Gas6-Axl signal promotes indoor VOCs exposure-induced pulmonary fibrosis via pulmonary microvascular endothelial cells-fibroblasts cross-talk.

Volatile organic compounds (VOCs) as environmental pollutants were associated with respiratory diseases. Pulmonary fibrosis (PF) was characterized by an increase of extracellular matrix, leading to deterioration of lung function. The adverse effects on lung and the potential mechanism underlying VOCs induced PF had not been elucidated clearly. In this study, the indoor VOCs exposure mouse model along with an ex vivo biosensor assay was established. Based on scRNA-seq analysis, the adverse effects on lung and potential molecular mechanism were studied. Herein, the results showed that VOCs exposure from indoor decoration contributed to decreased lung function and facilitated pulmonary fibrosis in mice. Then, the whole lung cell atlas after VOCs exposure and the heterogeneity of fibroblasts were revealed. We explored the molecular interactions among various pulmonary cells, suggesting that endothelial cells contributed to fibroblasts activation in response to VOCs exposure. Mechanistically, pulmonary microvascular endothelial cells (MPVECs) secreted Gas6 after VOCs-induced PANoptosis phenotype, bound to the Axl in fibroblasts, and then activated fibroblasts. Moreover, Atf3 as the key gene negatively regulated PANoptosis phenotype to ameliorate fibrosis induced by VOCs exposure. These novel findings provided a new perspective about MPVECs could serve as the initiating factor of PF induced by VOCs exposure.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Oxymatrine Modulation of TLR3 Signaling: A Dual-Action Mechanism for H9N2 Avian Influenza Virus Defense and Immune Regulation.

In our research, we explored a natural substance called Oxymatrine, found in a traditional Chinese medicinal plant, to fight against a common bird flu virus known as H9N2. This virus not only affects birds but can also pose a threat to human health. We focused on how this natural compound can help in stopping the virus from spreading in cells that line the lungs of birds and potentially humans. Our findings show that Oxymatrine can both directly block the virus and boost the body's immune response against it. This dual-action mechanism is particularly interesting because it indicates that Oxymatrine might be a useful tool in developing new ways to prevent and treat this type of bird flu. Understanding how Oxymatrine works against the H9N2 virus could lead to safer and more natural ways to combat viral infections in animals and humans, contributing to the health and well-being of society. The H9N2 Avian Influenza Virus (AIV) is a persistent health threat because of its rapid mutation rate and the limited efficacy of vaccines, underscoring the urgent need for innovative therapies. This study investigated the H9N2 AIV antiviral properties of Oxymatrine (OMT), a compound derived from traditional Chinese medicine, particularly focusing on its interaction with pulmonary microvascular endothelial cells (PMVECs). Employing an array of in vitro assays, including 50% tissue culture infectious dose, Cell Counting Kit-8, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot, we systematically elucidated the multifaceted effects of OMT. OMT dose-dependently inhibited critical antiviral proteins (PKR and Mx1) and modulated the expression of type I interferons and key cytokines (IFN-α, IFN-ß, IL-6, and TNF-α), thereby affecting TLR3 signaling and its downstream elements (NF-κB and IRF-3). OMT's antiviral efficacy extended beyond TLR3-mediated responses, suggesting its potential as a versatile antiviral agent. This study not only contributes to the growing body of research on the use of natural compounds as antiviral agents but also underscores the importance of further investigating the broader application of OMT for combating viral infections.

