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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Endothelial dysfunction is one of the main pathogenetic mechanisms of pulmonary hypertension (PH). In the pathological microenvironment, excess reactive oxygen species (ROS) further induce pulmonary microvascular endothelial cell (PMEC) dysfunction. Recently, there has been an extreme lack of drugs that specifically target and eliminate ROS within the microenvironment. Although some nanomaterials are known as effective ROS-scavenging agents and can further alleviate disease progression, few nanomaterials have been reported in the endothelial dysfunction of PH. Based on the above situation, we synthesized a kind of novel nanoclusters (cerium dioxide/bovine serum albumin, CeO2/BSA), which can not only effectively scavenge ROS but also inhibit hypoxia-induced PMEC overproliferation, migration, and apoptosis resistance. CeO2/BSA nanoclusters were proved to be enriched in the lung, which effectively relieved pulmonary vascular remodeling, reduced the pressure of the pulmonary artery, and finally significantly improved cardiac function. Through in-depth mechanism analysis, we found that CeO2/BSA nanoclusters may be involved in Th1 and Th2 differentiation pathways mainly by effectively scavenging high levels of ROS in the pulmonary artery. In addition, the good biocompatibility of CeO2/BSA nanoclusters increased the possibility of their clinical application in the treatment of PH. In conclusion, this work provides a novel and effective solution to improve the dilemma of the ROS target and elimination in the treatment of PH. [ABSTRACT FROM AUTHOR]

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

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 CSEinduced 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 CSEinduced 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.

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)-1β, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective The incidence rate of heat stroke (HS) has increased, with high morbidity and mortality rates, in recent years. Previous studies have suggested that vascular endothelial cell injury is one of the main pathological features of HS. Uncoupling protein 2 (UCP2) exhibits antioxidant activity under various stress conditions. This study aims to investigate the role of UCP2 in HS-induced vascular endothelial injury. Method To explore the mechanisms mediating vascular endothelial cell injury induced by HS, we established an HS model of HUVECs in vitro. The percentage of cell death and viability induced by HS were assessed using annexin V-FITC/PI staining and CCK8 assays. HS-induced mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. HS-induced mitochondrial superoxide was measured by MitoSOX staining, and analyzed by flow cytometry. UCP2, Drp1, phosphorylated Drp1, OPA1, and Mfn2 expression levels were measured by western blotting. Results HS triggered mitochondrial fragmentation and UCP2 upregulation in a time-dependent manner in HUVECs. As a specific Drp1 inhibitor, Mdivi‐1 pretreatment significantly promoted mitochondrial fission and apoptosis in HS-induced HUVECs. In addition, siRNA-mediated UCP2 knockdown further aggravated mitochondrial fragmentation and ΔΨm depolarization and increased mitochondrial ROS production and cell apoptosis in HS-induced HUVECs, which were abolished by Drp1 inhibition. Conclusion Our results indicate that UCP2 protects against HS-induced vascular endothelial damage and that it enhances mitochondrial function. These findings reveal that UCP2 can be a potential contributor to mechanism-based therapeutic strategies for HS.

Abstract Background Our previous study showed that circular RNA-gamma-secretase-activating protein (circGSAP) was down-regulated in pulmonary microvascular endothelial cells (PMECs) in response to hypoxia, and regulated the cell cycle of PMECs via miR-942-5p sponge in pulmonary hypertension (PH). However, the mechanism whether circGSAP affects the dysfunction of PEMCs through other microRNAs (miRNAs) remains largely unknown. Therefore, we aimed to demonstrate the underlying mechanisms of circGSAP regulating PMECs dysfunction by absorbing other miRNAs to regulate target genes in idiopathic pulmonary arterial hypertension (IPAH). Methods Quantitative real-time polymerase chain reaction, immunofluorescence staining, Cell Counting Kit-8, Calcein-AM/PI staining, Transwell assay, dual-luciferase reporter assay, and ELISA were used to elucidate the roles of circGSAP. Results Here we showed that plasma circGSAP levels were significantly decreased in patients with IPAH and associated with poor outcomes. In vivo, circGSAP overexpression improved survival, and alleviated pulmonary vascular remodeling of monocrotaline-induced PH (MCT-PH) rats. In vitro, circGSAP overexpression inhibited hypoxia-induced PMECs proliferation, migration and increased mortality by absorbing miR-27a-3p. BMPR2 was identified as a miR-27a-3p target gene. BMPR2 silencing ameliorated the effect of the miR-27a-3p inhibitor on PMECs proliferation,migration and mortality. The levels of BMPR2 were upregulated in circGSAP-overexpressed PMECs and lung tissues of MCT-PH rats. Conclusion Our findings demonstrated that circGSAP alleviated the dysfunction of PMECs via the increase of BMPR2 by competitively binding with miR-27a-3p, and mitigated pulmonary vascular remodeling of MCT-PH rats, providing potential therapeutic strategies for IPAH.

Abstract Small extracellular vesicles (sEVs) have been recognized to be more effective than direct stem cell differentiation into functional target cells in preventing tissue injury and promoting tissue repair. Our previous study demonstrated the protective effect of adipose-derived stem cells (ADSCs) on lipopolysaccharide (LPS)-induced acute lung injury and the effect of autophagy on ADSC functions, but the role of ADSC-derived sEVs (ADSC-sEVs) and autophagy-mediated regulation of ADSC-sEVs in LPS-induced pulmonary microvascular barrier damage remains unclear. After treatment with sEVs from ADSCs with or without autophagy inhibition, LPS-induced human pulmonary microvascular endothelial cell (HPMVECs) barrier damage was detected. LPS-induced acute lung injury in mice was assessed in vivo after intravenous administration of sEVs from ADSCs with or without autophagy inhibition. The effects of autophagy on the bioactive miRNA components of ADSC-sEVs were assessed after prior inhibition of cell autophagy. We found that ADSC-sEV effectively alleviated LPS-induced apoptosis, tight junction damage and high permeability of PMVECs. Moreover, in vivo administration of ADSC-sEV markedly inhibited LPS-triggered lung injury. However, autophagy inhibition, markedly weakened the therapeutic effect of ADSC-sEVs on LPS-induced PMVECs barrier damage and acute lung injury. In addition, autophagy inhibition, prohibited the expression of five specific miRNAs in ADSC-sEVs -under LPS-induced inflammatory conditions. Our results indicate that ADSC-sEVs protect against LPS-induced pulmonary microvascular barrier damage and acute lung injury. Autophagy is a positive mediator of sEVs function, at least in part through controlling the expression of bioactive miRNAs in sEVs.

