonstrated that hsa_circ_101555 might function as a competing endogenous RNA of miR-145-5p to upregulate CDCA3 expression in HCC. These findings suggest that hsa_circ_101555 may be a potential therapeutic target for patients with HCC.

Clinical Differences between Eosinophilic and Noneosinophilic Acute Exacerbation of Chronic Obstructive Pulmonary Disease: A Multicenter Cross-Sectional Study.

METHODS: A total of 643 AECOPD patients were enrolled in this multicenter cross-sectional study. Finally, 455 were included, 214 in the normal-eosinophil AECOPD (NEOS-AECOPD) group, 63 in the mild increased-eosinophil AECOPD (MEOS-AECOPD) group, and 138 in the severe increased-eosinophil AECOPD (SEOS-AECOPD) group. Demographic data, underlying diseases, symptoms, and laboratory findings were collected. Multiple logistic regression analysis was performed to identify the independent factors associated with blood eosinophils (EOS). Correlations between blood EOS and its associated independent factors were evaluated. RESULTS: The significant differences in 19 factors, including underlying diseases, clinical symptoms, and laboratory parameters, were identified by univariate analysis. Subsequently, multiple logistic regression analysis revealed that lymphocyte%, neutrophil% (NS%), procalcitonin (PCT), and anion gap (AG) were independently associated with blood EOS in AECOPD. Both blood EOS counts and EOS% were significantly correlated with lymphocyte%, NS%, PCT, and AG. CONCLUSIONS: Collectively, blood EOS was independently associated with lymphocyte%, NS%, PCT, and AG in AECOPD patients. Lymphocyte% was lower, and NS%, PCT, and AG were higher in eosinophilic AECOPD. Our results indicate that viral-dominant infections are the probable major etiologies of eosinophilic AECOPD. Noneosinophilic AECOPD is more likely associated with bacterial-dominant infections. The systemic inflammation in noneosinophilic AECOPD was more severe.

Monocyte chemotactic protein-inducing protein 1 negatively regulating asthmatic airway inflammation and mucus hypersecretion involving γ-aminobutyric acid type A receptor signaling pathway in vivo and in vitro.

BACKGROUND: Mounting evidence, consistent with our previous study, showed that γ-aminobutyric acid type A receptor (GABAAR) played an indispensable role in airway inflammation and mucus hypersecretion in asthma. Monocyte chemotactic protein-inducing protein 1 (MCPIP1) was a key negative regulator of inflammation. Recent studies showed that inflammation was largely suppressed by enhanced MCPIP1 expression in many inflammatory diseases. However, the role and potential mechanism of MCPIP1 in airway inflammation and mucus hypersecretion in asthma were still not well studied. This study was to explore the role of MCPIP1 in asthmatic airway inflammation and mucus hypersecretion in both mice and BEAS-2B cells, and its potential mechanism. METHODS: In vivo, mice were sensitized and challenged by ovalbumin (OVA) to induce asthma. Airway inflammation and mucus secretion were analyzed. In vitro, BEAS-2B cells were chosen. Interleukin (IL)-13 was used to stimulate inflammation and mucus hypersecretion in cells. MCPIP1 Lentiviral vector (LA-MCPIP1) and plasmid-MCPIP1 were used to up-regulate MCPIP1 in lung and cells, respectively. MCP-1, thymic stromal lymphopoietin (TSLP), mucin 5AC (MUC5AC), MCPIP1, and GABAARß2 expressions were measured in both lung and BEAS-2B cells. Immunofluorescence staining was performed to observe the expression of GABAARß2 in cells. RESULTS: MCPIP1 was up-regulated by LA-MCPIP1 (P < 0.001) and plasmid-MCPIP1 (P < 0.001) in lung and cells, respectively. OVA-induced airway inflammation and mucus hypersecretion, OVA-enhanced MCP-1, TSLP, MUC5AC, and GABAARß2 expressions, and OVA-reduced MCPIP1 were significantly blunted by LA-MCPIP1 in mice (all P < 0.001). IL-13-enhanced MCP-1, TSLP, MUC5AC, and GABAARß2 expressions, and IL-13-reduced MCPIP1 were markedly abrogated by plasmid-MCPIP1 in BEAS-2B cells (all P < 0.001). CONCLUSION: The results of this study suggested that OVA and IL-13-induced airway inflammation and mucus hypersecretion were negatively regulated by MCPIP1 in both lung and BEAS-2B cells, involving GABAAR signaling pathway.

NFATc3 pathway participates in the process that 15-LO/15-HETE protects pulmonary artery smooth muscle cells against apoptosis during hypoxia.

