Apoptosis Of Pulmonary Microvascular Endothelial Cells Research Articles

LRG1 promotes the apoptosis of pulmonary microvascular endothelial cells through KLK10 in chronic obstructive pulmonary disease.

Cigarette smoking is one of the most important causes of COPD and could induce the apoptosis of pulmonary microvascular endothelial cells (PMVECs). The conditional knockout of LRG1 from endothelial cells reduced emphysema in mice. However, the mechanism of the deletion of LRG1 from endothelial cells rescued by cigarette smoke (CS) induced emphysema remains unclear. This research aimed to demonstrate whether LRG1 promotes the apoptosis of PMVECs through KLK10 in COPD. Nineteen patients were divided into three groups: control non-COPD (n=7), smoker non-COPD (n=7), and COPD (n=5). The emphysema mouse model defined as the CS exposure group was induced by CS exposure plus cigarette smoke extract (CSE) intraperitoneal injection for 28 days. Primary PMVECs were isolated from the mouse by magnetic bead sorting method via CD31-Dynabeads. Apoptosis was detected by western blot and flow cytometry. LRG1 was increased in lung tissue of COPD patients and CS exposure mice, and CSE-induced PMVECs apoptosis model. KLK10 was over-expressed in lung tissue of COPD patients and CS exposure mice, and CSE-induced PMVECs apoptosis model. LRG1 promoted apoptosis in PMVECs. LRG1 knockdown reversed CSE-induced apoptosis in PMVECs. The mRNA and protein expression of KLK10 were increased after over-expressed LRG1 in PMVECs isolated from mice. Similarly, both the mRNA and protein levels of KLK10 were decreased after LRG1 knockdown in PMVECs. The result of co-immunoprecipitation revealed a protein-protein interaction between LRG1 and KLK10 in PMVECs. KLK10 promoted apoptosis via the down-regulation of Bcl-2/Bax in PMVECs. KLK10 knockdown could reverse CSE-induced apoptosis in PMVECs. LRG1 promotes apoptosis via up-regulation of KLK10 in PMVECs isolated from mice. KLK10 promotes apoptosis via the down-regulation of Bcl-2/Bax in PMVECs. There was a direct protein-protein interaction between LRG1 and KLK10 in PMVECs. Our novel findings provide insights into the understanding of LRG1/KLK10 function as a potential molecule in COPD.

MiR-216a reduces apoptosis of pulmonary microvascular endothelial cells in COPD by targeting DNMT1.

Abnormal apoptosis of pulmonary microvascular endothelial cells (PMVECs) participates in the pathogenesis of COPD. Studies have shown that microRNAs (miRNAs) contribute to the pathogenesis of pulmonary diseases by regulating cell apoptosis. The present study aimed to investigate the effects of miR-216a in cigarette smoke extract (CSE)-induced apoptosis of PMVECs in COPD and explore the potential mechanisms. The emphysema model mice were treated with CSE and CS exposure. The expression of miR-216a and DNA methyltransferase 1 (DNMT1) was assessed in emphysema mice and COPD patients. The miR-216a mimic and Lenti-DNMT1 were transfected into PMVECs to identify the underlying mechanisms. The expression levels of miR-216a and DNMT1 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) or Western blot. Moreover, cell apoptosis was examined by flow cytometry assays. The results show that the expression of miR-216a was decreased, whereas the expression of DNMT1 was increased in the lung tissue of emphysema mice and COPD patients. In addition, the expression of miR-216a was significantly reduced in CSE-treated PMVECs, and the overexpression of miR-216a attenuated CSE-induced PMVEC apoptosis. Furthermore, the expression of DNMT1 was increased in the CSE-induced PMVECs and then was reduced after the overexpression of miR-216a in the CSE-stimulated PMVECs. Luciferase reporter assays confirmed the target reaction between miR-216a and DNMT1. Also, the overexpression of DNMT1 was able to reverse the anti-apoptotic effect of miR-216a in CSE-induced PMVECs. The results indicate that miR-216a may play a crucial role in CSE-induced apoptosis by directly regulating its target gene DNMT1 in COPD. It provides insights into the function of MiR-216a/DNMT1 as a potential molecule in COPD.