Background: Our previous study showed that circular RNA-gamma-secretase-activating protein (circGSAP) was down-regulated in pulmonary microvascular endothelial cells (PMECs) in response to hypoxia, and regulated the cell cycle of PMECs via miR-942-5p sponge in pulmonary hypertension (PH). However, the mechanism whether circGSAP affects the dysfunction of PEMCs through other microRNAs (miRNAs) remains largely unknown. Therefore, we aimed to demonstrate the underlying mechanisms of circGSAP regulating PMECs dysfunction by absorbing other miRNAs to regulate target genes in idiopathic pulmonary arterial hypertension (IPAH).Methods: Quantitative real-time polymerase chain reaction, immunofluorescence staining, Cell Counting Kit-8, Calcein-AM/PI staining, Transwell assay, dual-luciferase reporter assay, and ELISA were used to elucidate the roles of circGSAP.Results: Here we showed that plasma circGSAP levels were significantly decreased in patients with IPAH and associated with poor outcomes. In vivo, circGSAP overexpression improved survival, and alleviated pulmonary vascular remodeling of monocrotaline-induced PH (MCT-PH) rats. In vitro, circGSAP overexpression inhibited hypoxia-induced PMECs proliferation, migration and increased mortality by absorbing miR-27a-3p. BMPR2 was identified as a miR-27a-3p target gene. BMPR2 silencing ameliorated the effect of the miR-27a-3p inhibitor on PMECs proliferation,migration and mortality. The levels of BMPR2 were upregulated in circGSAP-overexpressed PMECs and lung tissues of MCT-PH rats.Conclusion: Our findings demonstrated that circGSAP alleviated the dysfunction of PMECs via the increase of BMPR2 by competitively binding with miR-27a-3p, and mitigated pulmonary vascular remodeling of MCT-PH rats, providing potential therapeutic strategies for IPAH. [ABSTRACT FROM AUTHOR]

Objective To study the change of serum bone morphogenetic protein (BMP)-2 and BMP-4 during the development of sepsis and its mechanisms. Methods Blood was collected for quantifying serum BMP-2 and BMP-4 from patients with sepsis (sepsis group), patients without sepsis (non-sepsis group) in 24 hours of ICU admission and from healthy volunteers (control group). Concentration of serum BMP-2 and BMP-4 was determined by ELISA. The expression of BMP-2, pSmad1/5, and BMPRⅡ of lip polysaccharide (LPS)-treated rat pulmonary microvascular endothelial cells (RPMECs) was detected by Western blot. Results Serum BMP-2 and BMP-4 were significantly increased in sepsis group (P＜0.05) and the level of BMP-2 was significantly higher in sepsis group than that in non-sepsis group (P＜0.05). BMP-2 was found to be a marker for differential diagnosis of separating sepsis LPS may significantly increase the expression of BMP-2 of RPMECs but decrease the expression of phosphorylated Smad1/5 (pSmad1/5) and BMP receptor Ⅱ (BMPRⅡ) (P＜0.05). Conclusions BMP-2 is a potential diagnostic marker and act as apro-inflammatory factor in early phase of sepsis.

Abstract Background Inflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated. Methods LPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer’s instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65. Results Fasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB. Conclusions The present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway.

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. [Display omitted] • VOCs exposure from indoor decoration caused pulmonary fibrosis in mice. • The heterogeneity of fibroblasts after VOCs exposure were revealed. • The ex vivo biosensor assay could observe the mixed effects of VOCs exposure. • Gas6-Axl ligand-receptor pair facilitated cross-talk between MPVECs and fibroblasts. • Atf3 as the key gene negatively regulated VOCs induced-PANoptosis in MPVECs. [ABSTRACT FROM AUTHOR]

Objective: Acute 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.Methods: Mice 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.Results: H19 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.Conclusion: LncRNA 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. [ABSTRACT FROM AUTHOR]

Background We recently demonstrated that circGSAP was diminished in lung tissues from patients with pulmonary arterial hypertension and in hypoxia-induced pulmonary microvascular endothelial cells (PMECs). However, the underlying role of circGSAP in PMECs remains unknown. The study aimed to investigate the contribution of circGSAP to proliferation, apoptosis and cell cycle of PMECs in hypoxic environment and explore the mechanism.Methods The expression of circGSAP was quantified by real-time PCR or immunofluorescence in human lung tissue and PMECs. CircGSAP plasmid, circGSAP small interfering RNA (siRNA), miRNA inhibitor and target gene siRNA were synthesized to verify the role of circGSAP on regulating the proliferation, apoptosis, and cell cycle of PMECs.Results CircGSAP levels were decreased in lungs and plasma of patients with pulmonary hypertension second to chronic obstructive pulmonary disease (COPD-PH) and were associated with poor outcomes of COPD-PH patients. Upregulation of circGSAP inhibited proliferation, apoptosis resistance and G1/S transition of PMECs. Dual luciferase reporter assays showed that circGSAP acted as a competitive endogenous RNA regulating miR-942-5p, and identified SMAD4 as a target gene of miR-942-5p, Then, we verified the functions of miR-942-5p and SMAD4 in PMECs. In addition, the effect of circGSAP siRNA on PMECs was mitigated by transfection of miR-942-5p inhibitor, and the effect of miR-942-5p inhibitor on PMECs was inhibited by SMAD4 siRNA.Conclusion Our findings demonstrated that diminished circGSAP accelerated cell cycle to facilitate cell proliferation and apoptosis resistance through competitively binding miR-942-5p to modulate SMAD4 expressions in hypoxia-induced PMECs, indicating potential therapeutic strategies for PH.