Elevated circulating BMP9 aggravates pulmonary angiogenesis in hepatopulmonary syndrome rats through ALK1-Endoglin-Smad1/5/9 signalling.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) is a hepatokine that plays a pivotal role in the progression of liver diseases. Moreover, an increasing number of studies have shown that BMP9 is associated with hepatopulmonary syndrome (HPS), but its role in HPS is unclear. Here, we evaluated the influence of CBDL on BMP9 expression and investigated potential mechanisms of BMP9 signalling in HPS. METHODS: We profiled the circulating BMP9 levels in common bile duct ligation-induced HPS rat model, and then investigated the effects and mechanisms of HPS rat serum on pulmonary vascular endothelial dysfunction in rat model, as well as in primarily cultured rat pulmonary microvascular endothelial cells. RESULTS: Our data revealed that circulating BMP9 levels were significantly increased in the HPS rats compared to control group. Besides, the elevated BMP9 in HPS rat serum was not only crucial for promoting endothelial cell proliferation and tube formation through the activin receptor-like kinase1 (ALK1)-Endoglin-Smad1/5/9 pathway, but also important for accumulation of monocytes. Treatments with ALK1-Fc or silencing ALK1 expression to inhibit the BMP9 signalling pathway effectively eliminated these effects. In agreement with these observations, increased circulating BMP9 was associated with an increase in lung vessel density and accumulation of pro-angiogenic monocytes in the microvasculature in HPS rats. CONCLUSIONS: This study provided evidence that elevated circulating BMP9, secreted from the liver, promote pulmonary angiogenesis in HPS rats via ALK1-Endoglin-Smad1/5/9 pathway. In addition, BMP9-regulated pathways are also involved in accumulation of pro-angiogenic monocytes in the pulmonary microvasculature in HPS rats.

CircALMS1 Alleviates Pulmonary Microvascular Endothelial Cell Dysfunction in Pulmonary Hypertension.

BACKGROUND: Circular RNAs can serve as regulators influencing the development of pulmonary hypertension (PH). However, their function in pulmonary vascular intimal injury remains undefined. Thus, we aimed to identify specifically expressed circular RNAs in pulmonary microvascular endothelial cells (PMECs) under hypoxia and PH. METHODS AND RESULTS: Deep RNA sequencing and quantitative real-time polymerase chain reaction revealed that circALMS1 (circular RNA Alstrom syndrome protein 1) was reduced in human PMECs under hypoxia (P<0.0001). Molecular biology and histopathology experiments were used to elucidate the roles of circALMS1 in regulating PMEC dysfunction among patients with PH. The circALMS1 expression was decreased in the plasma of patients with PH (P=0.0315). Patients with lower circALMS1 levels had higher risk of death (P=0.0006). Moreover, the circALMS1 overexpression of adeno-associated viruses improved right ventricular function and reduced pulmonary vascular remodeling in monocrotaline-PH and sugen/hypoxia-PH rats (P<0.05). Furthermore, circALMS1 overexpression promoted apoptosis and inhibited PMEC proliferation and migration under hypoxia by directly downregulating miR-17-3p (P<0.05). Dual luciferase assay confirmed the direct binding of circALMS1 to miR-17-3p and miR-17-3p binding to its target gene YT521-B homology domain-containing family protein 2 (YTHDF2) (P<0.05). The YTHDF2 levels were also downregulated in hypoxic PMECs (P<0.01). The small interfering RNA YTHDF2 reversed the effects of miR-17-3p inhibitors on PMEC proliferation, migration, and apoptosis. Finally, the results indicated that, although YTHDF2, as an N(6)-methyladenosine reader protein, contributes to the degradation of many circular RNAs, it could not regulate the circALMS1 levels in PMECs (P=0.9721). CONCLUSIONS: Our study sheds new light on circALMS1-regulated dysfunction of PMECs by the miR-17-3p/YTHDF2 pathway under hypoxia and provides insights into the underlying pathogenesis of PH.

Long noncoding RNA ANRIL alleviates hypoxia-induced pulmonary microvascular endothelial cell damage.

BACKGROUND: High-altitude pulmonary oedema (HAPE) is a form of noncardiogenic pulmonary oedema. Studies have found that long noncoding RNA (lncRNA) plays an important role in HAPE. ANRIL is significant in pulmonary illnesses, which implies that alterations in ANRIL expression levels may be involved in the beginning and development of HAPE. However, the specific mechanism is indistinct. The present study is meant to explore the effect and mechanism of ANRIL on hypoxic-induced injury of pulmonary microvascular endothelial cells (PMEVCs). METHODS: In the hypoxic model of PMVECs, overexpression of ANRIL or knockdown of miR-181c-5p was performed to assess cell proliferation, apoptosis, and migration. Furthermore, the levels of apoptosis-related proteins, inflammatory factors, and vascular active factors were also measured. RESULTS: The results showed that, after 24 h of hypoxia, PMVECs proliferation and migration were suppressed in comparison to the control group, along with an increase in apoptosis, a decrease in the expression of ANRIL, and an increase in the expression of miR-181c-5p (all p < .05). The damage caused by hypoxia in PMVECs can be lessened by overexpressing ANRIL, which also inhibits the production of TNF-α, iNOS, and VEGF as well as BAX and cleaved caspase-3 (all p < .05). Further experimental results showed that overexpression of ANRIL and knockdown of miR-181c-5p had the same protection against hypoxic injury in PMVECs (all p < .05). CONCLUSIONS: Our study suggests that ANRIL may prevent hypoxia injury to PMVECs in HAPE through the negative regulation of miR-181c-5p.

