Neutrophil extracellular traps induce pyroptosis of pulmonary microvascular endothelial cells by activating the NLRP3 inflammasome.

Excessive formation of neutrophil extracellular traps (NETs) may lead to myositis-related interstitial lung disease (ILD). There is evidence that NETs can directly injure vascular endothelial cells and play a pathogenic role in the inflammatory exudation of ILD. However, the specific mechanism is unclear. This study aimed to investigate the specific mechanism underlying NET-induced injury to human pulmonary microvascular endothelial cells (HPMECs). HPMECs were stimulated with NETs (200 ng/ml) in vitro. Cell death was detected by propidium iodide staining. The morphological changes of the cells were observed by transmission electron microscopy (TEM). Pyroptosis markers were detected by western blot, immunofluorescence, and quantitative real-time polymerase chain reaction, and the related inflammatory factor Interleukin-1ß (IL-1ß) was verified by enzyme-linked immunosorbent assay (ELISA). Compared with the control group, HPMECs mortality increased after NET stimulation, and the number of pyroptosis vacuoles in HPMECs was further observed by TEM. The pulmonary microvascular endothelial cells (PMECs) of the experimental autoimmune myositis mouse model also showed a trend of pyroptosis in vivo. Cell experiment further confirmed the significantly high expression of the NLRP3 inflammasome and pyroptosis-related markers, including GSDMD and inflammatory factor IL-1ß. Pretreated with the NLRP3 inhibitor MCC950, the activation of NLRP3 inflammasome and pyroptosis of HPMECs were effectively inhibited. Our study confirmed that NETs promote pulmonary microvascular endothelial pyroptosis by activating the NLRP3 inflammasome, suggesting that NETs-induced pyroptosis of PMECs may be a potential pathogenic mechanism of inflammatory exudation in ILD.

MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury.

The role of autophagy in pulmonary microvascular endothelial cells (PMVECs) is controversial in LPS-induced acute lung injury (ALI). Mixed lineage kinase domain-like pseudokinase (MLKL) has recently been reported to maintain cell survival by facilitating autophagic flux in response to starvation rather than its well-recognized role in necroptosis. Using a mouse PMVEC and LPS-induced ALI model, we showed that in PMVECs, MLKL was phosphorylated (p-MLKL) and autophagic flux was accelerated at the early stage of LPS stimulation (1-3 h), manifested by increases in concentrations of lipidated MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß; LC3-II), decreases in concentrations of SQSTM1/p62 (sequestosome 1), and fusion of the autophagosome and lysosome by pHluorin-mKate2-human LC3 assay, which were all reversed by either MLKL inhibitor or siRNA MLKL. In mice, the inhibition of MLKL increased vascular permeability and aggravated mouse ALI upon 3-hour LPS stimulation. The p-MLKL induced by short-term LPS formed multimers to facilitate the closure of the phagophore by HaloTag-LC3 autophagosome completion assay. The charged multivesicular body protein 2A (CHMP2A) is essential in the process of phagophore closure into the nascent autophagosome. In agreement with the p-MLKL change, CHMP2A concentrations markedly increased during 1-3-hour LPS stimulation. CHMP2A knockdown blocked autophagic flux upon LPS stimulation, whereas CHMP2A overexpression boosted autophagic flux and attenuated mouse ALI even in the presence of MLKL inhibitor. We propose that the activated MLKL induced by short-term LPS facilitates autophagic flux by accelerating the closure of the phagophore via CHMP2A, thus protecting PMVECs and alleviating LPS-induced ALI.

Cigarette Smoke Extract and Lipopolysaccharide Induce Pyroptosis in Pulmonary Microvascular Endothelial Cells of Rats.