Small extracellular vesicles (sEVs) have been recognized to be more effective than direct stem cell differentiation into functional target cells in preventing tissue injury and promoting tissue repair. Our previous study demonstrated the protective effect of adipose-derived stem cells (ADSCs) on lipopolysaccharide (LPS)-induced acute lung injury and the effect of autophagy on ADSC functions, but the role of ADSC-derived sEVs (ADSC-sEVs) and autophagy-mediated regulation of ADSC-sEVs in LPS-induced pulmonary microvascular barrier damage remains unclear. After treatment with sEVs from ADSCs with or without autophagy inhibition, LPS-induced human pulmonary microvascular endothelial cell (HPMVECs) barrier damage was detected. LPS-induced acute lung injury in mice was assessed in vivo after intravenous administration of sEVs from ADSCs with or without autophagy inhibition. The effects of autophagy on the bioactive miRNA components of ADSC-sEVs were assessed after prior inhibition of cell autophagy. We found that ADSC-sEV effectively alleviated LPS-induced apoptosis, tight junction damage and high permeability of PMVECs. Moreover, in vivo administration of ADSC-sEV markedly inhibited LPS-triggered lung injury. However, autophagy inhibition, markedly weakened the therapeutic effect of ADSC-sEVs on LPS-induced PMVECs barrier damage and acute lung injury. In addition, autophagy inhibition, prohibited the expression of five specific miRNAs in ADSC-sEVs -under LPS-induced inflammatory conditions. Our results indicate that ADSC-sEVs protect against LPS-induced pulmonary microvascular barrier damage and acute lung injury. Autophagy is a positive mediator of sEVs function, at least in part through controlling the expression of bioactive miRNAs in sEVs. [ABSTRACT FROM AUTHOR]

Background: Inflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated. Methods: LPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer's instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65. Results: Fasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB. Conclusions: The present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Pulmonary microvascular endothelial cells (PMECs) and the extracellular vesicles (EVs) derived from PMECs participate in maintaining pulmonary homeostasis and mediating the inflammatory response. However, obtaining a high-purity population of PMECs and their EVs from mouse is still notoriously difficult. Herein we provide a method to isolate primary mouse PMECs (pMPMECs) and to transduce SV40 lentivirus into pMPMECs to establish an immortalized cell line (iMPMECs), which provides sufficient quantities of EVs for further studies. pMPMECs and iMPMECs can be identified using morphologic criteria, a phenotypic expression profile (e. g., CD31, CD144, G. simplicifolia lectin binding), and functional properties (e. g., Dil-acetylated low-density protein uptake, Matrigel angiogenesis). Furthermore, pMPMEC–EVs and iMPMEC–EVs can be identified and compared. The characteristics of pMPMEC–EVs and iMPMEC–EVs are ascertained by transmission electron microscopy, nanoparticle tracking analysis, and specific protein markers. iMPMECs produce far more EVs than pMPMECs, while their particle size distribution is similar. Our detailed protocol to isolate and immortalize MPMECs will provide researchers with an in vitro model to investigate the specific roles of EVs in pulmonary physiology and diseases. [ABSTRACT FROM AUTHOR]

Pulmonary microvascular endothelial cells (PMECs) and the extracellular vesicles (EVs) derived from PMECs participate in maintaining pulmonary homeostasis and mediating the inflammatory response. However, obtaining a high-purity population of PMECs and their EVs from mouse is still notoriously difficult. Herein we provide a method to isolate primary mouse PMECs (pMPMECs) and to transduce SV40 lentivirus into pMPMECs to establish an immortalized cell line (iMPMECs), which provides sufficient quantities of EVs for further studies. pMPMECs and iMPMECs can be identified using morphologic criteria, a phenotypic expression profile (e.g., CD31, CD144, G. simplicifolia lectin binding), and functional properties (e.g., Dil-acetylated low-density protein uptake, Matrigel angiogenesis). Furthermore, pMPMEC–EVs and iMPMEC–EVs can be identified and compared. The characteristics of pMPMEC–EVs and iMPMEC–EVs are ascertained by transmission electron microscopy, nanoparticle tracking analysis, and specific protein markers. iMPMECs produce far more EVs than pMPMECs, while their particle size distribution is similar. Our detailed protocol to isolate and immortalize MPMECs will provide researchers with an in vitro model to investigate the specific roles of EVs in pulmonary physiology and diseases.

Previously, our study has demonstrated that porcine pulmonary microvascular endothelial cells (PPMVECs) were susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and produced a significant non-specific immune response to it. The significance of microvascular endothelial glycocalyx is increasingly attracting attention, and its rich carbohydrate components are not only important signaling molecules, but also remarkably influence the signaling of most proteins. Comprehending changes in the carbohydrate chains contributes to understanding cell functions. This study aimed to reveal the effects of HP-PRRSV infection on the surface carbohydrate chains of PPMVECs. PPMVECs were isolated and cultured in vitro and infected with HP-PRRSV HN and JXA1 strains. Scanning electron microscopy analysis indicated that at 48 h post-infection, some broken holes were in their cell membranes, and that the surface fibrous glycocalyx was obviously reduced or even disappeared. Lectin microarray analysis indicated that the fluorescence intensities of 8 and 7 lectin sites were significantly changed by the HP-PRRSV HN and JXA1 strains, respectively, among which there were 6 common lectin sites. The up-regulation of common lectins (RCA-I, LEL, and STL) and the down-regulation of common lectins (LCA, DSA, and PHA-E) were confirmed by lectin fluorescence staining and lectin flow cytometry, respectively. Together, the results show that the HP-PRRSV infection can induce the glycocalyx disruption of PPMVECs and their surface glycoprofiling changes, and that the poly-N-acetyllactosamine and complex N-glycan are the main up-regulated and down-regulated carbohydrate chains, respectively. Our findings may provide insights into revealing the pathogenesis of HP-PRRSV from the perspective of glycobiology.