Sema3A inactivates the ERK/JNK signalling pathways to alleviate inflammation and oxidative stress in lipopolysaccharide-stimulated rat endothelial cells and lung tissues

Semaphorin 3A (Sema3A) is a secretory member of the semaphorin family of immune response regulators. This research focuses on its effects on inflammation and oxidative stress in acute respiratory distress syndrome (ARDS). By analysing the GEO dataset GSE57011, we obtained Sema3A as the most downregulated gene in ARDS samples. Lipopolysaccharide (LPS) was used to stimulate rat pulmonary microvascular endothelial cells (PMVECs) and rats to induce ARDS-like symptoms in vitro and in vivo, respectively. LPS induced severe damage in rat lung tissues, in which reduced immunohistochemical staining of Sema3A was detected. Sema3A overexpression reduced apoptosis and angiogenesis of LPS-induced PMVECs and alleviated lung injury and pulmonary edoema of rats. Moreover, ELISA results showed that Sema3A overexpression downregulated the levels of inflammatory cytokines and oxidative stress markers both in PMVECs and the rat lung. Activation of ERK/JNK signalling aggravated LPS-induced damage on PMVECs; however, the aggravation was partly blocked by Sema3A, which suppressed phosphorylation of ERK/JNK. Overall, this study demonstrates that Sema3A inactivates the ERK/JNK signalling to ameliorate inflammation and oxidative stress in LPS-induced ARDS models. Sema3A might therefore represent a candidate option for ARDS treatment.

Sulforaphane alleviated vascular remodeling in hypoxic pulmonary hypertension via inhibiting inflammation and oxidative stress

Hypoxic pulmonary hypertension (HPH) is a cardiopulmonary disease featured by pulmonary vascular remodeling, which is due to abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) and dysfunction of endothelial cells (ECs). Sulforaphane (SFN) is a natural isothiocyanate extracted from cruciferous vegetables with promising anti-inflammatory and anti-oxidative activities. This study aimed to explore the effect and mechanism of SFN on HPH. Male mice were exposed to persistent chronic hypoxia for 4 weeks to induce HPH. The results demonstrated that SFN repressed the increased right ventricular systolic pressure (RVSP) and attenuated the right ventricular hypertrophy and pulmonary arteries remodeling in HPH mice. In particular, after SFN treatment, the CD68 positive cells in lung sections were reduced; TNF-α and IL-6 levels in lungs and serum declined; activation of NF-κB in PASMCs was inhibited in response to hypoxia. Besides, SFN enhanced the superoxide dismutase (SOD) activity in serum, SOD2 expression, total glutathione levels, and GSH/GSSG ratio in PASMCs, along with a decrease in malondialdehyde (MDA) contents in serum and ROS production in PASMCs after hypoxia exposure. Notably, SFN, as an Nrf2 activator, reversed the reduction in Nrf2 expression in hypoxic PASMCs. In vitro, SFN treatment inhibited hyperproliferation and promoted apoptosis of PASMCs under hypoxia conditions. SFN also prevented the apoptosis of pulmonary microvascular ECs caused by hypoxia. Therefore, these data suggested that SFN could significantly restrain the inflammation and oxidative stress, thereby inhibiting PASMCs proliferation, promoting PASMCs apoptosis, and reversing hypoxia injury in ECs to improve pulmonary vascular remodeling.

LncRNA H19 alleviates sepsis-induced acute lung injury by regulating the miR-107/TGFBR3 axis