Transcriptomics and proteomics revealed sex differences in human pulmonary microvascular endothelial cells.

Marked sexual dimorphism is displayed in the onset and progression of pulmonary hypertension (PH). Females more commonly develop pulmonary arterial hypertension, yet females with pulmonary arterial hypertension and other types of PH have better survival than males. Pulmonary microvascular endothelial cells play a crucial role in pulmonary vascular remodeling and increased pulmonary vascular resistance in PH. Given this background, we hypothesized that there are sex differences in the pulmonary microvascular endothelium basally and in response to hypoxia that are independent of the sex hormone environment. Human pulmonary microvascular endothelial cells (HPMECs) from healthy male and female donors, cultured under physiological shear stress, were analyzed using RNA sequencing and label-free quantitative proteomics. Gene set enrichment analysis identified a number of sex-different pathways in both normoxia and hypoxia, including pathways that regulate cell proliferation. In vitro, the rate of proliferation in female HPMECs was lower than in male HPMECs, a finding that supports the omics results. Interestingly, thrombospondin-1, an inhibitor of proliferation, was more highly expressed in female cells than in male cells. These results demonstrate, for the first time, important differences between female and male HPMECs that persist in the absence of sex hormone differences and identify novel pathways for further investigation that may contribute to sexual dimorphism in pulmonary hypertensive diseases.NEW & NOTEWORTHY There is marked sexual dimorphism in the development and progression of pulmonary hypertension. We show differences in RNA and protein expression between female and male human pulmonary microvascular endothelial cells grown under conditions of physiological shear stress, which identify sex-different cellular pathways both in normoxia and hypoxia. Importantly, these differences were detected in the absence of sex hormone differences. The pathways identified may provide novel targets for the development of sex-specific therapies.

Irbesartan eases lipopolysaccharide-induced lung injury In Vitro and In Vivo.

Acute lung injury (ALI) is classified as a devastating pulmonary disorder contributing to significant incidence and fatality rate. Irbesartan (IRB) is an angiotensin II receptor blocker that has been proposed to protect against oleic acid-induced ALI. To this end, the current study is concentrated on ascertaining the role of IRB in ALI and figuring out the probable action mechanism. First, cell counting kit-8 (CCK-8) appraised the viability of human pulmonary microvascular endothelial cells (HPMVECs) exposed to ascending concentrations of IRB. HPMVEC injury model and a mouse model of ALI induced by lipopolysaccharide (LPS) were pretreated by IRB. In vitro, cell viability was estimated by CCK-8 assay, and lactate dehydrogenase (LDH) release was tested by LDH assay kit. Enzyme-linked immunosorbent assay (ELISA) and Western blotting estimated the expression levels of inflammatory factors. Fluorescein isothiocyanate-dextran was used to assess HPMVEC permeability. Western blotting examined the expression of adherent and tight junction proteins. In vivo, hematoxylin and eosin staining evaluated lung tissue damage and lung wet/dry (W/D) weight was measured. ELISA analyzed the levels of inflammatory factors in the serum and bronchoalveolar lavage fluid (BALF), and Western blotting examined the expression of inflammatory factors. The total cell, neutrophil, and macrophage numbers in BALF were determined using a cell counter. Lung capillary permeability was assayed by Evans blue albumin and total protein concentration in BALF was measured using bicinchoninic acid method. Immunofluorescence assay and Western blotting examined the expression of adherent and tight junction proteins in lung tissues. It was observed that IRB dose-dependently enhanced the viability while reduced LDH release, inflammatory response as well as permeability in LPS-challenged HPMVECs in vitro. In addition, LPS-stimulated lung tissue damage, pulmonary edema, inflammatory response as well as lung capillary permeability in vivo were all reversed following IRB treatment. Collectively, IRB treatment might elicit protective behaviors against LPS-triggered ALI.