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., Competing Interests: The authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors declare that they have no competing interests, financial or otherwise, related to the current work. All authors report that since the initial planning of the work they received grants from the National Natural Science Foundation of China (Grant numbers: 82270045 and 81970044) and payments were made to P. Chen., (© 2024 Cheng W. et al.)

Capillary endothelial cells possess a specialized metabolism necessary to adapt to the unique alveolar-capillary environment. Here, we highlight how endothelial metabolism preserves the integrity of the pulmonary circulation by controlling vascular permeability, defending against oxidative stress, facilitating rapid migration and angiogenesis in response to injury, and regulating the epigenetic landscape of endothelial cells. Recent reports on single-cell RNA-sequencing reveal subpopulations of pulmonary capillary endothelial cells with distinctive reparative capacities, which potentially offer new insight into their metabolic signature. Lastly, we discuss broad implications of pulmonary vascular metabolism on acute respiratory distress syndrome, touching on emerging findings of endotheliitis in coronavirus disease 2019 (COVID-19) lungs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: The dysfunction of pulmonary microvascular endothelial cells (PMVECs) is one of the critical characteristics of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) induced by severe infection. PIM1 is a constitutively active serine/threonine kinase that is involved in multiple biological processes. However, the underlying correlation between PIM1 and PMVECs injury remains unclear. The main purpose of this study was to reveal roles of PIM1 and explore the potential mechanisms during the development of endotoxin-induced ALI induced by intraperitoneal LPS administration. Materials and methods: PIM1 level in the lung tissues of endotoxin-induced ALI mice or plasma derived from cardiopulmonary bypass (CPB)-induced ALI patients were measured. The protective roles of PIM1 specific inhibitor SMI-4a on endotoxin-induced lung injuries were evaluated through histological, permeability, neutrophil infiltration and survival assessment. The relationship between PIM1 and ELK3/ICAM-1 axis was validated in vivo and vitro. The correlation between plasma PIM1 and indicative vascular endothelium injury biomarkers (PaO2/FiO2 ratio, Ang-II, E-selectin and PAI-1) levels derived from CPB-induced ALI patient were analyzed. Results: PIM1 expression in the lung tissues was increased in the mice of endotoxin-induced ALI. The PIM1 specific inhibitor SMI-4a administration relieved the severity of endotoxin-induced ALI. More importantly, PIM1 modulates ICAM1 expression through regulating transcription factor ELK3 expression in vitro. Eventually, plasma PIM1 level was positively correlated with Ang-II and PAI-1 levels but negatively correlated with SpO2/FiO2 ratio among CPB induced ALI patients. Conclusion: Our results indicated that PIM1 inhibition carried a protective role against endotoxin-induced ALI by modulating the ELK3/ICAM1 axis on PMVECs. PIM1 may be a potential therapeutic target for endotoxin-induced ALI. [ABSTRACT FROM AUTHOR]

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.

Objective To investigate the effect of pulmonary microvascular endothelial cells (PMVECs) induced by interleron-γ (IFN-γ) on the proliferation of co-cultured T lymphocytes, and to explore its possible mechanism. Methods The lung tissues of female Balb/C mice were isolated, and mouse PMVECs were cultivated by tissue block method. After the identification of inverted microscope, transmission electron microscope observation and endothelial cell surface marker CD31 fluorescence detection identification, PMVECs with high purity were coufirmed. PMVECs were added to the lower chamber of the Transwell plate and then were randomly divided into five groups: the control group, 10 ng/mL group, 20 ng/mL group, 50 ng/mL group, and 80 ng/mL group, which were added with 0, 10, 20, 50, and 80 ng/mL IFN-γ, respectively. T-lymphocytes were added to the upper chamber of the transwell plate for co-culture, and flow cytometry was used to detect the T-lymphocyte proliferation index (PI) of the upper chamber. PMVECs of the third generation were randomly divided into two groups: the observation group and control group, which were added with 80 ng/mL IFN-γ and an equal amount of PBS, respectively; reverse-phase high-performance liquid chromatography was used to detect tryptophan, kynurenine and indoleamine 2, 3-dioxygenase (IDO) activity in the cell culture supernatant. The expression levels of IDO mRNA and protein were detected by RT-PCR and Western blotting, respectively. Results The T-lymphocyte PI of the control group, 10 ngl mL group, 20 ngmL group, 50 ngmL group, and 80 nglmL group were 3. 06 土0. 07, 2. 93 土 0. 09, 2. 46 士0. 28, 2. 21 士0. 41, and 2. 20 土0. 05, respectively; T-lymphocyte PI of the 20 nglmL group, 50 ng/mL group, and 80 ng/mL group was lower than that of control group (all P < 0. 01), but there was no significant difference in T lymphocyte Pl among 20 ng/mL group, 50 ngmL group, and 80 ng/mL group (all P > 0. 05). The observation group had a significantly lower tryptophan level in the cell culture supernatant than the control group, and the kynurenine level and !DO activity were higher than those of the control group (all P <0. 01). The relative expression levels of !DO mRNA in the observation group and the control group were 0. 68 土0. 02 and O, and the relative expression levels of !DO protein were 0.49 士0. 02 and O, respectively, with significant difference between every two groups (all P < 0. 01). Conclusion PMVECs treated with IFN-γ can inhibit the proliferation of co-cultured T lymphocytes by promoting tryptophan degradation, kynurenine accumulation, and !DO expression. [ABSTRACT FROM AUTHOR]

