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Introduction: The pulmonary endothelium is the primary target of lung ischemia-reperfusion injury leading to primary graft dysfunction after lung transplantation. We hypothesized that treating damaged rat lungs by a transient heat stress during ex-vivo lung perfusion (EVLP) to elicit a pulmonary heat shock response could protect the endothelium from severe reperfusion injury. Methods: Rat lungs damaged by 1h warm ischemia were reperfused on an EVLP platform for up to 6h at a constant temperature (T°) of 37°C (EVLP37°C group), or following a transient heat stress (HS) at 41.5°C from 1 to 1.5h of EVLP (EVLPHS group). A group of lungs exposed to 1h EVLP only (pre-heating conditions) was added as control (Baseline group). In a first protocol, we measured lung heat sock protein expression (HSP70, HSP27 and Hsc70) at selected time-points (n=5/group at each time). In a second protocol, we determined (n=5/group) lung weight gain (edema), pulmonary compliance, oxygenation capacity, pulmonary artery pressure (PAP) and vascular resistance (PVR), the expression of PECAM-1 (CD31) and phosphorylation status of Src-kinase and VE-cadherin in lung tissue, as well as the release in perfusate of cytokines (TNFα, IL-1β) and endothelial biomarkers (sPECAM, von Willebrand Factor -vWF-, sE-selectin and sICAM-1). Histological and immunofluorescent studies assessed perivascular edema and formation of 3-nitrotyrosine (a marker of peroxinitrite) in CD31 lung endothelium. Results: HS induced an early (3h) and persisting expression of HSP70 and HSP27, without influencing Hsc70. Lungs from the EVLP37°C group developed massive edema, low compliance and oxygenation, elevated PAP and PVR, substantial release of TNFα, IL-1β, s-PECAM, vWF, E-selectin and s-ICAM, as well as significant Src-kinase activation, VE-cadherin phosphorylation, endothelial 3-NT formation and reduced CD31 expression. In marked contrast, all these alterations were either abrogated or significantly attenuated by HS treatment. Conclusion: The therapeutic application of a transient heat stress during EVLP of damaged rat lungs reduces endothelial permeability, attenuates pulmonary vasoconstriction, prevents src-kinase activation and VE-cadherin phosphorylation, while reducing endothelial peroxinitrite generation and the release of cytokines and endothelial biomarkers. Collectively, these data demonstrate that therapeutic heat stress may represent a promising strategy to protect the lung endothelium from severe reperfusion injury. [ABSTRACT FROM AUTHOR]

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia. [ABSTRACT FROM AUTHOR]

IntroductionThe pulmonary endothelium is the primary target of lung ischemia-reperfusion injury leading to primary graft dysfunction after lung transplantation. We hypothesized that treating damaged rat lungs by a transient heat stress during ex-vivo lung perfusion (EVLP) to elicit a pulmonary heat shock response could protect the endothelium from severe reperfusion injury.MethodsRat lungs damaged by 1h warm ischemia were reperfused on an EVLP platform for up to 6h at a constant temperature (T°) of 37°C (EVLP37°C group), or following a transient heat stress (HS) at 41.5°C from 1 to 1.5h of EVLP (EVLPHS group). A group of lungs exposed to 1h EVLP only (pre-heating conditions) was added as control (Baseline group). In a first protocol, we measured lung heat sock protein expression (HSP70, HSP27 and Hsc70) at selected time-points (n=5/group at each time). In a second protocol, we determined (n=5/group) lung weight gain (edema), pulmonary compliance, oxygenation capacity, pulmonary artery pressure (PAP) and vascular resistance (PVR), the expression of PECAM-1 (CD31) and phosphorylation status of Src-kinase and VE-cadherin in lung tissue, as well as the release in perfusate of cytokines (TNFα, IL-1β) and endothelial biomarkers (sPECAM, von Willebrand Factor -vWF-, sE-selectin and sICAM-1). Histological and immunofluorescent studies assessed perivascular edema and formation of 3-nitrotyrosine (a marker of peroxinitrite) in CD31 lung endothelium.ResultsHS induced an early (3h) and persisting expression of HSP70 and HSP27, without influencing Hsc70. Lungs from the EVLP37°C group developed massive edema, low compliance and oxygenation, elevated PAP and PVR, substantial release of TNFα, IL-1β, s-PECAM, vWF, E-selectin and s-ICAM, as well as significant Src-kinase activation, VE-cadherin phosphorylation, endothelial 3-NT formation and reduced CD31 expression. In marked contrast, all these alterations were either abrogated or significantly attenuated by HS treatment.ConclusionThe therapeutic application of a transient heat stress during EVLP of damaged rat lungs reduces endothelial permeability, attenuates pulmonary vasoconstriction, prevents src-kinase activation and VE-cadherin phosphorylation, while reducing endothelial peroxinitrite generation and the release of cytokines and endothelial biomarkers. Collectively, these data demonstrate that therapeutic heat stress may represent a promising strategy to protect the lung endothelium from severe reperfusion injury.