We studied the effect of cigarette smoke extract (CSE), LPS, or their combination on the activity and pyroptosis of pulmonary microvascular endothelial cells (PMVEC) in rats. PMVEC were cultured without treatment, with CSE in different concentrations (1-25%), with 20 ng/ml LPS, or with 20% CSE+20 ng/ml LPS. Cell viability was determined using the CCK8 kit, apoptosis was evaluated by flow cytometry, and cell morphology was evaluated using light microscopy. The content of IL-1ß and IL-18 was measured by ELISA. CSE decreased cell viability in a dose-dependent manner. The morphology of cells in the CSE+LPS group showed the most significant cytomorphological changes and the highest pyroptosis rate. Flow cytometry showed that the apoptosis rates in the CSE and LPS groups were higher than in the control group, but the highest rate of apoptosis was revealed in the CSE+LPS group (p<0.01). The levels of IL-18 and IL-1ß in the cell supernatant of the CSE, LPS, and CSE+LPS groups were significantly (p<0.01) increased in comparison with the control. These levels in the CSE+LPS group were higher (p<0.01) than in other groups. There were no differences between the CSE and LPS groups. Thus, the effect of CSE on cell viability is dose-dependent. Combined treatment with CSE+LPS can induce cell pyroptosis and increase the levels of inflammatory cytokines in PMVEC. These observations demonstrated that pyroptosis caused by CSE and LPS can play an important role in pulmonary vascular remodeling.

Fibroblast growth factor 21 improves lipopolysaccharide-induced pulmonary microvascular endothelial cell dysfunction and inflammatory response through SIRT1-mediated NF-κB deacetylation.

Pneumonia is a common infectious disease of the respiratory system in children. It often leads to death in children by causing acute lung injury. Fibroblast growth factor 21 (FGF21) is a peptide hormone that plays an important role in the regulation of energy homeostasis. This study aimed to investigate the role of FGF21 in alleviating the lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cell (HPMEC) injury, as well as the underlying mechanism. The expression of sirtuin 1 (SIRT1), NF-κB p65, Ac-NF-κB p65, apoptosis-related proteins, tight junction proteins and adhesion molecules in HPMECs were analyzed by Western blotting. The viability and apoptosis of HPMECs were detected by CCK-8 and TUNEL assays. Lactate dehydrogenase level and levels of inflammatory factors were respectively determined by assay kits. The mRNA expression of adhesion molecules in HPMECs was analyzed by RT-qPCR. As a result, SIRT1 expression was decreased and the expression of NF-κB p65 and Ac-NF-κB p65 were increased in LPS-induced HPMECs, which were reversed by recombinant FGF21 (rFGF21). rFGF21 increased the viability and inhibited the apoptosis, inflammatory response, permeability, and release of cell adhesion molecules of LPS-induced HPMECs. In addition, EX527 as SIRT1 inhibitor could reverse the effect of rFGF21 on LPS-induced HPMECs. In conclusion, FGF21 improved LPS-induced HPMEC dysfunction and inflammatory response through SIRT1-mediated NF-κB deacetylation.

Endothelial Microparticles Derived from Primary Pulmonary Microvascular Endothelial Cells Mediate Lung Inflammation in Chronic Obstructive Pulmonary Disease by Transferring microRNA-126.

BACKGROUND: Extracellular vesicles (EVs) are considered to new types of intercellular communication media, and microRNA is one of the most common transferring components of EVs. This study aimed to explore the potential role of endothelial microparticles (EMPs) derived from primary pulmonary microvascular endothelial cells in regulating lung inflammation of chronic obstructive pulmonary disease (COPD) through transferring microRNA-126 (miR-126). METHODS: EMPs generated from primary pulmonary microvascular endothelial cells were isolated by gradient centrifugation and characterized by transmission electron microscopy, flow cytometry and Western blotting. EMPs were treated to in vitro and in vivo COPD models induced by cigarette smoke extract (CSE). miR-126 mimics or inhibitors were transfected into EMPs by calcium chloride. Pathological changes of lung tissue, mRNA and protein levels of inflammation-related factors were measured to explore the effect of EMPs transferring miR-126 on CSE-induced inflammation. RESULTS: Both in vitro and in vivo studies demonstrated that mRNA and protein levels of inflammation-related factors were significantly increased in COPD group, while EMPs could dramatically reverse these increases. In vitro, overexpression of miR-126 in EMPs decreased HMGB1 expression and magnified the decreasing effect of EMPs on inflammation-related factors. CONCLUSION: The present study reveals that EMPs are capable of alleviating lung inflammation and transferring miR-126 can magnify the anti-inflammatory effect of EMPs, which may provide a novel therapeutic alternative for COPD.