Lung ischemia-reperfusion (I/R) injury severely endangers human health, and recent studies have suggested that certain microRNAs (miRNAs) play important roles in this pathological phenomenon. The current study aimed to ascertain the ability of miR-223 to influence lung I/R injury by targeting hypoxia-inducible factor-2α (HIF2β). First, mouse models of lung I/R injury were established: during surgical procedures, pulmonary arteries and veins and unilateral pulmonary portal vessels were blocked and resuming bilateral pulmonary ventilation, followed by restoration of bipulmonary ventilation. In addition, a lung I/R injury cell model was constructed by exposure to hypoxic reoxygenation (H/R) in mouse pulmonary microvascular endothelial cells (PMVECs). Expression of miR-223, HIF2α, and β-catenin in tissues or cells was determined by RT-qPCR and Western blot analysis. Correlation between miR-223 and HIF2α was analyzed by dual luciferase reporter gene assay. Furthermore, lung tissue injury and mouse PMVEC apoptosis was evaluated by hematoxylin and eosin (H&E), TUNEL staining, and flow cytometry. Autophagosomes in cells were detected by light chain 3 immunofluorescence assay. miR-223 was expressed at a high level while HIF2α/β-catenin was downregulated in tissues and cells with lung I/R injury. Furthermore, miR-223 targeted and repressed HIF2α expression to downregulate β-catenin expression. The miR-223/HIF2α/β-catenin axis aggravated H/R injury in mouse PMVECs and lung I/R injury in mice by enhancing autophagy. Taken together, miR-223 inhibits HIF2α to repress β-catenin, thus contributing to autophagy to complicate lung I/R injury. These findings provide a promising therapeutic target for treating lung I/R injury. [ABSTRACT FROM AUTHOR]

The balance between Ang II/AT1R and Ang‐(1‐7)/Mas plays a pivotal role in the development of lipopolysaccharides (LPS)‐induced acute respiratory distress syndrome. However, the mechanisms underlying the balancing process still remain unclear. Here we investigated the roles of nuclear factor (NF)‐κB and p53 in regulating AT1R and Mas expression. The results demonstrated that Ang II pretreatment resulted in downregulation of Mas and upregulation of AT1R, phosphorylated p65, and apoptosis in LPS‐treated Human pulmonary microvascular endothelial cells (HPMVECs), but had no effect on p53 expression. Lentiviral vector‐mediated P65 knockdown, but not a P53 knockdown, reversed all these effects of Ang II. On the other hand, Ang‐(1‐7) pretreatment lead to an increased in Mas expression and a decrease in AT1R, p53, and phosphorylated p65 expressions with suppressed apoptosis in LPS‐treated cells. P65 knockdown promoted the protein expression of both AT1R and Mas while inhibiting p53 expression. P53 knockdown, but not a p65 knockdown, reversed all these effects of Ang‐(1‐7). Interestingly, p65 overexpression upregulated p53 and AT1R but downregulated Mas. P53 knockdown activated p65. These results suggest that there is a two‐way feedback regulation between AT1R and Mas receptor via the NF‐kB p65/P53 pathway, which may play a key role in LPS‐induced HPMVECs apoptosis. [ABSTRACT FROM AUTHOR]

Impaired mitochondrial function is a key factor attributing to lung ischaemia‐reperfusion (IR) injury, which contributes to major post‐transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3‐MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia‐reoxygenation (H/R) were monitored after 3‐MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3‐MA inhibited cell apoptosis of donor lung to alleviate I/R‐induced lung injury as well as inhibited H/R‐induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R‐ and H/R‐mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R‐induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury. [ABSTRACT FROM AUTHOR]

Abstract The present study sought to determine the pharmacological effects of penehyclidine, an anticholinergic agent, on the angiogenic capacity of pulmonary microvascular endothelial cells (PMVECs). In vitro Matrigel network formation assay, cell proliferation assay, cell-matrix adhesion assay, and wound-healing assay were performed in PMVECs with or without exposure to penehyclidine or, in some cases, glycopyrrolate or acetylcholine, over a concentration range. In addition, the phosphorylation state of Akt and ERK, as well as the endogenous level of mTOR and RICTOR were examined in PMVECs by Western blot following the cells exposure to penehyclidine or, for some proteins, glycopyrrolate or acetylcholine. Finally, Western blot for Akt phosphorylation and in vitro Matrigel network formation assay were performed in PMVECs following their exposure to penehyclidine with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or mTOR inhibitor torin-1. We found that, in PMVECs, penehyclidine affected the network formation and cell migration, but not proliferation or cell-matrix adhesion, in a concentration-specific manner, i.e., penehyclidine increased the network formation and cell migration at lower concentrations but increased these processes at higher concentrations. Coincidentally, we observed that penehyclidine concentration-specifically affected the phosphorylation state of Akt in PMVECs, i.e., increased Akt phosphorylation at lower concentrations and decreased it at higher concentrations. In contrast, glycopyrrolate was found straightly to decrease network formation and Akt phosphorylation in a concentration-dependent manner. Further, we demonstrated that PI3K or mTOR blockade abolished both the enhanced network formation and the increased Akt phosphorylation by penehyclidine. Hence, penehyclidine may differentially alter the angiogenic capacity of PMVECs through affecting the Akt signaling pathway downstream of PI3K and mTOR. Findings from this study suggest a unique pharmacological feature of penehyclidine, which may imply its clinical and therapeutic value in modulating angiogenesis. [ABSTRACT FROM AUTHOR]