ObjectiveAcute lung injury (ALI) increases sepsis morbidity and mortality. LncRNA H19 plays a critical role in sepsis. miR-107 is highly-expressed and TGFβ type III receptor (TGFBR3) is poorly-expressed in sepsis, yet their roles in sepsis development require further investigation. This study aimed to investigate the mechanism of H19 in alleviating sepsis-induced ALI through the miR-107/TGFBR3 axis.MethodsMice were intravenously injected with Ad-H19 adenovirus vector or control vector one week before establishing the mouse model of cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with oe-H19 or oe-NC plasmids and then stimulated by lipopolysaccharide (LPS). Lung injury was assessed via hematoxylin–eosin staining, measurement of wet-to-dry (W/D) ratio, and TUNEL staining. Levels of H19, miR-107, and TGFBR3 were determined by RT-qPCR. Apoptosis of PMVECs was evaluated by flow cytometry. Levels of Bax and Bcl-2 in lung tissues and PMVECs were measured using Western blot. Total protein concentration and the number of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) were quantified. Levels of TNF-α, IL-1β, IL-6, and IL-10 in BALF, lung tissues, and PMVECs were measured by ELISA. Cross-linking relationships among H19, miR-107 and TGFBR3 were verified by dual-luciferase and RIP assays.ResultsH19 was poorly-expressed in CLP-operated mice. H19 overexpression attenuated sepsis-induced ALI, which was manifested with complete alveolar structure, decreased lung injury score and lung W/D ratio, and inhibited apoptosis in CLP-operated mice, which was manifested with decreased number of TUNEL-positive cells and Bax level and increased Bcl-2 level. CLP-operated mice had increased concentration of total protein and number of total cells, neutrophils, and macrophages in BALF, which was nullified by H19 overexpression. H19 overexpression declined levels of TNF-α, IL-1β, and IL-6 and elevated IL-10 levels. H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production. H19 targeted TGFBR3 as the ceRNA of miR-107. miR-107 overexpression or silencing TGFBR3 partially averted the inhibition of H19 overexpression on LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production.ConclusionLncRNA H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production and attenuated sepsis-induced ALI by targeting TGFBR3 as the ceRNA of miR-107.

MiR-141 exacerbates lung ischemia-reperfusion injury by targeting EGFR/β-catenin axis-mediated autophagy.

Some microRNAs (miRNAs) play important roles in lung ischemia-reperfusion injury (LIRI) injury. Here, this study aimed to examine whether miR-141 was related to lung ischemia-reperfusion injury (IRI) via regulating autophagy and the epidermal growth factor receptor (EGFR), and to explore the underlying signal transduction pathways. To this end, we constructed the LIRI cell model and mouse models, separately. According to RT-qPCR and Western blotting (WB) analysis results, miR-141 up-regulation together with β-catenin and EGFR down-regulation within mouse pulmonary microvascular endothelial cells (PMVECs) or lung tissues was related to lung IRI. Besides, we conducted dual-luciferase reporter assay, which suggested the binding of EGFR to miR-141. In addition, we carried out TUNEL staining, HE staining, and flow cytometric analysis to assess the apoptosis of PMVECs and the injury to mouse lung tissues. Furthermore, we performed light-chain immunofluorescence assay to examine autophagosomes within PMVECs. According to our results, miR-141 suppressed β-catenin level through reducing EGFR level. Besides, the miR-141/EGFR/β-catenin axis enhanced autophagy to aggravate LIRI. To sum up, miR-141 suppresses EGFR expression to inhibit β-catenin level, which subsequently aggravates autophagy and complicates LIRI. The above results offer the candidate therapeutic target for the treatment of lung IRI.

Protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells.

To investigate the protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells (PMVECs). Malondialdehyde and superoxide dismutase (SOD) were used to determine the optimal modeling of lung hypoxic injury cells. CCK-8 method was used to detect cell viability for determining the effective dose of areca nut polyphenols. Rat PMVECs were divided into control group, hypoxia model group and areca nut polyphenols group. BCA method was used to detect the protein concentration of each group, and the oxidative stress level in PMVECs was measured. Western blotting was used to detect the expression of inflammatory and apoptosis-related proteins. Immunofluorescence staining was used to detect the expression of occludin and zonula occludens (ZO) 1. Transwell chamber was used to detect transendothelial electrical resistance, and rhodamine fluorescent dye was used to detect PMVECs barrier permeability. The hypobaric hypoxia-induced cell injury model was established by culturing PMVECs for 48 h at 1% oxygen concentration. The 20 μg/mL areca nut polyphenols significantly reversed the survival rate and the oxidative stress of PMVECs in hypoxia model group (all P<0.05). Areca nut polyphenols had significant inhibitory effect on the up-regulation of inflammation-related proteins, including nuclear factor-κB (NF-κB) and nuclear factor-E2-related factor (Nrf) 2 in hypoxia model group (all P<0.05). And areca nut polyphenols could reduce hypoxia-induced PMVECs apoptosis by down-regulating the expressions of apoptosis-related proteins, including cysteine aspartic acid specific protease (caspase) 3, Bcl-2 associated X protein (Bax) in PMVECs (all P<0.05). In addition, areca nut polyphenols effectively improves the transendothelial electrical resistance and barrier permeability of PMVECs through elevating the expression of occludin and ZO-1 (all P<0.05). Areca nut polyphenols can inhibit the hypoxic damage of PMVECs by reducing oxidative stress and apoptosis down-regulating the expression of inflammatory proteins and reducing membrane permeability.