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.

Protective effect of adrenomedullin on hyperoxia-induced lung injury. / è‚¾ä¸Šè ºé«“è´¨ç´ å¯¹é«˜æ°§è‚ºæŸä¼¤çš„ä¿æŠ¤ä½œç”¨ç ”ç©¶.

OBJECTIVES: To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions. METHODS: HPMECs were randomly divided into an air group and a hyperoxia group (n=3 each).The HPMECs in the hyperoxia group were cultured in an atmosphere of 92% O2 (3 L/minute) +5% CO2. RT-qPCR and Western blot were used to measure the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB. Other HPMECs were divided into a non-interference group and an interference group (n=3 each), and the mRNA and protein expression levels of ADM, ERK1/2, and PKB were measured after the HPMECs in the interference group were transfected with ADM siRNA. RESULTS: Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (P<0.05). Compared with the non-interference group, the interference group had significant reductions in the mRNA and protein expression levels of ADM, ERK1/2, and PKB (P<0.05). CONCLUSIONS: ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.

Anti-high mobility group box 1 monoclonal antibody suppressed hyper-permeability and cytokine production in human pulmonary endothelial cells infected with influenza A virus.

OBJECTIVE: High mobility group box-1 (HMGB1) has been reported to be involved in influenza A virus-induced acute respiratory distress syndrome (ARDS). We studied the efficacy of an anti-HMGB1 mAb using an in vitro model of TNF-α stimulation or influenza A virus infection in human pulmonary microvascular endothelial cells (HMVECs). METHODS: Vascular permeability of HMVECs was quantified using the Boyden chamber assay under tumor necrosis factor-α (TNF-α) stimulation or influenza A virus infection in the presence of anti-HMGB1 mAb or control mAb. The intracellular localization of HMGB1 was assessed by immunostaining. Extracellular cytokine concentrations and intracellular viral mRNA expression were quantified by the enzyme-linked immunosorbent assay and quantitative reverse transcription PCR, respectively. RESULTS: Vascular permeability was increased by TNF-α stimulation or influenza A infection; HMVECs became elongated and the intercellular gaps were extended. Anti-HMGB1 mAb suppressed both the increase in permeability and the cell morphology changes. Translocation of HMGB1 to the cytoplasm was observed in the non-infected cells. Although anti-HMGB1 mAb did not suppress viral replication, it did suppress cytokine production in HMVECs. CONCLUSION: Anti-HMGB1 mAb might be an effective therapy for severe influenza ARDS.

ZMPSTE24 Regulates SARS-CoV-2 Spike Protein-enhanced Expression of Endothelial PAI-1.

Endothelial dysfunction is implicated in the thrombotic events reported in patients with coronavirus disease (COVID-19), but the underlying molecular mechanisms are unknown. Circulating levels of the coagulation cascade activator PAI-1 are substantially higher in patients with COVID-19 with severe respiratory dysfunction than in patients with bacterial sepsis and acute respiratory distress syndrome. Indeed, the elevation of PAI-1 is recognized as an early marker of endothelial dysfunction. Here, we report that the rSARS-CoV-2-S1 (recombinant severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] viral envelope spike) glycoprotein stimulated robust production of PAI-1 by human pulmonary microvascular endothelial cells (HPMECs). We examined the role of protein degradation in this SARS-CoV-2-S1 induction of PAI-1 and found that the proteasomal degradation inhibitor bortezomib inhibited SARS-CoV-2-S1-mediated changes in PAI-1. Our data further show that bortezomib upregulated KLF2, a shear-stress-regulated transcription factor that suppresses PAI-1 expression. Aging and metabolic disorders are known to increase mortality and morbidity in patients with COVID-19. We therefore examined the role of ZMPSTE24 (zinc metallopeptidase STE24), a metalloprotease with a demonstrated role in host defense against RNA viruses that is decreased in older individuals and in metabolic syndrome, in the induction of PAI-1 in HPMECs by SARS-CoV-2-S1. Indeed, overexpression of ZMPSTE24 blunted enhancement of PAI-1 production in spike protein-exposed HPMECs. In addition, we found that membrane expression of the SARS-CoV-2 entry receptor ACE2 was reduced by ZMPSTE24-mediated cleavage and shedding of the ACE2 ectodomain, leading to accumulation of ACE2 decoy fragments that may bind SARS-CoV-2. These data indicate that decreases in ZMPSTE24 with age and comorbidities may increase vulnerability to vascular endothelial injury by SARS-CoV-2 viruses and that enhanced production of endothelial PAI-1 might play role in prothrombotic events in patients with COVID-19.