Heat stress combined with lipopolysaccharide induces pulmonary microvascular endothelial cell glycocalyx inflammatory damage in vitro.

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Transcriptomic analysis of human pulmonary microvascular endothelial cells treated with LPS.

Acute respiratory distress syndrome (ARDS) has a rapid onset and progression, which lead to the severity and complexity of the primary disease and significantly increase the fatality rate of patients. Transcriptomics provides some ideas for clarifying the mechanism of ARDS, exploring prevention and treatment targets, and searching for related specific markers. In this study, RNA-Seq technology was used to observe the gene expression of human pulmonary microvascular endothelial cells (PMVECs) induced by LPS, and to excavate the key genes and signaling pathways in ARDS process. A total of 2300 up-regulated genes were detected, and a corresponding 1696 down-regulated genes were screened. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) were also used for functional annotation of key genes. TFDP1 was identified as a cell cycle-dependent differentially expressed gene, and its reduced expression was verified in LPS-treated PMVECs and lung tissues of CLP-induced mice. In addition, the inhibition of TFDP1 on inflammation and apoptosis, and the promotion of proliferation were confirmed. The decreased expression of E2F1, Rb, CDK1 and the activation of MAPK signaling pathway were substantiated in the in vivo and in vitro models of ARDS. Moreover, SREBF1 has been demonstrated to be involved in cell cycle arrest in PMVECs by inhibiting CDK1. Our study shows that transcriptomics combined with basic research can broaden the investigation of ARDS mechanisms and may provide a basis for future mechanistic innovations.

Endothelial to mesenchymal transition in neonatal hyperoxic lung injury: role of sex as a biological variable.

Bronchopulmonary dysplasia (BPD) is characterized by an arrest in alveolarization, abnormal vascular development, and variable interstitial fibroproliferation in the premature lung. Endothelial to mesenchymal transition (EndoMT) may be a source of pathological fibrosis in many organ systems. Whether EndoMT contributes to the pathogenesis of BPD is not known. We tested the hypothesis that pulmonary endothelial cells will show increased expression of EndoMT markers upon exposure to hyperoxia and that sex as a biological variable will modulate differences in expression. Wild-type (WT) and Cdh5-PAC CreERT2 (endothelial reporter) neonatal male and female mice (C57BL6) were exposed to hyperoxia (0.95 [Formula: see text]) either during the saccular stage of lung development (95% [Formula: see text]; postnatal day 1-5 [PND1-5]) or through the saccular and early alveolar stages of lung development (75% [Formula: see text]; PND1-14). Expression of EndoMT markers was measured in whole lung and endothelial cell mRNA. Sorted lung endothelial cells (from room air- and hyperoxia-exposed lungs) were subjected to bulk RNA-Seq. We show that exposure of the neonatal lung to hyperoxia leads to upregulation of key markers of EndoMT. Furthermore, using lung sc-RNA-Seq data from neonatal lung we were able to show that all endothelial cell subpopulations including the lung capillary endothelial cells show upregulation of EndoMT-related genes. Markers related to EndoMT are upregulated in the neonatal lung upon exposure to hyperoxia and show sex-specific differences. Mechanisms mediating EndoMT in the injured neonatal lung can modulate the response of the neonatal lung to hyperoxic injury and need further investigation.NEW & NOTEWORTHY We show that neonatal hyperoxia exposure increased EndoMT markers in the lung endothelial cells and this biological process exhibits sex-specific differences.

MiR26-5p inhibits pathological pulmonary microvascular angiogenesis via down-regulating WNT5A.