In vitro and vivo study of tranilast protects from acute respiratory distress syndrome and early pulmonary fibrosis induced by smoke inhalation

BackgroundTranilast (N-[3＇,4＇-dimethoxycinnamoyl]-anthranilic acid) is an analog of a tryptophan metabolite. It was identified with anti-inflammatory and antifibrotic activities, and used in the treatment of a variety of diseases, such as anti - allergy, bronchial asthma, and hypertrophic scars. As a drug with few adverse reactions, tranilast has attracted great attention, but its application is limited due to the uncertainty of dosages and mechanisms. In this study, the protection effects of different doses of tranilast on smoke inhalation mediated lung injury on rats, and on the damage of three kinds of lung cells in vitro were investigated. MethodIn vivo, Sprague-Dawley rats were randomly divided into sham group, smoke group (rats were exposed to pine sawdust smoke three times, each time for 5 min), different doses of tranilast treatment group (doses were 100 mg/kg, 200 mg/kg and 300 mg/kg, ip.) and placebo group. After 1, 3 and 7 days, pulmonary function, pathologic injury by HE staining, cytokines and oxidative stress level by kits were determined. At 7days, lung fibrosis was assessed by Masson’s trichrome staining and the level of hydroxyproline (HYP). In vitro, three kinds of lung cells from normal rats were isolated: type II alveolar epithelial cells (AT-II), pulmonary microvascular endothelial cells (PMVECs) and pulmonary fibroblasts (PFs). To investigate the potential effects of tranilast on cell proliferation, cell cycle and cytokine production of three kinds of lung cells exposed to smoke. ResultsCompared with smoke group and placebo group, tranilast treatment significantly reduced histopathological changes (such as pulmonary hemorrhage, edema and inflammatory cell infiltration, etc.), significantly reduced histopathological score (p < 0.05), increased arterial oxygen partial pressure, and decreased the levels of IL-1β, TNF-α, TGF-β1 (p < 0.05), oxidative stress and the expression of nuclear transcription factor κB (NF-κB) smoke exposed rats (p < 0.01). In particular, the effect of 200 mg/kg dose was more prominent. In vitro, smoke induced AT-II and PMVECs apoptosis, improved PFs proliferation (p < 0.01), activity of SOD and decreased the content of MDA (p < 0.01). However, tranilast seems to be turning this trend well. The inflammatory factor IL-11β, TNF-α and TGF-β1, and the expression of NF-κB were significantly lower in the tranilast treatment than in the smoke group (p < 0.01). ConclusionThis study indicates that tranilast had a protective effect on acute respiratory distress syndrome and early pulmonary fibrosis of rats in vivo. In addition, tranilast promotes proliferation of AT-II and PMVECs but inhibits PFs proliferation, down-regulates secretion of inflammatory cytokines and alleviates oxidative stress of AT-II, PMVECs and PFs after smoke stimuli in vitro.

LPS induces pulmonary microvascular endothelial cell barrier dysfunction by upregulating ceramide production

The specific role of ceramides in pulmonary microvascular endothelial cell (PMVEC) barrier dysfunction remains unclear. In the present study, pretreatment with pan-caspase inhibitors significantly reduced LPS-induced PMVEC apoptosis and helped to stimulate PMVEC barrier reconstruction after 12 h but had no effect on PMVEC barrier dysfunction in the first 8 h. Further studies showed that imipramine, an acid sphingomyelinase (ASMase) inhibitor, significantly inhibited LPS-induced barrier dysfunction, while an siRNA targeting serine palmityl transferase subunit 1 (SPTLC1) and the pharmacological inhibitor myriocin did not inhibit early acute barrier dysfunction but significantly inhibited PMVEC apoptosis and apoptosis-dependent delayed barrier dysfunction. In addition, LPS was shown to activate RhoA by inducing transient receptor potential channel 6 (TRPC6) overexpression and calcium influx through the ASMase/ceramide pathway, and activation of RhoA further induced the cytoskeletal rearrangement of PMVECs and destruction of intercellular junctions, ultimately leading to early acute PMVEC barrier dysfunction. However, regarding apoptosis-dependent delayed barrier dysfunction, the ceramide-induced de novo synthesis pathway in paracellular cells induced the apoptosis of PMVECs, in which Txnip overexpression inhibited Trx activity and subsequently activated ASK1 in the context of LPS-induced PMVEC apoptosis, acting upstream of the ceramide-induced activation of p38 MAPK and JNK. At the same time, in rats with LPS- or exogenous C8 ceramide-induced ALI, ceramide was demonstrated to play an important role in lung injury by inducing the Txnip/TRX/ASK1/P38 and JNK pathways. Thus, the Txnip/TRX/ASK1/p38 and JNK pathways might be involved in ceramide-mediated PMVEC apoptosis in LPS-induced ALI.