Angiopoietin-2 (Ang-2) is a Tie-2 ligand that destabilizes vascular structures, enhances vascular permeability and induces vascular regression and endothelial cell apoptosis. Although there is evidence for the involvement of the Ang/Tie2 axis in acute lung injury (ALI), the underlying mechanisms involved in Ang-2-induced cell apoptosis are not well understood. In this study, whether Ang-2 contributes to microvascular endothelial cell injury and mediates lipopolysaccharide (LPS)-induced endothelial cell apoptosis and its associated signaling pathways was investigated. Exposure of rat pulmonary microvascular endothelial cells (RPMVECs) to LPS, Ang-2 and related inhibitors was performed to measure the expression levels of Ang-2, the activation of mitogen-activated protein kinases (MAPKs), the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, and expression of the apoptosis-related proteins Bax and Bcl-2 using western blotting, reverse transcription-quantitative polymerase chain reaction, flow cytometry and fluorescence microscopy. The expression of Ang-2 in the RPMVECs was increased by LPS independent of time. The phosphorylation of p38 MAPK and ERK1/2 was significantly upregulated and the activation of apoptosis-related proteins Bax and Bcl was mediated by Ang-2. In addition, inhibition of the p38 pathway by SB203580 attenuated the Ang-2-mediated cell apoptosis, but inhibition of the ERK1/2 pathway by PD98059 exerted an anti-apoptotic effect against Ang-2. In conclusion, LPS-induced apoptosis is partly mediated via stimulation of p38 and ERK1/2 signaling pathways, where Ang-2 acts an inflammation-related factor to participate in the course of cell apoptosis in RPMVECs. [ABSTRACT FROM AUTHOR]

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI. [ABSTRACT FROM AUTHOR]

Objective: It has been documented that ezrin/radixin/moesin (ERM) phosphorylation by the p38 mitogen-activated protein kinase (MAPK), Rho/ROCK, and protein kinase C (PKC) pathways leads to filamentous actin (F-actin) reorganization and microvascular endothelial cell hyperpermeability. In this study, we investigated the effects of Xijiao Dihuang Decoction combined with Yinqiao Powder (XDY) on influenza virus (IV)-induced F-actin restructuring and ERM phosphorylation regulated by the Rho/Rho kinase 1 (ROCK), p38 MAPK, and PKC signaling pathways in pulmonary microvascular endothelial cells (PMVECs). Methods: Serum containing XDY (XDY-CS; 13.8 g/kg) was acquired using standard protocols for serum pharmacology. Primary PMVECs were obtained from male Wistar rats and cultured. After adsorption of IV A (multiplicity of infection, 0.01) for 1 h, medium with 20% XDY-CS was added to the PMVECs. The distributions of F-actin and phosphorylated ERM were determined by confocal microscopy, and F-actin expression was measured by flow cytometry. The expression levels of ROCK1, phosphorylated myosin phosphatase target-subunit (p-MYPT), phosphorylated MAPK kinase, phosphorylated p38 (p-p38), phosphorylated PKC (p-PKC), and phosphorylated ERM (p-ERM) were determined by western blotting. Results: F-actin reorganization in IV-infected PMVECs was reversed by XDY-CS treatment, which was accompanied by reduced p-ERM production. The p-ERM protein accumulated at plasma membrane of PMVECs infected with IV, which was also inhibited by XDY-CS treatment. In addition, XDY-CS treatment drastically reduced the levels of p-p38, ROCK1, p-MYPT, and p-PKC induced by IV infection in PMVECs. Conclusion: These results show that XDY-CS inhibited influenza-induced F-actin reorganization in PMVECs by down-regulating p-ERM expression via inhibition of the Rho/ROCK, p38 MAPK, and PKC pathways. In conclusion, XDY could reduce the damage to endothelial cytoskeleton induced by IV infection, thus protecting the barriers of PMVECs.

Background/Aims: Pulmonary microvascular endothelial cell (PMVEC) proliferation and angiogenesis contribute to the development of hepatopulmonary syndrome (HPS). MicroRNA-199a-5p (miR-199a-5p) has emerged as a potent regulator of angiogenesis, and its expression levels significantly decrease in the serum of patients with hepatopathy. However, it has not been reported about whether miR-199a-5p might control PMVEC proliferation. Here, we described the miR-199a-5p governing PMVEC proliferation in HPS. Methods: PMVECs were treated with rat serum from common bile duct ligation (CBDL) or sham. MiR-199a-5p mimic or inhibitor was used to change the miR-199a-5p expression. Knockdown of caveolin-1 (Cav-1) was performed using siRNA. NSC-23766 was used to inhibit Rac1 activity. Gene and protein expressions were quantified by qRT-PCR and western blot. Cell proliferation was analyzed by 3H-TdR incorporation and CCK-8 assays. Stress fibers were detected by immunofluorescence. Results: CBDL rat serum induced the down-regulation of miR-199a-5p. Delivery of miR-199a-5p suppressed the CBDL rat serum-induced PMVEC proliferation whereas knockdown of miR-199a-5p promoted PMVEC proliferation. This was accompanied by a decrease and an increase in Cav-1 expression, respectively. Cav-1 siRNA abolished the enhancement of PMVEC proliferation induced by the miR-199a-5p inhibition. Although stress fibers were disrupted in Cav-1 deficient cells, NSC-23766 increased stress fibers and contributed to cell proliferation. Conclusions: CBDL rat serum induced down-regulation of miR-199a-5p in PMVECs, which led to an increase of Cav-1 gene expression. Increased Cav-1 expression, by inhibiting Rac1 activity, led to the formation of stress fibers, which contribute to PMVEC proliferation and thus the pathogenesis of HPS.

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., Competing Interests: The authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. The authors declare that they have no competing interests, financial or otherwise, related to the current work. P. Chen reports that since the initial planning of the work he received support from the National Natural Science Foundation of China (Grant numbers: 82270045)., (© 2023 Lin L. et al.)