Hypoxia activates nuclear factor of activated T cells isoforms c3 (NFATc3), a Ca(2+)-dependent transcription factor in murine pulmonary arteries (PAs), and NFATc3 has been proved to be implicated in hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation, but it remains unclear whether NFATc3 acts on the apoptosis of PASMCs, an important step in PAs remodeling. Our laboratory has demonstrated that 15-hydroxyeicosatetraenoic acid (15-HETE) is a key factor in hypoxia-induced PA remodeling and can increase PASMC intracellular Ca(2+) ([Ca(2+)](i)) in rats. It is possible that NFATc3 is related with the function of 15-HETE anti-apoptosis during hypoxia. Our results identified that NFATc3 was mainly localized in rat PASMCs and was upregulated in PAs during hypoxia-induced rat pulmonary hypertension (PH), while this effect was inhibited by administration of nordihydroguaiaretic acid (NDGA), a 15-lipoxygenase (15-LO) inhibitor. Moreover, hypoxia and exogenous 15-HETE promoted the expression and nuclear translocation of NFATc3 in PASMCs, which was inhibited by NDGA or small interfering RNA targeted to rat 15-LO1 or 15-LO2. Furthermore, endogenous 15-HETE induced by hypoxia and exogenous 15-HETE suppressed serum deprivation-induced loss of rat PASMCs survival and prevented annexin V binding, mitochondrial membrane potential depolarization, DNA nick end labeling and chromatin condensation. Although all these effects were suppressed after the cells were treated with cyclosporin A (a calcineurin/NFAT inhibitor), it aggravated the apoptosis induced by serum deprivation. Thus, all these results indicate that 15-HETE-mediated PASMCs anti-apoptosis in hypoxic PH via the Ca(2+)-NFATc3 pathway.

Enhancement of the HIF-1α/15-LO/15-HETE axis promotes hypoxia-induced endothelial proliferation in preeclamptic pregnancy.

Preeclampsia (PE) is an extremely serious condition in pregnant women and the leading cause of maternal and fetal morbidity and mortality. Despite active research, the etiological factors of this disorder remain elusive. The increased release of 15-hydroxyeicosatetraenoic acid (15-HETE) in the placenta of preeclamptic patients has been studied, but its exact role in PE pathogenesis remains unknown. Mounting evidence shows that PE is associated with placental hypoxia, impaired placental angiogenesis, and endothelial dysfunction. In this study, we confirmed the upregulated expression of hypoxia-inducible factor 1α (HIF-1α) and 15-lipoxygenase-1/2 (15-LO-1/2) in patients with PE. Production of the arachidonic acid metabolite, 15-HETE, also increased in the preeclamptic placenta, which suggests enhanced activation of the HIF-1α-15-LO-15-HETE axis. Furthermore, this study is the first to show that the umbilical cord of preeclamptic women contains significantly higher serum concentrations of 15-HETE than that of healthy pregnant women. The results also show that expression of 15-LO-1/2 is upregulated in both human umbilical vein endothelial cells (HUVECs) collected from preeclamptic women and in those cultured under hypoxic conditions. Exogenous 15-HETE promotes the migration of HUVECs and in vitro tube formation and promotes cell cycle progression from the G0/G1 phase to the G2/M + S phase, whereas the 15-LO inhibitor, NDGA, suppresses these effects. The HIF-1α/15-LO/15-HETE pathway is therefore significantly associated within the pathology of PE.

MicroRNA-190 regulates hypoxic pulmonary vasoconstriction by targeting a voltage-gated K⁺ channel in arterial smooth muscle cells.

Pulmonary arterial hypertension (PAH) is associated with sustained vasoconstriction, profound structural remodeling of vasculatures and alterations in Ca(2+) homeostasis in arterial smooth muscle cells (SMCs), while the underlying mechanisms are still elusive. By regulating the expression of proteins, microRNAs (miRNAs) are known to play an important role in cell fates including differentiation, apoptosis and proliferation, and may be involved in the development of PAH. Based on our previous study, hypoxia produced a significant increase of the miR-190 level in the pulmonary artery (PA), here, we used synthetic miR-190 to mimic the increase in hypoxic conditions and showed evidence for the effects of miR-190 on pulmonary arterial vasoconstriction and Ca(2+) influx in arterial SMCs. Synthetic miR-190 remarkably enhanced the vasoconstriction responses to phenylephrine (PE) and KCl. The voltage-gated K(+) channel subfamily member, Kcnq5, mRNA was shown to be a target for miR-190. Meanwhile, miR-190 antisense oligos can partially reverse the effects of miR-190 on PASMCs and PAs. Therefore, these results suggest that miR-190 appears to be a positive regulator of Ca(2+) influx, and plays an important role in hypoxic pulmonary vascular constriction.