Objectives: Pathological micro angiogenesis is a key pathogenic factor in pulmonary diseases such as pulmonary hypertension and hepatopulmonary syndrome. More and more pieces of evidence show that excessive proliferation of pulmonary microvascular endothelial cells is the key event of pathological micro angiogenesis. The purpose of this research is to reveal the mechanism of miR26-5p regulating pulmonary microvascular hyperproliferation. Materials and Methods: Hepatopulmonary syndrome rat model was made by common bile duct ligation. HE and IHC staining were used for analysis of the pathology of the rat. CCK8, transwell, and wound healing assay were used to assess miR26-5p or target gene WNT5A functioned toward PMVECs. microRNA specific mimics and inhibitors were used for up/down-regulated miR26-5p expression in PMVECs. Recombinant lentivirus was used for overexpression/knockdown WNT5A expression in PMVECs. And the regulation relationship of miR26-5p and WNT5A was analyzed by dual-luciferase reporter assay. Results: qPCR showed that miR26-5p was significantly down-regulated in the course of HPS disease. Bioinformatics data showed that WNT5A was one of the potential key target genes of miR26-5p. Immunohistochemistry and qPCR analysis showed that WNT5A was largely expressed in pulmonary microvascular endothelial cells, in addition, this molecule was significantly up-regulated with the progression of the disease. Furthermore, dual luciferase reporter assay showed that miR26-5p could bind to WNT5A 3 'UTR region to inhibit WNT5A synthesis. Conclusion: The results suggested MiR26-5p negatively regulated PMVECs proliferation and migration by WNT5A expression. Overexpression of miR26-5p may be a potentially beneficial strategy for HPS therapy.

Differential responses of pulmonary vascular cells from PAH patients and controls to TNFα and the effect of the BET inhibitor JQ1.

BACKGROUND: Pulmonary arterial hypertension (PAH) encompasses a group of diseases characterized by raised pulmonary vascular resistance, resulting from vascular remodelling and inflammation. Bromodomain and extra-terminal (BET) proteins are required for the expression of a subset of NF-κB-induced inflammatory genes which can be inhibited by the BET mimic JQ1+. We hypothesised that JQ+ would supress TNFα-driven inflammatory responses in human pulmonary vascular cells from PAH patients. METHODS: Immunohistochemical staining of human peripheral lung tissue (N = 14 PAH and N = 12 non-PAH) was performed for the BET proteins BRD2 and 4. Human pulmonary microvascular endothelial cells (HPMEC) and pulmonary artery smooth muscle cells (HPASMC) from PAH patients (N = 4) and non-PAH controls (N = 4) were stimulated with TNFα in presence or absence of JQ1+ or its inactive isomer JQ1-. IL-6 and -8 mRNA was measured by RT-qPCR and protein levels by ELISA. Chromatin immunoprecipitation analysis was performed using EZ-ChIP™ and NF-κB p65 activation determined using a TransAm kit. MTT assay was used to measure cell viability. RESULTS: Nuclear staining of BRD2 and BRD4 was significantly (p < 0.0001) increased in the lung vascular endothelial and smooth muscle cells from PAH patients compared to controls with normal lung function. TNFα-driven IL-6 release from both HPMECs and HPASMCs was greater in PAH cells than control cells. Levels of CXCL8/IL-8 protein release was higher in PAH HPASMCs than in control cells with similar release observed in HPMECs. TNFα-induced recruitment of activated NF-κB p65 to the IL-6 and CXCL8/IL-8 promoters were similar in both cell types and between subject groups. JQ1+ suppressed TNFα-induced IL-6 and CXCL8/IL-8 release and mRNA expression to a comparable extent in control and PAH HPMECs and HPASMCs. JQ1 had a greater efficacy on IL-6 release in HPMEC and on CXCL8/IL-8 release in HPASMC. CONCLUSION: BET inhibition decreases TNFα driven inflammation in primary pulmonary vascular cells. The anti-inflammatory actions of JQ1 suggests distinct cell-specific regulatory control of these genes. BET proteins could be a target for future therapies for PAH.