Identification and Functional Analysis of Long Non-coding RNAs in Human Pulmonary Microvascular Endothelial Cells Subjected to Cyclic Stretch.

Background: Despite decades of intense research, the pathophysiology and pathogenesis of acute respiratory distress syndrome (ARDS) are not adequately elucidated, which hamper the improvement of effective and convincing therapies for ARDS patients. Mechanical ventilation remains to be one of the primary supportive approaches for managing ARDS cases. Nevertheless, mechanical ventilation leads to the induction of further aggravating lung injury which is known as leading to ventilator-induced lung injury (VILI). It has been reported that lncRNAs play important roles in various cellular process through transcriptional, posttranscriptional, translational, and epigenetic regulations. However, to our knowledge, there is no investigation of the expression profile and functions of transcriptome-level endothelium-related lncRNAs in VILI yet. Methods: To screen the differential expression of lncRNAs and mRNAs in Human pulmonary microvascular endothelial cells (HPMECs) subjected to cyclic stretch, we constructed a cellular model of VILI, followed by transcriptome profiling using Affymetrix Human Transcriptome Array 2.0. Bioinformatics analyses, including functional and pathway enrichment analysis, protein-protein interaction network, lncRNA-mRNA coexpression network, and cis-analyses, were performed to reveal the potential functions and underlying mechanisms of differentially expressed lncRNAs. Results: In total, 199 differentially expressed lncRNAs (DELs) and 97 differential expressed mRNAs were screened in HPMECs subjected to 20% cyclic stretch for 2 h. The lncRNA-mRNA coexpression network suggested that DELs mainly enriched in response to hypoxia, response to oxidative stress, inflammatory response, cellular response to hypoxia, and NF-kappa B signaling pathway. LncRNA n335470, n406639, n333984, and n337322 might regulate inflammation and fibrosis induced by cyclic stretch through cis- or trans-acting mechanisms. Conclusion: This study provides the first transcriptomic landscape of differentially expressed lncRNAs in HPMECs subjected to cyclic stretch, which provides novel insights into the molecular mechanisms and potential directions for future basic and clinical research of VILI.

Mitomycin C induces pulmonary vascular endothelial-to-mesenchymal transition and pulmonary veno-occlusive disease via Smad3-dependent pathway in rats.

BACKGROUND AND PURPOSE: Pulmonary veno-occlusive disease (PVOD) is a rare disease characterized by the obstruction of small pulmonary veins leading to pulmonary hypertension. However, the mechanisms underlying pulmonary vessel occlusion remain largely unclear. EXPERIMENTAL APPROACH: A mitomycin C (MMC)-induced PVOD rat model was used as in vivo animal model, and primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as in vitro cell model. KEY RESULTS: Our data suggested an endothelial-to-mesenchymal transition (EndoMT) may be present in the pulmonary microvessels isolated from either PVOD patients or MMC-induced PVOD rats. In comparison to the control vessels, vessels from both PVOD patients and PVOD rats had co-localized staining of specific endothelial marker von Willebrand factor (vWF) and mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the presence of cells that co-express endothelial and mesenchymal markers. In both the lung tissues of MMC-induced PVOD rats and MMC-treated rat PMVECs there were decreased levels of endothelial markers (e.g. VE-cadherin and CD31) and increased mesenchymal markers (e.g. vimentin, fibronectin and α-SMA) were detected indicating EndoMT. Moreover, MMC-induced activation of the TGFß/Smad3/Snail axis, while blocking this pathway with either selective Smad3 inhibitor (SIS3) or small interfering RNA (siRNA) against Smad3, dramatically abolished the MMC-induced EndoMT. Notably, treatment with SIS3 remarkably prevented the pathogenesis of MMC-induced PVOD in rats. CONCLUSIONS AND IMPLICATIONS: Our data indicated that targeted inhibition of Smad3 leads to a potential, novel strategy for PVOD therapy, likely by inhibiting the EndoMT in pulmonary microvasculature.