MicroRNA-138 plays a role in hypoxic pulmonary vascular remodelling by targeting Mst1.

Unbalanced apoptosis is a major cause of structural remodelling of vasculatures associated with PAH (pulmonary arterial hypertension), whereas the underlying mechanisms are still elusive. miRNAs (microRNAs) regulate the expression of several proteins that are important for cell fate, including differentiation, proliferation and apoptosis. It is possible that these regulatory RNA molecules play a role in the development of PAH. To test this hypothesis, we studied the effect of several miRNAs on the apoptosis of cultured PASMCs (pulmonary artery smooth muscle cells) and identified miR-138 to be an important player. miR-138 was expressed in PASMCs, and its expression was subjected to regulation by hypoxia. Expression of exogenous miR-138 suppressed PASMC apoptosis, prevented caspase activation and disrupted Bcl-2 signalling. The serine/threonine kinase Mst1, an amplifier of cell apoptosis, seemed to be a target of miR-138, and the activation of the Akt pathway was necessary for the anti-apoptotic effect of miR-138. Therefore the results of the present study suggest that miR-138 appears to be a negative regulator of PASMC apoptosis, and plays an important role in HPVR (hypoxic pulmonary vascular remodelling).

Effect of Mitofusin 2 on smooth muscle cells proliferation in hypoxic pulmonary hypertension.

Mitofusin 2 (Mfn2) is an important mitochondrial protein in maintaining mitochondrial network and bioenergetics. Recently, Mfn2 has been reported to have a potential role in regulating cell proliferation, apoptosis, and differentiation in many cell types. In this study, we performed immunohistochemistry, pulmonary artery smooth muscle cells (PASMCs) DNA analysis, proliferating cell nuclear antigen expression and cell cycle analysis to determine the role of Mfn2 in hypoxia-induced pulmonary vascular remodeling. Our results showed that hypoxia promoted the proliferation of pulmonary artery smooth muscle cells, including regulating more cells at G(2)/M+S phase, increasing proliferating cell nuclear antigen and Cyclin A expression, whereas all these effects of hypoxia were suppressed after the cells were treated with siRNA against Mfn2. Our results also proved that PI3K/Akt signaling pathway was involved in Mfn2-induced smooth muscle cell proliferation. Thus, these results indicate that Mfn2 mediates PASMC proliferation in hypoxic pulmonary hypertension via the PI3K/Akt signaling pathway.

Platelet-derived growth factor (PDGF) induces pulmonary vascular remodeling through 15-LO/15-HETE pathway under hypoxic condition.

15-lipoxygenase (15-LO) is known to play an important role in chronic pulmonary hypertension. Accumulating evidence for its down-stream participants in the vasoconstriction and remodeling processes of pulmonary arteries, while how hypoxia regulates 15-LO/15-hydroxyeicosatetraenoic acid (15-HETE) to mediate hypoxic pulmonary hypertension is still unknown. Platelet-derived growth factor (PDGF) is an important vascular regulator whose concentration increases under hypoxic condition in the lungs of both humans and mice with pulmonary hypertension. The present study was carried out to determine whether hypoxia advances the pulmonary vascular remodeling through the PDGF/15-LO/15-HETE pathway. We found that pulmonary arterial medial thickening caused by hypoxia was alleviated after a treatment of the hypoxic rats with imatinib, which was associated with down-regulations of 15-LO-2 expression and 15-HETE production. Moreover, the increases in cell proliferation and endogenous 15-HETE content by hypoxia were attenuated by the inhibitors of PDGF-ß receptor in pulmonary artery smooth muscle cells (PASMCs). The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of 15-LO inhibitors or 15-LO RNA interference. These results suggest that hypoxia promotes PASMCs proliferation and survival, contributing to pulmonary vascular medial hypertrophy, which is likely to be mediated via the PDGF-BB/15-LO-2/15-HETE pathway.

Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells.

Pulmonary artery endothelial plexiform lesion is responsible for pulmonary vascular remodeling (PVR), a basic pathological change of pulmonary arterial hypertension (PAH). Recent evidence suggests that epoxyeicosatrienoic acid (EET), which is derived from arachidonic acid by cytochrome p450 (CYP) epoxygenase, has an essential role in PAH. However, until now, most research has focused on pulmonary vasoconstriction; it is unclear whether EET produces mitogenic and angiogenic effects in pulmonary artery endothelial cells (PAEC). Here we found that 500 nM/l 8,9-EET, 11,12-EET, and 14,15-EET markedly augmented JNK and c-Jun activation in PAECs and that the activation of c-Jun was mediated by JNK, but not the ERK or p38 MPAK pathway. Moreover, treatment with 8,9-EET, 11,12-EET, and 14,15-EET promoted cell proliferation and cell-cycle transition from the G0/G1 phase to S phase and stimulated tube formation in vitro. All these effects were reversed after blocking JNK with Sp600125 (a JNK inhibitor) or JNK1/2 siRNA. In addition, the apoptotic process was alleviated by three EET region isomers through the JNK/c-Jun pathway. These observations suggest that 8,9-EET, 11,12-EET, and 14,15-EET stimulate PAEC proliferation and angiogenesis, as well as protect the cells from apoptosis, via the JNK/c-Jun pathway, an important underlying mechanism that may promote PAEC growth and angiogenesis during PAH.