PDK1 upregulates PINK1-mediated pulmonary endothelial cell mitophagy during hypoxia-induced pulmonary vascular remodeling.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a complication of lung diseases with pulmonary vascular remodeling, although the underlying molecular mechanisms have not been fully elucidated. This study investigated the underlying molecular events by using a rat HPH model and primary pulmonary microvascular endothelial cells (PMVECs). METHODS AND RESULTS: This study first established a rat HPH model and cultured PMVECs for transmission electron microscopic analysis and manipulation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or phosphatase and tensin homolog-induced kinase 1 (PINK1) expression in vitro. After that, the cell viability was assessed and the expression of different proteins was assayed using cell viability and western blot assays, respectively. Reactive oxygen species production, apoptosis, NLR family pyrin domain containing 3 (NLRP3) expression, and the levels of interleukin (IL)-1ß, IL-6, and IL-8 were also assessed, while the interaction of PDK1 and PINK1 was determined using co-immunoprecipitation/western blot assays. Hypoxia induced mitophagy in the PMVECs and upregulated PINK1/Parkin expression, whereas knockdown of PINK1 expression under hypoxic conditions inhibited cell proliferation but induced endothelial cell apoptosis in vitro, decreased reactive oxygen species production and NLRP3 expression, and reduced the levels of inflammatory factors in PMVECs. However, hypoxia induced PDK1 expression, whereas knockdown of PDK1 downregulated PINK1 expression. Furthermore, treatment of the model rats with the PDK1 inhibitor dichloroacetate (DCA) was able to decrease PINK1 expression. In addition, the PDK1 and PINK1 proteins could interact with each other in the mitochondria of PMVECs to regulate the cell viability. CONCLUSIONS: This study revealed that PDK1 induced PMVEC proliferation but inhibited their apoptosis to participate in pulmonary vascular remodeling, ultimately leading to HPH through regulation of PINK1-mediated mitophagy signaling. Therefore, PINK1 is a novel therapeutic target for the control of HPH.

Extracellular Vesicle ITGAM and ITGB2 Mediate Severe Acute Pancreatitis-Related Acute Lung Injury.

Integrins expressed on extracellular vesicles (EVs) secreted by various cancers are reported to mediate the organotropism of these EVs. Our previous experiment found that pancreatic tissue of mice with severe cases of acute pancreatitis (SAP) overexpresses several integrins and that serum EVs of these mice (SAP-EVs) can mediate acute lung injury (ALI). It is unclear if SAP-EV express integrins that can promote their accumulation in the lung to promote ALI. Here, we report that SAP-EV overexpress several integrins and that preincubation of SAP-EV with the integrin antagonist peptide HYD-1 markedly attenuates their pulmonary inflammation and disrupt the pulmonary microvascular endothelial cell (PMVEC) barrier. Further, we report that injecting SAP mice with EVs engineered to overexpress two of these integrins (ITGAM and ITGB2) can attenuate the pulmonary accumulation of pancreas-derived EVs and similarly decrease pulmonary inflammation and disruption of the endothelial cell barrier. Based on these findings, we propose that pancreatic EVs can mediate ALI in SAP patients and that this injury response could be attenuated by administering EVs that overexpress ITGAM and/or ITGB2, which is worthy of further study due to the lack of effective therapies for SAP-induced ALI.

Shear Stress Markedly Alters the Proteomic Response to Hypoxia in Human Pulmonary Endothelial Cells.

Blood flow produces shear stress that homeostatically regulates the phenotype of pulmonary endothelial cells, exerting antiinflammatory and antithrombotic actions and maintaining normal barrier function. Hypoxia due to diseases, such as chronic obstructive pulmonary disease (COPD), causes vasoconstriction, increased vascular resistance, and pulmonary hypertension. Hypoxia-induced changes in endothelial function play a central role in the development of pulmonary hypertension. However, the interactive effects of hypoxia and shear stress on the pulmonary endothelial phenotype have not been studied. Human pulmonary microvascular endothelial cells were cultured in normoxia or hypoxia while subjected to physiological shear stress or in static conditions. Unbiased proteomics was used to identify hypoxia-induced changes in protein expression. Using publicly available single-cell RNA sequencing datasets, differences in gene expression between the alveolar endothelial cells from COPD and healthy lungs were identified. Sixty proteins were identified whose expression changed in response to hypoxia in conditions of physiological shear stress but not in static conditions. These included proteins that are crucial for endothelial homeostasis (e.g., JAM-A [junctional adhesion molecule A], ERG [ETS transcription factor ERG]) or implicated in pulmonary hypertension (e.g., thrombospondin-1). Fifty-five of these 60 have not been previously implicated in the development of hypoxic lung diseases. mRNA for 5 of the 60 (ERG, MCRIP1 [MAPK regulated corepressor interacting protein 1], EIF4A2 [eukaryotic translation initiation factor 4A2], HSP90AA1 [heat shock protein 90 alpha family class A member 1], and DNAJA1 [DnaJ Hsp40 (heat shock protein family) member A1]) showed similar changes in the alveolar endothelial cells of COPD compared with healthy lungs in females but not in males. These data show that the proteomic responses of the pulmonary microvascular endothelium to hypoxia are significantly altered by shear stress and suggest that these shear-hypoxia interactions are important in the development of hypoxic pulmonary vascular disease.