Protective effect of adrenomedullin on hyperoxia-induced lung injury / 中国当代儿科杂志

OBJECTIVES@#To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions.@*METHODS@#HPMECs were randomly divided into an air group and a hyperoxia group (@*RESULTS@#Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (@*CONCLUSIONS@#ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.

Vascular endothelial growth factor contributes to lung vascular hyperpermeability in sepsis-associated acute lung injury.

Vascular endothelial growth factor (VEGF) is a prime regulator of vascular permeability. Acute lung injury (ALI) is characterized by high-permeability pulmonary edema in addition to refractory hypoxemia and diffuse pulmonary infiltrates. In this study, we examined whether VEGF can be implicated as a pulmonary vascular permeability factor in sepsis-associated ALI. We found that a great increase in lung vascular leak occurred in mice instilled intranasally with lipopolysaccharide (LPS), as assessed by IgM levels in bronchoalveolar lavage fluid. Treatment with the VEGF-neutralizing monoclonal antibody bevacizumab significantly reduced this hyperpermeability response, suggesting active participation of VEGF in non-cardiogenic lung edema associated with LPS-induced ALI. However, this was not solely attributable to excessive levels of intrapulmonary VEGF. Expression levels of VEGF were significantly reduced in lung tissues from mice with both intranasal LPS administration and cecal ligation and puncture (CLP)-induced sepsis, which may stem from decreases in non-endothelial cells-dependent VEGF production in the lungs. In support of this assumption, stimulation with LPS and interferon-γ (IFN-γ) significantly increased VEGF in human pulmonary microvascular endothelial cells (HPMECs) at mRNA and protein levels. Furthermore, a significant rise in plasma VEGF levels was observed in CLP-induced septic mice. The increase in VEGF released from HPMECs after LPS/IFN-γ challenge was completely blocked by either specific inhibitor of mitogen-activated protein kinase (MAPK) subgroups. Taken together, our results indicate that VEGF can contribute to the development of non-cardiogenic lung edema in sepsis-associated ALI due to increased VEGF secretion from pulmonary vascular endothelial cells through multiple MAPK-dependent pathways.

Analysis of Long Noncoding RNA Expression Profile in Human Pulmonary Microvascular Endothelial Cells Exposed to Lipopolysaccharide.

BACKGROUND/AIMS: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are a continuum of life-threatening lung changes. Pulmonary vascular injury is one of the most important initial causes of ALI and ARDS. However, the functions of long noncoding RNAs (lncRNAs) in pulmonary endothelial injury remain largely unknown. The aim of the present study was to determine the lncRNA expression profile of human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS) and explore the potential functions of differentially expressed lncRNAs. METHODS: Microarray analysis was used to identify differentially expressed lncRNAs and mRNAs. Bioinformatics analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, lncRNA-mRNA coexpression network and transcription factor (TF)-lncRNA network analyses, were performed to predict the functions of significantly differentially expressed lncRNAs and mRNAs. Realtime polymerase chain reaction (PCR) was used to determine the expression of selected lncRNAs and mRNAs. RESULTS: In this study, we found that 213 lncRNAs and 212 mRNAs were significantly differentially expressed in HPMECs exposed to LPS (fold change > 2.0, p < 0.05). Furthermore, we found that mRNAs co-expressed with lncRNAs were significantly enriched in the TNF signaling pathway, the NF-κB signaling pathway, cell adhesion molecules (CAMs), cytokine-cytokine receptor interactions, and extracellular matrix (ECM)-receptor interactions. The expression levels of all but one of the selected lncRNAs and mRNAs detected by real-time PCR were similar to those detected by microarray analysis. CONCLUSION: Our data indicate that lncRNAs play an important role in LPS-induced pulmonary endothelial inflammation and barrier dysfunction and may be potential preventive and therapeutic targets for ALI and ARDS.