Lung inflammation is an essential inducer of various diseases and is closely related to pulmonary-endothelium dysfunction. Herein, we propose a pulmonary endothelium-targeted codelivery system of anti-inflammatory indomethacin (IND) and antioxidant superoxide dismutase (SOD) by assembling the biopharmaceutical SOD onto the “vector” of rod-like pure IND crystals, followed by coating with anti-ICAM-1 antibody (Ab) for targeting endothelial cells. The codelivery system has a 237 nm diameter in length and extremely high drug loading of 39% IND and 2.3% SOD. Pharmacokinetics and biodistribution studies demonstrate the extended blood circulation and the strong pulmonary accumulation of the system after intravenous injection in the lipopolysaccharide (LPS)-induced inflammatory murine model. Particularly, the system allows a robust capacity to target pulmonary endothelium mostly due to the rod-shape and Ab coating effect. In vitro, the preparation shows the synergistic anti-inflammatory and antioxidant effects in LPS-activated endothelial cells. In vivo, the preparation exhibits superior pharmacodynamic efficacy revealed by significantly downregulating the inflammatory/oxidative stress markers, such as TNF-α, IL-6, COX-2, and reactive oxygen species (ROS), in the lungs. In conclusion, the codelivery system based on rod-like pure crystals could well target the pulmonary endothelium and effectively alleviate lung inflammation. The study offers a promising approach to combat pulmonary endothelium-associated diseases.

Lung inflammation is an essential inducer of various diseases and is closely related to pulmonary-endothelium dysfunction. Herein, we propose a pulmonary endothelium-targeted codelivery system of anti-inflammatory indomethacin (IND) and antioxidant superoxide dismutase (SOD) by assembling the biopharmaceutical SOD onto the "vector" of rod-like pure IND crystals, followed by coating with anti-ICAM-1 antibody (Ab) for targeting endothelial cells. The codelivery system has a 237 nm diameter in length and extremely high drug loading of 39% IND and 2.3% SOD. Pharmacokinetics and biodistribution studies demonstrate the extended blood circulation and the strong pulmonary accumulation of the system after intravenous injection in the lipopolysaccharide (LPS)-induced inflammatory murine model. Particularly, the system allows a robust capacity to target pulmonary endothelium mostly due to the rod-shape and Ab coating effect. In vitro , the preparation shows the synergistic anti-inflammatory and antioxidant effects in LPS-activated endothelial cells. In vivo , the preparation exhibits superior pharmacodynamic efficacy revealed by significantly downregulating the inflammatory/oxidative stress markers, such as TNF- α , IL-6, COX-2, and reactive oxygen species (ROS), in the lungs. In conclusion, the codelivery system based on rod-like pure crystals could well target the pulmonary endothelium and effectively alleviate lung inflammation. The study offers a promising approach to combat pulmonary endothelium-associated diseases. The codelivery system Ab-INRplex attaches to the ICAM-1 overexpressed on inflamed endothelial cells, and synergistically relieves the inflammation by inhibiting COX-2 and reactive oxygen species production. [Display omitted] [ABSTRACT FROM AUTHOR]