Mechanisms of pulmonary microvascular endothelial cells barrier dysfunction induced by LPS: The roles of ceramides and the Txnip/NLRP3 inflammasome.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are characterized by pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction and proinflammatory cytokine influx into lung tissue, resulting in pulmonary oedema. Ceramide overproduction is an important mediator of pulmonary hyperinflammation and pulmonary oedema in Acute lung injury (ALI). Ceramides induce NLRP3 inflammasome activation are essential for the hyperinflammatory response. However, the roles and specific mechanisms of ceramide-induced NLRP3 inflammasome activation, proinflammatory cytokine manufacturing and PMVECs barrier dysfunction in ALI are unclear. Herein, pretreatment with the acid sphingomyelinase (ASMase) inhibitor imipramine (but not a neutral sphingomyelinase (NSMase) inhibitor or de novo pathway inhibitor) significantly inhibited ceramide early production in rats with lipopolysaccharide (LPS)-induced ALI; Furthermore, the Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury were significantly decreased. Verapamil, a Txnip inhibitor, substantially inhibited Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury in rats with C8-ceramide-induced ALI. In vitro, short hairpin RNA-mediated Txnip silencing significantly inhibited C8-ceramide-induced Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages (AMs) and early secretion of the proinflammatory cytokines IL-1ß (4-12 h) as well as IL-6 and TNF-α at subsequent times (later than 12 h). However, C8-ceramide significantly increased the early secretion (within 8 h) of the proinflammatory cytokines IL-1ß, IL-6 and TNF-α in a co-culture model of NR8383 AMs and PMVECs, and Txnip silencing of NR8383 AMs inhibited the secretion of pro-inflammatory cytokines and reduced cytoskeletal rearrangements, intercellular connection breakage and hyperpermeability in PMVECs. Overall, our results suggest that in LPS-induced ALI, ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 AMs leads to early IL-1ß release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI.

Mechanisms of 15-Epi-LXA4-Mediated HO-1 in Cytoprotection Following Inflammatory Injury.

INTRODUCTION: Heme oxygenase-1 (HO-1) is a protective protein in oxidative stress response. LXA4 is an "inflammatory braking signal" that is widely studied at present. The purpose of this study was to elucidate that LXA4 can protect cells by inducing HO-1 in human pulmonary microvascular endothelial cells (HPMECs) as in vitro model to explain acute lung injury after severe acute pancreatitis. METHODS: This study was performed in two parts: (1) To investigate the mechanisms of lipoxin A4-induced HO-1 expression in vitro, the study subjects were divided into four groups: a control group, LXA4 group (50 ng/mL LXA4), inhibitor group (50 ng/mL LXA4 + 20 µM LY294002 or 50 ng/mL LXA4 + 2 nmol/mL Bis II), and agonist group (50 ng/mL insulin-like growth factor 1, PMA). Western blotting was used to detect the expression of p-Akt, Akt, protein kinase C (PKC), p-Nrf2, Nrf2, and Keap1, and the location of Nrf2 was detected using immunofluorescence. The activation of antioxidant responsive element induced by Nrf2 was detected using Electrophoretic Mobility Shift Assay and (2) to investigate the cytoprotection of HO-1 induced by LXA4 in vitro, the subjects were divided into four groups: a control group, tumor necrosis factor α (TNF-α) group (50 ng/mL), LXA4 group (50 ng/mL TNF-α + 50 ng/mL LXA4), and Zinc protoporphyrin IX group (pretreated with 0.5 µM Zinc protoporphyrin IXfor 12 h, followed by 50 ng/mL TNF-α + 50 ng/mL LXA4). BCECF/AM-labeled THP-1 cells were used to analyze the adhesion of HPMECs, and a mitochondrial membrane potential assay kit with JC-1 was used to analyze the apoptosis of HPMECs. RESULTS: In part one, (1) LXA4 upregulated the expression of HO-1 in a dose-dependent manner and (2) LXA4 activated the PI3K/Akt and PKC pathways and modulated the phosphorylation and subsequent depolymerization of Nrf2 from Keap1, promoting the translocation of Nrf2 to the nucleus. In part two, (1) LXA4 reversed the changes in mitochondrial membrane potential to alleviate apoptosis in HPMECs and (2) LXA4 attenuated the adhesion of HPMECs induced by TNF-α. CONCLUSIONS: LXA4 can activate the PI3K/Akt and PKC pathways and induce the phosphorylation of Nrf2, resulting in the upregulation of HO-1. In addition, LXA4 alleviates adhesion and protects mitochondrial function by upregulating the expression of HO-1, which exerts cytoprotection in severe acute pancreatitis-induced lung injury.