MMI-0100 ameliorates lung inflammation in a mouse model of acute respiratory distress syndrome by reducing endothelial expression of ICAM-1.

PURPOSE: ICAM-1 plays a critical role in the development of acute respiratory distress syndrome (ARDS). MK2 regulates the expression of ICAM-1 in human pulmonary microvascular endothelial cells. To explore whether the inhibition of MK2 activation has the same effect in experimental animals, MMI-0100, a peptide-mediated inhibitor of MK2, was used to verify whether MMI-0100 can ameliorate lung inflammation in a mouse model of ARDS by reducing endothelial expression of ICAM-1. METHODS: In this study, C57BL/6 mice were randomly divided into three groups: a control group, an lipopolysaccharides (LPS) group, and an LPS plus MMI-0100 group. Mice were killed 24 hours after the administration of LPS and MMI-0100. The mouse lung tissue histopathology, wet/dry weight ratio (W/D), and the neutrophil count were used to measure the severity of lung inflammation in mice. The pulmonary microvascular endothelial cells (PMVECs) of the mice were isolated. The mRNA expression of ICAM-1 in mouse PMVECs was determined using RT-PCR, and the protein expression of MK2 and ICAM-1 in mouse PMVECs was analyzed using Western blotting and immunohistochemistry. RESULTS: We found that the level of phosphorylated MK2 in the LPS plus MMI-0100 group was reduced. Compared with the LPS group, the LPS plus MMI-0100 group of mice showed less severe inflammation, including a lower W/D and neutrophil count. The mRNA and protein expression of ICAM-1 in the LPS group was significantly higher than in the control group in mouse PMVECs, and the ICAM-1 level was reduced after the administration of MMI-0100. CONCLUSION: These data indicate that MMI-0100 ameliorates lung inflammation in a mouse model of ARDS by reducing endothelial expression of ICAM-1.

RhoA/mDia1 pathway involved in the expression of p-ERM in the pulmonary micro-vascular endothelial cell induced by lipopolysaccharide / 中华急诊医学杂志