HIV and Schistosoma infections have been individually associated with pulmonary vascular disease. Co-infection with these pathogens is very common in tropical areas, with an estimate of six million people co-infected worldwide. However, the effects of HIV and Schistosoma co-exposure on the pulmonary vasculature and its impact on the development of pulmonary vascular disease are largely unknown. Here, we have approached these questions by using a non-infectious animal model based on lung embolization of Schistosoma mansoni eggs in HIV-1 transgenic (HIV) mice. Schistosome-exposed HIV mice but not wild-type (Wt) counterparts showed augmented pulmonary arterial pressure associated with markedly suppressed endothelial-dependent vasodilation, increased endothelial remodeling and vessel obliterations, formation of plexiform-like lesions and a higher degree of perivascular fibrosis. In contrast, medial wall muscularization was similarly increased in both types of mice. Moreover, HIV mice displayed an impaired immune response to parasite eggs in the lung, as suggested by decreased pulmonary leukocyte infiltration, small-sized granulomas, and augmented residual egg burden. Notably, vascular changes in co-exposed mice were associated with increased expression of proinflammatory and profibrotic cytokines, including IFN-γ and IL-17A in CD4+ and γδ T cells and IL-13 in myeloid cells. Collectively, our study shows for the first time that combined pulmonary persistence of HIV proteins and Schistosoma eggs, as it may occur in co-infected people, alters the cytokine landscape and targets the vascular endothelium for aggravated pulmonary vascular pathology. Furthermore, it provides an experimental model for the understanding of pulmonary vascular disease associated with HIV and Schistosoma co-morbidity.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is linked to heterozygous mutations in the FOXF1 (Forkhead Box F1) gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed. Objectives: Identify-small molecule compounds that stimulate FOXF1 signaling. Methods: We used mass spectrometry, immunoprecipitation, and the in vitro ubiquitination assay to identify TanFe (transcellular activator of nuclear FOXF1 expression), a smallmolecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and acute lung injury and in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV. Measurements and Main Results: We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells in vitro. TanFe increased protein concentrations of FOXF1 and its target genes Flk1, Flt1, and Cdh5 in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein concentrations in lungs of Foxf11/2 embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of Foxf11/2 mice after birth. TanFe increased angiogenesis in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV with FOXF1 deletion. Conclusions: TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases. [ABSTRACT FROM AUTHOR]

The risk and progression of pulmonary vascular disease in patients with congenital heart disease is dependent on the hemodynamics associated with different lesions. However, the underlying mechanisms are not understood. Endothelin-1 is a potent vasoconstrictor that plays a key role in the pathology of pulmonary vascular disease. We utilized two ovine models of congenital heart disease: (1) fetal aortopulmonary graft placement (shunt), resulting in increased flow and pressure; and (2) fetal ligation of the left pulmonary artery resulting in increased flow and normal pressure to the right lung, to investigate the hypothesis that high pressure and flow, but not flow alone, upregulates endothelin-1 signaling. Lung tissue and pulmonary arterial endothelial cells were harvested from control, shunt, and the right lung of left pulmonary artery lambs at 3-7 weeks of age. We found that lung preproendothelin-1 mRNA and protein expression were increased in shunt lambs compared to controls. Preproendothelin-1 mRNA expression was modestly increased, and protein was unchanged in left pulmonary artery lambs. These changes resulted in increased lung endothelin-1 levels in shunt lambs, while left pulmonary artery levels were similar to controls. Pulmonary arterial endothelial cells exposed to increased shear stress decreased endothelin-1 levels by five-fold, while cyclic stretch increased levels by 1.5-fold. These data suggest that pressure or an additive effect of pressure and flow, rather than increased flow alone, is the principal driver of increased endothelin signaling in congenital heart disease. Defining the molecular drivers of the pathobiology of pulmonary vascular disease due to differing mechanical forces will allow for a more targeted therapeutic approach.

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1β, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated up-regulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 lM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) causes distant organ dysfunction through yet unknown mechanisms, leading to multiorgan failure and death. The lungs are one of the most common extrarenal organs affected by AKI, and combined lung and kidney injury has a mortality as high as 60%-80%. One mechanism that has been implicated in lung injury after AKI involves molecules released from injured kidney cells (DAMPs, or damage-associated molecular patterns) that promote a noninfectious inflammatory response by binding to pattern recognition receptors (PRRs) constitutively expressed on the pulmonary endothelium. To date there are limited data investigating the role of PRRs and DAMPs in the pulmonary endothelial response to AKI. Understanding these mechanisms holds great promise for therapeutics aimed at ameliorating the devastating effects of AKI. In this study, we stimulate primary human microvascular endothelial cells with DAMPs derived from injured primary renal tubular epithelial cells (RTECs) as an ex-vivo model of lung injury following AKI. We show that DAMPs derived from injured RTECs cause activation of Toll-Like Receptor and NOD-Like Receptor signaling pathways as well as increase human primary pulmonary microvascular endothelial cell (HMVEC) cytokine production, cell signaling activation, and permeability. We further show that cytokine production in HMVECs in response to DAMPs derived from RTECs is reduced by the inhibition of NOD1 and NOD2, which may have implications for future therapeutics. This paper adds to our understanding of PRR expression and function in pulmonary HMVECs and provides a foundation for future work aimed at developing therapeutic strategies to prevent lung injury following AKI., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