The effect of nuclear factor erythroid 2 related factor 2-induced inhibition of ferroptosis on hyperoxia lung injury / 中华实用儿科临床杂志

Objective:To observe the expression changes of nuclear factor erythroid 2 related factor 2 (Nrf2) and glutathione peroxidase (GPX4) in human pulmonary microvascular endothelial cells (HPMEC) under different experimental conditions, and to explore the role of Nrf2 in inhibiting ferroptosis in the process of alleviating hyperoxic lung injury(HLI).Methods:Hyperoxic model was established by hyperoxia exposure.HPMEC were treated with blank control (control group), oxygen exposure at the concentration of 950 mL/L (hyperoxia group), oxygen exposure at the concentration of 950 mL/L+ 10 μmol/L Ferrostatin (ferroptosis inhibitor group) and oxygen exposure at the concentration of 950 mL/L + 10 μmol/L ML385 (Nrf2 inhibitor group). Cell viability at 24 h and 48 h was tested by the Cell Counting Kit-8 assay, and reactive oxygen species (ROS) levels were detected by a commercial ROS kit.The mRNA and protein levels of Nrf2 and GPX4 were detected by real-time quantitative polymerase chain reaction and Western blot, respectively.Differences were analyzed using the Student′s t-test for a two-group comparison or one-way ANOVA test among groups. Results:(1)Compared with the control group, significantly decreased viability and increased ROS levels were detected in hyperoxia group.Meanwhile, the mRNA (24 h: 0.750±0.010 vs.1.010±0.160, 48 h: 0.690±0.050 vs.1.000±0.070) and protein levels of GPX4 (24 h: 0.160±0.010 vs.0.290±0.010, 48 h: 0.190±0.010 vs.0.250±0.010) at 24 h and 48 h were significantly downregulated, while the mRNA (24 h: 1.740±0.050 vs.1.000±0.050, 48 h: 2.130±0.020 vs.1.000±0.030) and protein levels of Nrf2 (24 h: 0.840±0.010 vs.0.480±0.010, 48 h: 0.840±0.010 vs.0.550±0.030) at 24 h and 48 h were significantly upregulated in hyperoxia group than those of control group (all P<0.05). (2)Compared with the hyperoxia group, significantly increased viability and decreased ROS levels were detected in ferroptosis inhibitor group.Meanwhile, the mRNA (24 h: 1.520±0.110, 48 h: 1.880±0.050) and protein levels of GPX4 (24 h: 0.290±0.010, 48 h: 0.250±0.004) at 24 h and 48 h were significantly upregulated, while the mRNA (24 h: 0.780±0.040, 48 h: 0.760±0.030) and protein levels of Nrf2 (24 h: 0.480±0.010, 48 h: 0.540±0.020) at 24 h and 48 h were significantly downregulated in ferroptosis inhibitor group than those of hyperoxia group (all P<0.05). (3)Compared with the hyperoxia group, significantly decreased viability and increased ROS levels were detected in Nrf2 inhibitor group.Meanwhile, the mRNA (24 h: 0.600±0.030, 48 h: 0.590±0.003) and protein levels of GPX4 (24 h: 0.150±0.001, 48 h: 0.180±0.001) at 24 h and 48 h were significantly downregulated, while the mRNA level of Nrf2 was significantly upregulated at 24 h (3.360±0.130), but downregulated at 48 h (1.430±0.130) (all P<0.05). No significant difference was detected in the protein level of Nrf2 at 24 h and 48 h between hyperoxia group and Nrf2 inhibitor group ( P>0.05). Conclusions:Ferroptosis is involved in the development of HLI, and Nrf2 is able to alleviate hyperoxic lung injury by inhibiting ferroptosis.Therefore, inhibition of ferroptosis by Nrf2 may provide a new therapeutic target for HLI.