Ferulic acid regulates miR-17/PTEN axis to inhibit LPS-induced pulmonary microvascular endothelial cells apoptosis through activation of PI3K/Akt pathway.

Acute lung injury (ALI) is mainly mediated by the damage of pulmonary microvascular endothelial cells (PMVECs). LPS is one of the pathogenic factors leading to microcirculatory abnormalities of ALI. Ferulic acid (FA) exhibits therapeutic effects against various diseases. During lipopolysaccharide-induced acute respiratory distress syndrome, FA, when given beforehand, could depress inflammation and oxidative stress. However, the concrete role and underlying mechanism of FA in ALI have not been well characterized. Ten μg/mL Lipopolysaccharide (LPS) was used to treat rat PMVECs for 24 hr. qRT-PCR was used to detect the level of miR-17 and phosphatase and tensin homolog deleted on chromosome ten (PTEN). Western blot was used to analyze the associated proteins in the PI3K/Akt pathway, and the apoptosis-related proteins. Flow cytometric analysis was performed to detect the apoptosis of PMVECs. MTT assay was constructed to detect the cell viability. Luciferase assay was conducted to detect the target gene of miR-17 and PTEN. A cell model for in vitro studying the role of FA in ALI was established using PMVECs. Our data demonstrate that FA up-regulates miR-17 and declines apoptosis induced by LPS. FA inhibits apoptosis mediated by up-regulating miR-17. Furthermore, we found miR-17 targeted PTEN negatively. FA inhibits cleaved caspase-3 and Bax expression through the PI3K/Akt pathway mediated by up-regulating miR-17. Over-expression of PTEN could contribute to the similar expression trend of the PI3K/Akt signal pathway protein compared to miR-17 inhibitor transfected cells. FA inhibits PMVECs apoptosis induced by LPS via miR-17/PTEN to further regulate the activation of the PI3K/Akt pathway in ALI. We anticipate that our data will provoke additional studies for ALI clinical therapy.

Effects of L-carnitine on autophagy and apoptosis of mouse pulmonary microvascular endothelial cells induced by lipopolysaccharide

Objective: To investigate the protective effects of L-carnitine (LC) on lipopolysaccharide (LPS) - injured mouse pulmonary microvascular endothelial cells (PMVECs) and its effects on autophagy and apoptosis. Methods: Cultured mouse PMVECs were divided into three groups: ① Control group, ② LPS group (10 μg/ml, 3, 6, 12, 24 h), ③ LPS (10 μg/ml, 24 h)+LC (2.5, 5.0, 10 μg/ml) (LPS+LC) group. PMVECs apoptosis was examined by Annexin V-FITC/PI double labeling method. Autophagosome was detected by immunofluorescence staining. Levels of autophagy-related protein LC3 and apoptosis-related protein caspase-3 were detected by Western blot. PMVECs viability was measured by CCK-8. Results: ① Compared with the control group, LPS treatment inhibited the PMVECs viability significantly, whereas the apoptosis rate and the expression of autophagy protein LC3 II were markedly increased after LPS treatment for 6 h, 12 h and 24 h. ② Compared with LPS group (10 μg/ml, 24 h), the PMVECs viability, levels of autophagy protein LC3 II and caspase-3 protein expression as well as apoptosis rate in LPS+LC group were increased significantly. Conclusion: LC can increase the activity of PMVECs injuried by LPS, promote autophagy and inhibit apoptosis of PMVECs.

Roles of VE-Cadherin in Hypoxia Induced Injury of Pulmonary Microvascular Endothelial Barrier.