Among various mRNA carrier systems, lipid nanoparticles (LNPs) stand out as the most clinically advanced. While current clinical trials of mRNA/LNP therapeutics mainly address liver diseases, the potential of mRNA therapy extends far beyond─yet to be unraveled. To fully unlock the promises of mRNA therapy, there is an urgent need to develop safe and effective LNP systems that can target extrahepatic organs. Here, we report on the development of sulfonium lipid nanoparticles (sLNPs) for systemic mRNA delivery to the lungs. sLNP effectively and specifically delivered mRNA to the lungs following intravenous administration in mice. No evidence of lung and systemic inflammation or toxicity in major organs was induced by sLNP. Our findings demonstrated that the newly developed lung-specific sLNP platform is both safe and efficacious. It holds great promise for advancing the development of new mRNA-based therapies for the treatment of lung-associated diseases and conditions.

Bronchopulmonary dysplasia (BPD) is a debilitating disease in premature infants resulting from lung injury that disrupts alveolar and pulmonary vascular development. Despite the use of lung-protective ventilation and targeted oxygen therapy, BPD rates have not significantly changed over the last decade. Recent evidence suggests that sepsis and conditions initiating the systemic inflammatory response syndrome in preterm infants are key risk factors for BPD. However, the mechanisms by which sepsis-associated systemic inflammation and microbial dissemination program aberrant lung development are not fully understood. Progress has been made within the last 5 years with the inception of animal models allowing mechanistic investigations into neonatal acute lung injury and alveolar remodeling attributable to endotoxemia and necrotizing enterocolitis. These recent studies begin to unravel the pathophysiology of early endothelial immune activation via pattern recognition receptors such as Toll-like receptor 4 and disruption of critical lung developmental processes such as angiogenesis, extracellular matrix deposition, and ultimately alveologenesis. Here we review scientific evidence from preclinical models of neonatal sepsis-induced lung injury to new data emerging from clinical literature. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) mediate intercellular communication by transferring genetic material, proteins and organelles between different cells types in both health and disease. Recent evidence suggests that these vesicles, more than simply diagnostic markers, are key mediators of the pathophysiology of acute respiratory distress syndrome (ARDS) and other lung diseases. In this review, we will discuss the contribution of EVs released by pulmonary structural cells (alveolar epithelial and endothelial cells) and immune cells in these diseases, with particular attention to their ability to modulate inflammation and alveolar-capillary barrier disruption, a hallmark of ARDS. EVs also offer a unique opportunity to develop new therapeutics for the treatment of ARDS. Evidences supporting the ability of stem cell-derived EVs to attenuate the lung injury and ongoing strategies to improve their therapeutic potential are also discussed. [ABSTRACT FROM AUTHOR]