Endothelial cell myofibroblast transition (EndoMT) is found during the process of bleomycin (BLM)-induced pulmonary fibrosis in rats, and plays a very important role in sustaining inflammation and collagen secretion. Moreover, some studies have suggested that the Notch1 signaling pathway may be involved in the expression of α-smooth muscle actin (α-SMA) in pulmonary microvascular endothelial cells (PMVECs), a protein marker of EndoMT. Therefore, we aimed to investigate the expression level of α-SMA and Notch1-related signaling molecules in PMVECs from BLM-induced rats and determine the relationship between the Notch1 signaling pathway and the expression of α-SMA in PMVECs. We found that the expression levels of α-SMA, Notch1, and Jagged1 were upregulated, while the expression levels of Dll4 were downregulated. Furthermore, there was a positive correlation between the expression of Jagged1 and the α-SMA proteins in PMVECs, and NF-κB was downregulated by decreasing the expression of Jagged1. In conclusion, the Jagged1/Notch1 signaling pathway is activated in PMVECs during the pathogenesis of BLM-induced pulmonary fibrosis in rats, and it may induce α-SMA expression via a non-canonical pathway involving NF-κB as the target molecule. The precise mechanism and the molecules involved in this signaling pathway need to be further elucidated. [ABSTRACT FROM AUTHOR]

The inhibition of mitogen-activated protein kinases (MAPKs), including c-Jun NH2-terminal protein kinase (JNK), p38 MAPK (p38) and extracellular signal-regulated protein kinase 1/2 (ERK1/2), have an important effect on lung ischemia-reperfusion injury (IRI) during lung transplantation (LT). However, the way in which combined MAPK inhibition exerts optimal protective effects on lung IRI remains to be elucidated. Therefore, the present study evaluated the therapeutic efficacy of the inhibition of MAPKs in rat pulmonary microvascular endothelial cells (PMVECs) in an IRI model of LT. The rat PMVECs were transfected with small interfering RNAs (siRNAs) against JNK, p38 or ERK1/2. Cotransfection was performed with siRNAs against JNK and p38 in the J+p group, JNK and ERK1/2 in the J+E group, p38 and ERK1/2 in the p+E group, or all three in the J+p+E group. Non-targeting (NT) siRNA was used as a control. The PMVECs were then treated to induce IRI, and the levels of inflammation, apoptosis and oxidative stress were detected. Differences between compared groups were determined using Tukey's honest significant difference test. In all groups, silencing of the MAPKs was shown to attenuate inflammation, apoptosis and oxidative stress to differing extents, compared with the NT group. The J+p and J+p+E groups showed lower levels of interleukin (IL)-1ß, IL-6 and malondialdehyde, a lower percentage of early-apoptotic cells, and higher superoxide dismutase (SOD) activity, compared with the other groups. No significant differences were observed in the inflammatory response, SOD activity or early apoptosis between the J+p and J+p+E groups. These findings suggested that the dual inhibition of JNK and p38 led to maximal amelioration of lung IRI in the PMVECs of the IRI model of LT, which occurred through anti-inflammatory, anti-oxidative and anti-apoptotic mechanisms. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. ALI and ARDS result are associated with high mortality in patients. At present, no effective treatments for ALI and ARDS exist. It is established that vitamin D exhibits anti‑inflammatory effects, however, the specific effect of vitamin D on ALI remains largely unknown. The aim of the present study was to investigate whether, and by which mechanism, vitamin D alleviates lipopolysaccharide (LPS)‑induced ALI. The results demonstrated that a vitamin D agonist, calcitriol, exhibited a beneficial effect on LPS‑induced ALI in rats; calcitriol pretreatment significantly improved LPS‑induced lung permeability, as determined using Evans blue dye. Results from reverse transcription‑quantitative polymerase chain reaction, western blotting and ELISA analysis demonstrated that calcitriol also modulated the expression of members of the renin‑angiotensin system (RAS), including angiotensin (Ang) I‑converting enzymes (ACE and ACE2), renin and Ang II, which indicates that calcitriol may exert protective effects on LPS‑induced lung injury, at least partially, by regulating the balance between the expression of members of the RAS. The results of the present study may provide novel targets for the future treatment of ALI. [ABSTRACT FROM AUTHOR]

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-1β (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-1β, 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-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI. • LPS results in the early production of large amounts of ceramides through the ASMase pathway (rather than the NSMase pathway or de novo synthesis pathway), and leads to pro-inflammatory cytokines secretion, PMVECs hyperpermeability and lung injury in rats. • Ceramide-induced upregulation of Txnip expression activates the NLRP3 inflammasome and plays a central role in pro-inflammatory cytokines secretion, PMVECs hyperpermeability and lung injury in rats. • Ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages leads to early IL-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of mycophenolic acid (MPA) on the proliferation of rat pulmonary microvascular endothelial cells (PMVECs) and on the secretion of endothelin-1 (ET-1) and interleukin-6 (IL-6) by these cells. Methods: Rat PMVECs were treated with five different final concentrations of MPA (0, 0.1, 1, 10 and 100 μM). The fetal bovine serum-induced proliferation of the PMVECs was detected using the Cell Counting Kit-8 (CCK-8). The levels of ET-1 and IL-6 secretion were assessed by radioimmunoassays. Results: At the 24 h time point, the optical density (OD) values at 450 nm for the five groups showed that there were no significant differences between the groups treated with 0 and 0.1 μM MPA (P=0.388) or between the groups treated with 10 and 100 μM MPA (P=0.292), but the OD values were significantly different between all other pairs of groups (PP=0.094), but the OD values were significantly different between all the other groups (PP=0.931), but the OD values were significantly different between all other pairs of groups (10 μM MPA vs 100 μM MPA: P=0.037; 0 μM MPA vs 1 μM MPA: P=0.006; 1 μM MPA vs 10 μM MPA: P=0.005; all other comparisons: PP=0.156) or the groups treated with 10 and 100 μM MPA (P=0.262), but there were significant differences between all other pairs of groups (PP>0.05). Conclusions: MPA inhibits fetal bovine serum-induced rat PMVEC proliferation and ET-1 secretion in a dose-dependent manner.