Role of 15-lipoxygenase/15-hydroxyeicosatetraenoic acid in hypoxia-induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease with a complex aetiology characterized by elevated pulmonary artery resistance, which leads to right heart ventricular afterload and ultimately progressing to right ventricular failure and often death. In addition to other factors, metabolites of arachidonic acid cascade play an important role in the pulmonary vasculature, and disruption of signaling pathways of arachidonic acid plays a central role in the pathogenesis of PAH. 15-Lipoxygenase (15-LO) is upregulated in pulmonary artery endothelial cells and smooth muscle cells of PAH patients, and its metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) in particular seems to play a central role in the contractile machinery, and in the initiation and propagation of cell proliferation via its effects on signal pathways, mitogens, and cell cycle components. Here, we focus on our important research into the role played by 15-LO/15-HETE, which promotes a proliferative, antiapoptotic, and vasoconstrictive physiological milieu leading to hypoxic pulmonary hypertension.

15-HETE mediates sub-acute hypoxia-induced TRPC1 expression and enhanced capacitative calcium entry in rat distal pulmonary arterial myocytes.

Sub-acute hypoxia causes pulmonary vasoconstriction (HPV) is associated with increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of pulmonary arterial smooth muscle cells (PASMCs). We previous have demonstrated that 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of arachidonic acid by 15-lipoxygenase (15-LO), causes elevated [Ca(2+)](i) in PASMCs partly through Ca(2+) entry via other than L-type Ca(2+) channels. In this study, we used SKF96365/La(3+) (SOCC antagonists) and Nordihydro-guiairetic acid (NDGA, a blockage of 15-LO) to examine the effect of 15-HETE on capacitative Ca(2+) entry and activity/expression of store-operated Ca(2+) channels (SOCCs) during sub-acute hypoxic procedure and the contribution of SOCCs on the maintenance of vascular tones. The results showed that the 15-HETE induced constriction of PA rings from normoxic and sub-acute hypoxic rats can be abolished by SKF96365 and La(3+). Capacitative Ca(2+) entry (CCE) was also enhanced in PASMCs cultured with 15-HETE under sub-acute hypoxic condition (3% O(2), 48h) and incubation with NDGA in PASMCs can greatly suppress this enhancement. Moreover, TRPC1, not TRPC4 and TRPC6, mRNA and protein expression were increased in PASMCs during these procedures. Meanwhile, the effect of 15-HETE on CCE and TRPC1 expression under sub-acute hypoxic cultivation were greatly suppressed in 15-LO knockdown PASMCs and PAs. These results suggest that 15-HETE mediated HPV through increased TRPC1 expression, leading to enhanced CCE, contributing to the maintenance of vascular tone.

15-Hydroxyeicosatetraenoic acid (15-HETE) protects pulmonary artery smooth muscle cells against apoptosis via HSP90.

AIMS: 15-Hydroxyeicosatetraenoic acid (15-HETE), generated by hypoxia, is a product of arachidonic acid and mainly catalyzed by 15-lipoxygenase (15-LO) in pulmonary artery. As HSP90 is known to be involved in apoptosis in other tissues and cells, we aim to test whether anti-apoptotic effect of 15-HETE is regulated by the molecular chaperone in pulmonary artery smooth muscle cells. MAIN METHODS: To test this hypothesis, we performed cell viability analysis, mitochondrial potential assay, caspase-3 activity measurement, Western blot, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling with and without HSP90 inhibitor. KEY FINDINGS: Our results showed that both exogenous and endogenous 15-HETE up-regulated HSP90 expression and prevented PASMC from serum deprivation-induced apoptosis. Serum deprivation lead to mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, and activation of caspase-3 and caspase-9 in PASMCs. 15-HETE reversed all these effects in a HSP90-dependent manner. SIGNIFICANCE: This study establishes the factor involved in 15-HETE-protecting PASMC from apoptosis and the regulation of HSP90 by 15-HETE may be an important mechanism underlying the treatment of pulmonary artery hypertension and provide a novel therapeutic target in future.