Abstract Background Calcium release-activated calcium (CRAC) channel inhibitors stabilize the pulmonary endothelium and block proinflammatory cytokine release, potentially mitigating respiratory complications observed in patients with COVID-19. This study aimed to investigate the safety and efficacy of Auxora, a novel, intravenously administered CRAC channel inhibitor, in adults with severe or critical COVID-19 pneumonia. Methods A randomized, controlled, open-label study of Auxora was conducted in adults with severe or critical COVID-19 pneumonia. Patients were randomized 2:1 to receive three doses of once-daily Auxora versus standard of care (SOC) alone. The primary objective was to assess the safety and tolerability of Auxora. Following FDA guidance, study enrollment was halted early to allow for transition to a randomized, blinded, placebo-controlled study. Results In total, 17 patients with severe and three with critical COVID-19 pneumonia were randomized to Auxora and nine with severe and one with critical COVID-19 pneumonia to SOC. Similar proportions of patients receiving Auxora and SOC experienced ≥ 1 adverse event (75% versus 80%, respectively). Fewer patients receiving Auxora experienced serious adverse events versus SOC (30% versus 50%, respectively). Two patients (10%) receiving Auxora and two (20%) receiving SOC died during the 30 days after randomization. Among patients with severe COVID-19 pneumonia, the median time to recovery with Auxora was 5 days versus 12 days with SOC; the recovery rate ratio was 1.87 (95% CI, 0.72, 4.89). Invasive mechanical ventilation was needed in 18% of patients with severe COVID-19 pneumonia receiving Auxora versus 50% receiving SOC (absolute risk reduction = 32%; 95% CI, − 0.07, 0.71). Outcomes measured by an 8-point ordinal scale were significantly improved for patients receiving Auxora, especially for patients with a baseline PaO2/FiO2 = 101–200. Conclusions Auxora demonstrated a favorable safety profile in patients with severe or critical COVID-19 pneumonia and improved outcomes in patients with severe COVID-19 pneumonia. These results, however, are limited by the open-label study design and small patient population resulting from the early cessation of enrollment in response to regulatory guidance. The impact of Auxora on respiratory complications in patients with severe COVID-19 pneumonia will be further assessed in a planned randomized, blinded, placebo-controlled study. Trial registration ClinicalTrials.gov, NCT04345614 . Submitted on 7 April 2020. Graphical abstract

Extracellular vesicles (EVs) mediate intercellular communication by transferring genetic material, proteins and organelles between different cells types in both health and disease. Recent evidence suggests that these vesicles, more than simply diagnostic markers, are key mediators of the pathophysiology of acute respiratory distress syndrome (ARDS) and other lung diseases. In this review, we will discuss the contribution of EVs released by pulmonary structural cells (alveolar epithelial and endothelial cells) and immune cells in these diseases, with particular attention to their ability to modulate inflammation and alveolar-capillary barrier disruption, a hallmark of ARDS. EVs also offer a unique opportunity to develop new therapeutics for the treatment of ARDS. Evidences supporting the ability of stem cell-derived EVs to attenuate the lung injury and ongoing strategies to improve their therapeutic potential are also discussed.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV. [ABSTRACT FROM AUTHOR]

Purpose/aimAngiogenesis is a central component of normal wound healing but it has not been fully characterized in lung repair following acute inflammatory injury. The current literature lacks vital information pertaining to the extent, timing, and location of this process. This information is necessary for examining mechanisms that drive normal lung repair in resolving acute inflammatory injury. The goal of our study was to formally characterize lung angiogenesis over a time course of bleomycin-induced lung injury.Materials and methodsFemale C57BL/6 mice age 8-12 weeks were treated with a single dose of intratracheal bleomycin. Total lung endothelial cells were quantified with flow cytometry 0, 7, 14, 21, and 28 days following bleomycin administration, and endothelial cell replication was assessed using bromodeoxyuridine (BrdU) incorporation.ResultsEndothelial cell replication was maximal 14 days after bleomycin administration, while total lung endothelial cells peaked at day 21. Tissue analysis with stereology was performed to measure total lung vascular surface area in bleomycin at day 21 relative to controls and demonstrated a trend toward increased vasculature in the bleomycin group.ConclusionsAngiogenesis begins shortly after injury in the bleomycin model and leads to an expansion in the lung endothelial cell population that peaks at day 21. This study offers the first longitudinal examination of angiogenesis following acute inflammatory lung injury induced by bleomycin. Information provided in this study will be vital for further investigating mechanisms of angiogenesis in both normal and abnormal lung repair.