Hypoxia induced injury of pulmonary microvascular endothelial barrier is closely related to the pathogenesis of acute lung injury after lung transplantation. VE-cadherin is an important structural molecule for pulmonary microvascular endothelial barrier. In this study, we aim to investigate the roles of VE-cadherin in hypoxia induced injury of pulmonary microvascular endothelial barrier. Rat model of hypoxia and cultured pulmonary microvascular endothelial cells (PMVECs) were utilized. Determination of PMVECs apoptosis, skeleton combination was conducted to verify the effects of hypoxia on injury of pulmonary microvascular endothelial barrier. In addition, VE-cadherin expression was modulated by administration of siRNA in order to investigate the roles of VE-cadherin in hypoxia induced PMVECs apoptosis and skeleton recombination. Our data indicated that expression of VE-cadherin was down-regulated in hypoxia-exposed PMVECs. Whereas, in the cells treated using siRNA, down-regulation of VE-cadherin did not trigger PMVECs apoptosis, but it increased the sensitivity of PMVECs to the hypoxia induced apoptosis. In cases of hypoxia, the expression of VE-cadherin was significantly down-regulated, together with endothelial skeleton recombination and increase of permeability, which then triggered endothelial barrier dysfunction. These data verify that VE-cadherin expression played an important role in hypoxia induced PMVECs apoptosis and cellular skeletal recombination.

Polymer Optical Waveguide Grating-Based Biosensor to Detect Effective Drug Concentrations of Ginkgolide A for Inhibition of PMVEC Apoptosis.

In this work, we successfully developed a fluorinated cross-linked polymer Bragg waveguide grating-based optical biosensor to detect effective drug concentrations of ginkgolide A for the inhibition of pulmonary microvascular endothelial cell (PMVEC) apoptosis. Fluorinated photosensitive polymer SU-8 (FSU-8) as the sensing core layer and polymethyl methacrylate (PMMA) as the sensing window cladding were synthesized. The effective drug concentration range (5–10 µg/mL) of ginkgolide A for inhibition of PMVEC apoptosis was analyzed and obtained by pharmacological studies. The structure of the device was optimized to be designed and fabricated by direct UV writing technology. The properties of the biosensor were simulated with various refractive indices of different drug concentrations. The actual sensitivity of the biosensor was measured as 1606.2 nm/RIU. The resolution and detection limit were characterized as 0.05 nm and 3 × 10−5 RIU, respectively. The technique is suitable for safe and accurate detection of effective organic drug dosages of Chinese herbal ingredients.

Heat shock protein 70 attenuates hypoxia‑induced apoptosis of pulmonary microvascular endothelialcells isolated from neonatal rats.

Pulmonary microvascular endothelial cell (PMVEC) apoptosis is the initial stage of adult pulmonary hypertension (PH), which involves high pulmonary arterial pressure and pulmonary vascular remodeling. However, the mechanism regulating PMVEC apoptosis and its involvement in the early stages of neonatal hypoxic PH (HPH) pathogenesis are currently unclear. The present study aimed to investigate the effects of heat shock protein 70 (HSP70) on hypoxia-induced apoptosis in PMVECs. PMVECs isolated from neonatal Sprague-Dawley rats were transfected with lentivirus with or without HSP70, or treated with the synthetic HSP70 inhibitor N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam under hypoxic conditions (5% O2) for 24, 48 or 72 h. PMVEC apoptosis was evaluated by performing flow cytometry and mitochondrial membrane potential (MMP) assays. The expression levels of HSP70, hypoxia-inducible factor-1α (HIF-1α) and apoptosis-associated proteins were determined by conducting reverse transcription-quantitative PCR and western blotting. Following 24, 48 or 72 h of hypoxia, the apoptotic rates of PMVECs were significantly elevated compared with cells under normoxic conditions. The MMP was significantly reduced, whereas the mRNA and protein expression levels of HIF-1α, cytochrome c (cyt C), caspase-3 and HSP70 were enhanced by hypoxia compared with those under normoxic conditions. Additionally, the mRNA and protein expression levels of B-cell lymphoma 2 (Bcl-2) were significantly downregulated in the hypoxia group compared with those in the normoxia group. In hypoxic PMVECs, HSP70 overexpression decreased the apoptotic rate and the expression levels of cyt C, downregulated the expression levels of caspase-3 and HIF-1α, and increased the MMP and the expression levels of Bcl-2. HSP70 inhibition resulted in the opposite outcomes compared with those of HSP70 overexpression. Therefore, the results of the present study suggested that HSP70 may inhibit mitochondrial pathway-mediated apoptosis in isolated neonatal rat PMVECs in early-stage hypoxia, which may be associated with HSP70-mediated HIF-1α downregulation. Overall, HSP70 may be protective against neonatal HPH through the HSP70/HIF-1α pathway.