Objective To investigate the possibility of the involvement of RhoA/mDia1 pathway to cause the expression of phosphorylate ezrin-radixin-moesin (p-ERM) in rat pulmonary micro-vascular endothelial cell (PMVEC) after the stimulation of lipopolysaccharide (LPS).Methods The specimens of lung tissue were taken from healthy male SPF grade SD rat with 100-120 g body weight which was purchased from the laboratory animal center of Anhui province.After culture,the PMVECs were randomly divided into dose-dependent groups (0,0.1,1,10 μg/mL LPS added in PMVECs and cultured for30 min,n =8 in each),time-dependent groups (10 μg/mL LPS added to PMVECs cultured for 0,15,30,60,120 min,n =8 in each) and intervention group (n =8).In the intervention group,PMVECs were cultured with 1 μg/mL C3 transferase in serum free media for 240 min,followed by treatment with 10 μg/mL LPS for 30 min.Meanwhile,two control groups in serum-free DMEM medium were made by adding 10.μg/mL LPS to PMVECs and 1 μg/mL C3 transferase to PMVECs respectively cultured for 30 min (n =8 in each).Western blot was used to detect the level of p-ERM,ERM and mDia1.Data were analyzed with SPSS 16.0 software,while one way analysis of variance (ANOVA) was used to compare multiple sets of variables,the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests,with P ＜0.05 for the statistically significant difference.Results ERM,p-ERM and mDia1 were presented in rat PMVEC.Stimulation with LPS up-regulated p-ERM,mDia1 in a dose-dependent manner:LPS [0 μg/mL LPS group:(0.520±0.101),0.1 μg/mL LPS group:(0.657 ±0.092),1 μg/mL LPS group:(0.891 ±0.167),10 μg/mL LPS group:(1.227 ±0.106);0 μg/mL group vs.0.1 μg/mL group,P ＞0.05;the rest P ＜0.01];and mDia1 [0 μg/mL LPS group:(0.200 ±0.102),0.1 μg/mL LPS group:(0.430 ±0.121),1 μg/mL LPS group:(0.603 ±0.154),10 μg/mL LPS group:(0.887 ±0.204);0.1 μg/mL group vs.1 μg/mL group,P ＞ 0.05;the rest P ＜ 0.05].In time-dependent group,the level of p-ERM protein increased at 15 min (0.670 ±0.149),peaked at 30 min (1.175 ±0.103),then decreased,at 60 min (0.959 ±0.189),90 min (0.842 ±0.129),but kept at higher level at 120 min (0.767 ±0.097) than that in control group (0.471 ±0.157,15 min group vs.120 min group,60 min group vs.90 min group and 90 min group vs.120 min group,P ＞ 0.05;the rest P ＜ 0.05);and the level of mDia1 increased at 15 min (0.779 ±0.035),peaked at 30 min (0.889 ±0.036) then decreased at 60 min (0.648 ±0.019),90 min (0.582 ±0.068),but kept at higher level at 120 min (0.526 ±0.059) than that in control group (0.284±0.118,all P ＜ 0.01).C3 transferase caused a marked attenuation of LPS induced p-ERM expression [control group:(0.339 ± 0.069);C3 + LPS group:(0.438 ± 0.07);C3 control group:(0.352 ± 0.071);LPS control group:(0.634 ± 0.191),C3 + LPS group vs.LPS control group,P =0.01],as the same in mDia1 [control group:(0.449 ±0.122);C3 + LPS group:(0.380 ±0.148);C3 control group:(0.404 ±0.164);LPS control group:(0.622 ±0.174),C3 + LPS group vs.LPS control group,P ＜ 0.01].Conclusions LPS could up-regulated the expression of p-ERM protein,and inhibition of RhoA/mDia1 signal pathway by C3 transferase could down-regulated the p-ERM levels.

Contribution of concentration-sensitive sodium channels to the absorption of alveolar fluid in mice.

The concentration-sensitive sodium channel (Nac) is activated by an increase in the extracellular sodium concentration. Although the expression of Nac in alveolar type II epithelial cells (AEC II) has been reported previously, the physiological role of Nac in the lung has not been established. We characterized Nac expression and examined amiloride-insensitive sodium transport mediated by Nac in mouse lung. Immunofluorescence studies revealed that Nac did not colocalize with either aquaporin 5 or cystic fibrosis transmembrane conductance regulator, but partially colocalized with the epithelial sodium channel γ-subunit. Immunoelectron microscopy studies showed that Nac localized at the basolateral membrane of pulmonary microvascular endothelial cells (PMVECs). Nac mRNA and protein were expressed in PMVECs isolated from the lungs of mice. Image analysis indicated that sodium influx into the alveolar wall was dependent on increases in extracellular sodium concentration. We conclude that Nac expressed in PMVECs and AEC II contributes to the reabsorption of sodium via an amiloride-insensitive pathway during alveolar fluid clearance.

Expressions of MK2, HuR, and ICAM-1 in the pulmonary microvascular endothelial cells of the mouse with acute respiratory distress syndrome / 医学研究生学报