HIV and Schistosoma infections have been individually associated with pulmonary vascular disease. Co-infection with these pathogens is very common in tropical areas, with an estimate of six million people co-infected worldwide. However, the effects of HIV and Schistosoma co-exposure on the pulmonary vasculature and its impact on the development of pulmonary vascular disease are largely unknown. Here, we have approached these questions by using a non-infectious animal model based on lung embolization of Schistosoma mansoni eggs in HIV-1 transgenic (HIV) mice. Schistosome-exposed HIV mice but not wild-type (Wt) counterparts showed augmented pulmonary arterial pressure associated with markedly suppressed endothelial-dependent vasodilation, increased endothelial remodeling and vessel obliterations, formation of plexiform-like lesions and a higher degree of perivascular fibrosis. In contrast, medial wall muscularization was similarly increased in both types of mice. Moreover, HIV mice displayed an impaired immune response to parasite eggs in the lung, as suggested by decreased pulmonary leukocyte infiltration, small-sized granulomas, and augmented residual egg burden. Notably, vascular changes in co-exposed mice were associated with increased expression of proinflammatory and profibrotic cytokines, including IFN-γ and IL-17A in CD4+ and γδ T cells and IL-13 in myeloid cells. Collectively, our study shows for the first time that combined pulmonary persistence of HIV proteins and Schistosoma eggs, as it may occur in co-infected people, alters the cytokine landscape and targets the vascular endothelium for aggravated pulmonary vascular pathology. Furthermore, it provides an experimental model for the understanding of pulmonary vascular disease associated with HIV and Schistosoma co-morbidity.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is an acute inflammatory process arises from a wide range of lung insults. A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier but the mechanisms involved are incompletely understood. The therapeutic potential of histone deacetylase (HDAC) inhibitors for the treatment of cardiovascular and inflammatory diseases is increasingly apparent, but the mechanisms by which HDACs regulate pulmonary vascular barrier function remain to be resolved. We found that specific Class IIa HDACs inhibitor, TMP269, significantly attenuated the lipopolysaccharide (LPS)‐induced human lung microvascular endothelial cells (HLMVEC) barrier compromise in vitro and improved vascular barrier integrity and lung function in murine model of ALI in vivo. TMP269 decreased LPS‐induced myosin light chain phosphorylation suggesting the role for Class IIa HDACs in LPS‐induced cytoskeleton reorganization. TMP269 did not affect microtubule structure and tubulin acetylation in contrast to the HDAC6‐specific inhibitor, Tubastatin A suggesting that Class IIa HDACs and HDAC6 (Class IIb) regulate endothelial cytoskeleton and permeability via different mechanisms. Furthermore, LPS increased the expression of ArgBP2 which has recently been attributed to HDAC‐mediated activation of Rho. Depletion of ArgBP2 abolished the ability of LPS to disrupt barrier function in HLMVEC and both TMP269 and Tubastatin A decreased the level of ArgBP2 expression after LPS stimulation suggesting that both Class IIa and IIb HDACs regulate endothelial permeability via ArgBP2‐dependent mechanism. Collectively, our data strongly suggest that Class IIa HDACs are involved in LPS‐induced ALI in vitro and in vivo via specific mechanism which involved contractile responses, but not microtubule reorganization. [ABSTRACT FROM AUTHOR]

Chronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease characterized by the presence of organized thromboembolic material in pulmonary arteries leading to increased vascular resistance, heart failure and death. Dysfunction of endothelial cells is involved in CTEPH. The present study describes for the first time the molecular processes underlying endothelial dysfunction in the development of the CTEPH. The advanced analytical approach and the protein network analyses of patient derived CTEPH endothelial cells allowed the quantitation of 3258 proteins. The 673 differentially regulated proteins were associated with functional and disease protein network modules. The protein network analyses resulted in the characterization of dysregulated pathways associated with endothelial dysfunction, such as mitochondrial dysfunction, oxidative phosphorylation, sirtuin signaling, inflammatory response, oxidative stress and fatty acid metabolism related pathways. In addition, the quantification of advanced oxidation protein products, total protein carbonyl content, and intracellular reactive oxygen species resulted increased attesting the dysregulation of oxidative stress response. In conclusion this is the first quantitative study to highlight the involvement of endothelial dysfunction in CTEPH using patient samples and by network medicine approach. [ABSTRACT FROM AUTHOR]