Human invasive pulmonary aspergillosis (IPA) is a serious infectious disease mainly caused by Aspergillus fumigatus ( A. fumigatus ). Pulmonary microvascular endothelial cells (PMVECs) are important ones in the human lung tissue. However, it remains unclear about the role of PMVECs in IPA. In the present study, we cocultured PMVECs with A. fumigatus . We observed that A. fumigatus induced dose- and time-dependent increases of interleukin 6 (IL-6), interleukin 1β (IL-1β) and intercellular adhesion molecule 1 (ICAM-1) concentration in the cultures. Significant increases in IL-6, IL-1β, E-selectin, and ICAM-1 mRNA expression were also observed in the cultures treated with A. fumigatus . While preincubation with SB203580 (10 μM) did not cause significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures, significant IL-6, IL-1β and ICAM-1 concentration decreases were observed in the cocultures preincubated with SB203580 (20 μM). Neither SP600125 (10-20 μM) nor PD98059 (10-20 μM) caused significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures. PCR results also showed that SB203580 (20 μM) (neither SP600125 (20 μM) nor PD98059 (20 μM)) preincubation significantly decreased IL-6, IL-1β, E-selectin and ICAM-1 mRNA expression in the cocultures. In addition, significant p38 MAPK phosphorylation increase was observed in the PMVECs cultures treated with A. fumigatus . Furthermore, silibinin pre-treatment and post-treatment were observed to significantly down-regulate mRNA and protein expression of proinflammatory factors and adhesion molecules in the cocultures. Finally, we observed that silibinin significantly inhibited A. fumigatus -induced p38 MAPK activation in PMVECs. Our results indicated that PMVECs might participate in IPA early inflammation which is mediated by p38 MAPK. Silibinin may inhibit A. fumigatus -induced inflammation in PMVECs through p38 MAPK. [ABSTRACT FROM AUTHOR]

Integrin αvβ5 mediates pulmonary endothelial barrier function and acute lung injury (LI), but its roles in cell apoptosis and autophagy are unclear. Thus, the aims of this study were to investigate the significance of αvβ5 in ischemia-reperfusion (I/R)-induced apoptosis and LI and to explore the relationship between αvβ5 and autophagy. Human pulmonary microvascular endothelial cells (HPMVECs) were pretreated with an αvβ5-blocking antibody (ALULA) and challenged with oxygen-glucose deprivation/oxygen-glucose restoration, which mimics I/R; then, cellular autophagy and apoptosis were detected, and cell permeability was assessed. In vivo, mice were pretreated with the autophagy inhibitor chloroquine (CLQ), followed by treatment with ALULA. The mice then underwent operative lung I/R. LI was assessed by performing a pathological examination, calculating the wet/dry lung weight ratio and detecting the bronchial alveolar lavage fluid (BALF) protein concentration. αvβ5 inhibition promoted HPMVEC autophagy under I/R in vitro, alleviated cell permeability, decreased the apoptosis ratio, and activated caspase-3 expression. These outcomes were significantly diminished when autophagy was inhibited with a small-interfering RNA construct targeting autophagy-related gene 7 (siATG7). Moreover, ALULA pretreatment alleviated I/R-induced LI (I/R-LI), which manifested as a decreased wet/dry lung weight ratio, an altered BALF protein concentration, and lung edema. Preinhibiting autophagy with CLQ, however, eliminated the protective effects of ALULA on I/R-LI. Therefore, inhibiting αvβ5 effectively ameliorated I/R-induced endothelial cell apoptosis and I/R-LI. This process was dependent on improved autophagy and its inhibitory effects on activated caspase-3. [ABSTRACT FROM AUTHOR]

Previous studies have suggested that the Notch signaling pathway plays a very important role in the proliferation and differentiation of pulmonary microvascular endothelial cells (PMVECs). Therefore, we aimed to investigate the expression level of Notch-related signaling molecules in PMVECs in bleomycin (BLM)-induced rat pulmonary fibrosis. Immunohistochemistry, immunofluorescence, Western blotting, and real-time PCR were used to analyze the differences in protein and mRNA expression levels of Notch-related signaling molecules, i.e. Notch1, Jagged1, Delta-like ligand 4 (Dll4), and hairy and enhancer of split homolog 1 (Hes1), between a control group treated with intratracheal instillation of saline and a study group treated with intratracheal instillation of BLM solution. Expression levels of the receptor Notch1 and one of its ligands, Jagged1, were upregulated, while the expression levels of the ligand Dll4 and the target molecule of the Notch signaling pathway, Hes1, were downregulated. The differences in protein and mRNA expression levels between the control and study groups were significant (p<0.001). The Jagged1/Notch1 signaling pathway is activated in the pathogenesis of BLM-induced rat pulmonary fibrosis, while the Dll4/Notch1 signaling pathway is inhibited, which inhibits the suppressive effect of Dll4/Notch1 signaling on PMVEC overproliferation, further causing PMVEC dysfunction in cell sprouting and maturation as well as abnormal differentiation of the cell phenotype. Conversely, the downexpression of Hes1 indicates that the Jagged1/Notch1 signaling pathway could be a non-canonical Notch signaling pathway independent of Hes1 activation, which differs from the canonical Dll4/Notch1 signaling pathway. [ABSTRACT FROM AUTHOR]

We studied the effect of baicalin, an extract from Radix Scutellariae (a traditional Chinese medicine) in inducing mouse pulmonary microvascular endothelial cells (MPMVECs) to produce interferons (IFNs). MPMVECs were cultured in vitro in the presence of different concentrations of baicalin (10, 20, and 30 μg mL−1), and the cells and the culture media were harvested at various time intervals. The proteins and mRNA levels (relative to β-actin) of IFN-α/β and IFN-γ were analyzed by RT-PCR and enzyme-linked immunosorbent assay (ELISA). It was observed that baicalin substantially up-regulated the expression of IFN-α/β and IFN-γ. In all baicalin-treated groups, the relative levels of IFN-α/β and IFN-γ mRNAs peaked after 12 h of culturing, and IFN-α/β and IFN-γ proteins peaked after 24 h of culturing. These results suggest that baicalin can effectively induce the expression of IFNs in pulmonary microvascular endothelial cells, and thus potentially act as an antiviral compound. This study may provide background information for developing new antiviral drugs based on baicalin.