Retraction.

Retraction: "MicroRNA-339-3p alleviates inflammation and edema and suppresses pulmonary microvascular endothelial cell apoptosis in mice with severe acute pancreatitis-associated acute lung injury by regulating Anxa3 via the Akt/mTOR signaling pathway", by Xing-Mao Wu, Kai-Qiang Ji, Hai-Yuan Wang, Yang Zhao, Jia Jia, Xiao-Peng Gao, Bin Zang, J Cell Biochem. 2018; 6704-6714: The above article, published online on 25 April 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.26859) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific context. Thus, the editors consider the conclusions of this article to be invalid.

LncRNA TUG1 regulates human pulmonary microvascular endothelial cell apoptosis via sponging of the miR-9a-5p/BCL2L11 axis in chronic obstructive pulmonary disease.

The aim of the present study was to investigate the function of long non-coding RNA taurine-upregulated gene 1 (lncRNA TUG1) in chronic obstructive pulmonary disease and further assess the underlying molecular mechanisms. Flow cytometry analysis was performed to detect cell apoptosis of human pulmonary microvascular endothelial cells (HPMECs) treated with 1% cigarette smoke extract (CSE). The activity of caspase-3 was measured using a Caspase-3 Activity assay kit and the protein expression of cleaved caspase-3, caspase-3 and Bcl-2 like 11 (BCL2L11) were measured using western blotting. Reverse transcription-quantitative PCR (RT-qPCR) was performed to measure the expression of TUG1 mRNA levels in the treated cells. The association between TUG1, the miR-9a-5p/BCL2L11 axis and with miR-9a-5p were predicted and verified using a dual luciferase reporter assay system. The mRNA expression of miR-9a-5p and BCL2L11, and the transfection efficiency were measured by RT-qPCR. The results showed that CSE induced cell apoptosis and increased lncRNA TUG1 expression in HPMECs. CSE significantly reduced the expression of miR-9a-5p in HPMECs compared with the control group. TUG1-short hairpin RNA relieved cell apoptosis induced by CSE by upregulating miR-9a-5p in HPMECs. The present study predicted and verified that BCL2L11 is a direct target of miR-9a-5p. The mRNA expression of BCL2L11 was increased in HPMECs following CSE treatment compared with the control group. miR-9a-5p mimic and BCL2L11-plasmid markedly increased the expression of miR-9a-5p and BCL2L11, respectively. miR-9a-5p mimic reversed the increase in cell apoptosis induced by CSE by inhibiting BCL2L11 expression in HPMECs. To conclude, the present study demonstrated that lncRNA TUG1 exerted roles in cell apoptosis induced by CSE through modulating the miR-9a-5p/BCL2L11 axis.

Methylation of miR-19b-3p promoter exacerbates inflammatory responses in sepsis-induced ALI via targeting KLF7.

Sepsis-induced acute lung injury is associated with dysregulated inflammatory reactions. MiR-19b-3p level was reported to be downregulated in patients with sepsis. To evaluate the role of miR-19b-3p in sepsis, cecum ligation and puncture-induced mouse sepsis model and lpopolysaccharide (LPS)-treated pulmonary microvascular endothelial cells (PMVECs) were used. For in vivo study, lung tissue was harvested for hematoxylin and eosin (H&E) staining, tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β, and p-p65, p-IκB measuring. Cell apoptosis was assessed by TUNEL assay. For in vitro study, cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Methylation of miR-19b-3p promoter was measured by methylation-specific PCR (MSP) assay. The target of miR-19b-3p was determined by dual-luciferase reporter gene assay. The level of miR-19b-3p was determined to be downregulated in vitro and in vivo. In addition, miR-19b-3p protected mice from inflammation injury through inhibiting NF-κB signaling pathway. Overexpression of miR-19b-3p increased cell viability, decreased apoptosis, and proinflammatory cytokines secretion in LPS-treated PMVECs. Besides these, Krüppel-like factor 7 (KLF7) was confirmed as the target of miR-19b-3p. And methylation of miR-19b-3p was the reason of decreased miR-19b-3p level. In conclusion, miR-19b-3p protected cells from sepsis-induced inflammation injury via inhibiting NF-κB signaling pathway, and KLF7 was a potential target.