Objective Intercellular adhesion molecule-1 (ICAM-1) plays an important role in mediating pulmonary infiltration of neutrophils .The aim of the study was to observe the expression of ICAM-1 and its potential regulators MK 2/HuR in pulmonary micro-vascular endothelial cells ( PMVEC ) in mice with acute respiratory distress syndrome ( ARDS) induced by lipopolysaccharide ( LPS) . Methods Ten 6-8 weeks old healthy C57BL/6 mice were randomly divided into an LPS and a control group of equal number , the former injected intraperitoneally with LPS diluted in 100 μL PBS while the latter with PBS only , both at 5 mg per kg of the body weight .At 24 hours after injection , all the mice were sacrificed .Real-time PCR was used to determine the mRNA expressions of HuR and ICAM-1 in the PMVECs, Western bolt employed to detect the protein expressions of MK2, HuR and ICAM-1, and flow cytometry adopted to measure the ICAM-1 expression on the surface of the PMVECs and pulmonary infiltration of neutrophils . Results The W/D ratio in the lung tissue of the mice was significantly lower in the LPS than in the control group (3.61 ±0.28 vs 6.16 ±0.40, P<0.05), while the rate of neutrophil infiltration markedly higher in the former than in the latter ([13.92 ±3.23]%vs [3.24 ±1.24]%, P<0.05).The mRNA and protein expressions of ICAM-1 in the PMVECs were significantly elevated in the LPS group as compared with that in the control (P<0.05), and so was the mRNA expression of HuR (P<0.05).No remarkable changes were observed in the expressions of total MK 2 and HuR proteins, but phosphorylated MK2 (p-MK2) and cytoplasmic HuR were increased in the LPS-stimulated mice. Conclusion Specific blockage or reduction of the HuR expression in PMVECs may lower the expression of ICAM-1, reduce neutrophil infiltration , and lessen pathophysiological changes in mice with ARDS .

Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling.

Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling.

Study of the level of ERM proteins in pulmonary microvascular endothelial cells induced by tumor necrosis factor-α / 中华急诊医学杂志

Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on the levels of ezrin-radixin-moesin (ERM) proteins and the phosphorylated ERM proteins (p-ERM) in rat pulmonary microvascular endothelial cells (PMVEC),and to explore Rho kinase (ROCK) influencing on modulation of the ERM proteins phosphorylation.Methods Cultured rat pulmonary microvascular endothelial cells were randomly divided into dose-dependent and time-dependent groups.In dose-dependent group,cells were cultured with different doses of TNF-α (0,0.1,1,10 μg/LTNF-α) for 60 min.In time-dependent group,cells were cultured with TNF-α (10 μg/L) for 0,15,30,60,90,120,180 min.In ROCK inhibitor (Y27632) intervention group,cells were cultured with TNF-α (10 μg/L) or Y27632 (30 μmol/L) + TNF-α (10 μg/L) for 60 min respectively.The levels of ERM proteins and p-ERM were determined by western blot.One way analysis of variance (ANOVA) was employed for statistical analysis by using SPSS version 16.0 software to compare values among all groups.A significant difference was presumed as a P value ＜ 0.05.Results Western blot revealed that ERM and p -ERM proteins were present in rat PMVEC.Stimulation withTNF-α gradually up-regulated the level of pERM proteins in a dose-dependent manner [0 μg/LTNF-α group:(0.648 ± 0.102),0.1 μg/LTNF-αgroup:(0.728-±0.082),1 μg/LTNF-α group:(0.926±0.121),10 μg/LTNF-α group:(1.245 ±0.134),all P =0.000].In time-dependent group,the level of p-ERM proteins rose at 15 min (0.777 ±0.151),peaked at 90 min (1.295 ±0.176),then decreased gradually at 120 min (0.802 ±0.139),but stayed higher level at 180 min (0.669 ±0.128) than that in un-stimulated 0 min group (0.631 ±0.123,P=0.004,0.000,0.001,0.016,respectively).When PMVEC pre-incubated with ROCK inhibitor and TNF-t,the level of p-ERM proteins caused a marked attenuation of TNF-αstimulation [(0.634 ± 0.112) vs.(0.875 ± 0.164),P =0.002],however,there are no significant differences compared to ROCK inhibitor alone group (0.661 ± 0.108) and no intervention group (0.654 ± 0.125),P =0.973,P =0.900,respectively).Conclusions TNF-α could induce up-regulation of the level of the phosphorylated ERM proteins in rat PMVEC,and ROCK signal molecules might involve in modulation of the ERM proteins phosphorylation.