Rationale: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysematous lungs exhibit decreased expression of HIF-2α (hypoxia-inducible factor-2α)-regulated genes, and tobacco smoke decreases pulmonary HIF-2α concentrations. These findings suggest that decreased HIF-2α expression is important in the development of emphysema.Objectives: The objective of this study was to evaluate the roles of endothelial-cell (EC) HIF-2α in the pathogenesis of emphysema in mice.Methods: Mouse lungs were examined for emphysema after either the loss or the overexpression of EC Hif-2α. In addition, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for HGF (hepatocyte growth factor), a protein involved in alveolar development and homeostasis. Lungs from patients with emphysema were measured for endothelial HIF-2α expression.Measurements and Main Results: EC Hif-2α deletion resulted in emphysema in association with fewer ECs and pericytes. After SU5416 exposure, EC Hif-2α-knockout mice developed more severe emphysema, whereas EC Hif-2α-overexpressing mice were protected. EC Hif-2α-knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2α expression.Conclusions: Here, we demonstrate a unique protective role for pulmonary endothelial HIF-2α and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2α may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2α may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients. [ABSTRACT FROM AUTHOR]

Background: Calcium release-activated calcium (CRAC) channel inhibitors stabilize the pulmonary endothelium and block proinflammatory cytokine release, potentially mitigating respiratory complications observed in patients with COVID-19. This study aimed to investigate the safety and efficacy of Auxora, a novel, intravenously administered CRAC channel inhibitor, in adults with severe or critical COVID-19 pneumonia.Methods: A randomized, controlled, open-label study of Auxora was conducted in adults with severe or critical COVID-19 pneumonia. Patients were randomized 2:1 to receive three doses of once-daily Auxora versus standard of care (SOC) alone. The primary objective was to assess the safety and tolerability of Auxora. Following FDA guidance, study enrollment was halted early to allow for transition to a randomized, blinded, placebo-controlled study.Results: In total, 17 patients with severe and three with critical COVID-19 pneumonia were randomized to Auxora and nine with severe and one with critical COVID-19 pneumonia to SOC. Similar proportions of patients receiving Auxora and SOC experienced ≥ 1 adverse event (75% versus 80%, respectively). Fewer patients receiving Auxora experienced serious adverse events versus SOC (30% versus 50%, respectively). Two patients (10%) receiving Auxora and two (20%) receiving SOC died during the 30 days after randomization. Among patients with severe COVID-19 pneumonia, the median time to recovery with Auxora was 5 days versus 12 days with SOC; the recovery rate ratio was 1.87 (95% CI, 0.72, 4.89). Invasive mechanical ventilation was needed in 18% of patients with severe COVID-19 pneumonia receiving Auxora versus 50% receiving SOC (absolute risk reduction = 32%; 95% CI, - 0.07, 0.71). Outcomes measured by an 8-point ordinal scale were significantly improved for patients receiving Auxora, especially for patients with a baseline PaO2/FiO2 = 101-200.Conclusions: Auxora demonstrated a favorable safety profile in patients with severe or critical COVID-19 pneumonia and improved outcomes in patients with severe COVID-19 pneumonia. These results, however, are limited by the open-label study design and small patient population resulting from the early cessation of enrollment in response to regulatory guidance. The impact of Auxora on respiratory complications in patients with severe COVID-19 pneumonia will be further assessed in a planned randomized, blinded, placebo-controlled study.Trial Registration: ClinicalTrials.gov, NCT04345614 . Submitted on 7 April 2020. [ABSTRACT FROM AUTHOR]

This study explored the impact of gold nanoparticles on the metabolic activity and morphology of human pulmonary endothelial cell monolayers. We developed a gold nanoparticle library of three different sizes and two surface chemistries that include anionic citrate and the cationic polyelectrolyte poly(allylamine hydrochloride). The nanoparticles were characterized in cell culture medium to assess how their physical properties are altered after exposure to biological fluids. A bovine serum albumin pretreatment protocol was developed to stabilize the nanoparticles in cell culture medium. Results of this study show that an 18 h exposure of human pulmonary artery endothelial cells to the different nanoparticles modestly affects cellular metabolic activity. However, nanoparticle exposure perturbs the cortical actin networks and induces the formation of intercellular gaps. In particular, exposure to the poly(allylamine hydrochloride)-coated particles reduces the area of cell–cell junctions—a change that correlates with increased leakiness of endothelial barriers. The presence of excess polyelectrolyte capping agents in the supernatant of poly(allylamine hydrochloride)-coated nanoparticles significantly impacts endothelial morphology. Pretreatment of the particle supernatant with bovine serum albumin mitigates the negative effects of free or bound polyelectrolytes on endothelial cell monolayers. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid–to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasomemediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH. [ABSTRACT FROM AUTHOR]

The article discusses research on the relationship between exercise by and physical fitness in children and their risks of cardiovascular disease as adults. There has been an increased focus on the health of the vascular endothelium, which may provide biomarkers for the prognosis of future cardiovascular disease. Ultrasound techniques have been developed to allow pediatric medical researched to actually visualize the malfunction of the endothelium. Evidence has been found that regular exercise minimizes endothelial dysfunction not just in children with other risk factors such as obesity, but in the general population.