Perfluorocarbon Protects against Lipopolysaccharide-Induced Apoptosis of Endothelial Cells in Pulmonary Microvessels.

This study was aimed to explore the effect and mechanisms of action of perfluorocarbon on LPS-induced apoptosis of pulmonary microvascular endothelial cells (PMVEC) isolated from Sprague-Dawley rats. Apoptosis rates were assessed by flow cytometry. Ultrastructural characteristics of PMVEC were evaluated by transmission electron microscopy. The protein expression of cleaved caspase-3 was measured using Western blotting. LPS significantly increased the level of apoptosis, induced the appearance of ultrastructural changes typical of apoptosis, up-regulated the expression of active caspase-3 protein. These effects of LPS were attenuated by co-administration of perfluorocarbon. These results suggest that perfluorocarbon can attenuate LPS-induced apoptosis of PMVEC by inhibiting TLR-4 signaling and caspase-3 activation.

Study on the Role of VE-cadherin/c-Abl Complex in Ang II-Associated Lung Injury

Our previous study found that Ang II-associated pulmonary microvascular endothelial cell apoptosis and skeletal remodeling are the main mechanisms leading to the impaired pulmonary microvascular endothelial dysfunction and leading to a series of disease-associated lung injury. Therefore, the purpose of this study is to explore the role of VE-cadherin/c-Abl complex in Ang II-associated lung injury. We constructed Ang II-associated lung injury models in vivo and in vitro. We found that in the Ang II-associated lung injury model, pulmonary microvascular endothelial cell apoptosis increased, cytoskeleton rearrangement, pVE-cadherin dephosphorylation, VE-cadherin/c-Abl complex depolymerization, and c-Abl expression increased then transfer to nucleus. After overexpression of c-Abl, although it cannot directly lead to pulmonary microvascular endothelial cells(PMVEC) apoptosis and cytoskeleton rearrangement, it improves the sensitivity of PMVEC to Ang II-associated apoptosis and backbone rearrangement. After inhibiting the expression of c-Abl, PMVEC apoptosis was reduced and cytoskeleton rearrangement was inhibited, partially reversing the Ang II-associated lung injury. The above results indicate that the VE-cadherin/c-Abl complex plays a protective role in Ang II-associated lung injury. Funding: This work is funded by the National Natural Science Foundation of Hubei Province (Grant No.2019CFB469) Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The Ethical Committee of Renmin Hospital of Wuhan University approved the animal study protocol.

MiR-642a-5p partially mediates the effects of lipopolysaccharide on human pulmonary microvascular endothelial cells via eEF2.

Inhalation or systemic administration of lipopolysaccharide (LPS) can induce acute pulmonary inflammation and lung injury. The pulmonary vasculature is composed of pulmonary microvascular endothelial cells (PMVECs), which form a semiselective membrane for gas exchange. The miRNA miR-642a-5p has previously been reported to be up-regulated in patients with acute respiratory distress syndrome; thus, here, we examined whether this miRNA is involved in the effects of LPS on PMVECs. The levels of miR-642a-5p and mRNA encoding eukaryotic elongation factor 2 (eEF2) were detected by quantitative RT-PCR. Moesin and eEF2 protein levels were tested by western blot assay. Dual-luciferase reporter assay was used to examine the relationship between miR-642a-5p and eEF2. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell permeability was analyzed using the transendothelial electrical resistance assay. We report that miR-642a-5p levels are significantly up-regulated in LPS-stimulated PMVECs, and miR-642a-5p contributes to LPS-induced hyperpermeability and apoptosis of PMVECs. LPS treatment results in down-regulation of eEF2 in PMVECs. Overexpression of eEF2, a direct target of miR-642a-5p, inhibited the effect of LPS on PMVECs. miR-642a-5p promoted LPS-induced hyperpermeability and apoptosis by targeting eEF2. Thus, miR-642a-5p and eEF2 may serve as potential targets for acute lung injury/acute respiratory distress syndrome diagnosis or treatment.