The pulmonary endothelium is dysfunctional in chronic obstructive pulmonary disease (COPD), a known risk factor for lung cancer. The pulmonary endothelium is altered in emphysema, which is disproportionately affected by cancers. Gene and microRNA expression differs between COPD and non-COPD lung. We hypothesised that the alteration in microRNA expression in the pulmonary endothelium contributes to its dysfunction. A total of 28 patients undergoing pulmonary resection were recruited and endothelial cells were isolated from healthy lung and tumour. MicroRNA expression was compared between COPD and non-COPD patients. Positive findings were confirmed by quantitative polymerase chain reaction (qPCR). Assays assessing angiogenesis and cellular migration were conducted in Human Umbilical Vein Endothelial Cells (n = 3–4) transfected with microRNA mimics and compared to cells transfected with negative control RNA. Expression of miR-181b-3p, miR-429 and miR-23c (all p < 0.05) was increased in COPD. Over-expression of miR-181b-3p was associated with reduced endothelial sprouting (p < 0.05). miR-429 was overexpressed in lung cancer as well and exhibited a reduction in tubular formation. MicroRNA-driven changes in the pulmonary endothelium thus represent a novel mechanism driving emphysema. These processes warrant further study to determine if they may be therapeutic targets in COPD and lung cancer.

Aims: Oxidant-induced endothelial injury plays a critical role in the pathogenesis of acute lung injury (ALI) and subsequent respiratory failure. Our previous studies revealed an endogenous antioxidant and protective pathway in lung endothelium mediated by heat shock protein 70 (Hsp70)–toll-like receptor 4 (TLR4) signaling. However, the downstream effector mechanisms remained unclear. Stanniocalcin 1 (STC1) has been reported to mediate antioxidant responses in tissues such as the lungs. However, regulators of STC1 expression as well as its physiological function in the lungs were unknown. We sought to elucidate the relationship between TLR4 and STC1 in hyperoxia-induced lung injury in vitro and in vivo and to define the functional role of STC1 expression in lung endothelium. Results: We identified significantly decreased STC1 expression in TLR4 knockout mouse lungs and primary lung endothelium isolated from TLR4 knockout mice. Overexpression of STC1 was associated with endothelial cytoprotection, whereas decreased or insufficient expression was associated with increased oxidant-induced injury and death. An Hsp70-TLR4-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signal mediates STC1 induction in the lungs and endothelial cells. We also demonstrated a previously unrecognized role for mitochondrial-associated STC1, via TLR4, in maintaining normal glycolysis, mitochondrial bioenergetics, and mitochondrial calcium levels. Innovation: To date, a physiological role for STC1 in oxidant-induced ALI has not been identified. In addition, our studies show that STC1 is regulated by TLR4 and exerts lung and endothelial protection in response to sterile oxidant-induced lung injury. Conclusions: Our studies reveal a novel TLR4-STC1-mediated mitochondrial pathway that has homeostatic as well as oxidant-induced cytoprotective functions in lung endothelium. [ABSTRACT FROM AUTHOR]

Abstract Background/aims Kidney ischemia and reperfusion injury could cause microvascular barrier dysfunction, lung inflammatory cascades activation, and programmed cell death of pulmonary endothelium, leading to acute lung injury. Our study aimed at determining whether erythropoietin (EPO) can ameliorate lung dysfunction following renal ischemia and reperfusion (IR) injury and explored the underlying mechanisms. Methods In vivo, C57BL/6 mice received EPO (6000 U/kg) before right renal vascular pedicles clamping for 30 minutes, followed by 24 hours of reperfusion. The lung histopathologic changes and inflammatory cytokines expression were assessed. In vitro, cultured human umbilical vein endothelial cells were treated with EPO, and apoptosis rate, proliferation capacity, and phosphorylation status of the Janus kinase-signal transducer and activator of transcription 3 (Jak-STAT3) pathway were measured respectively in the presence or absence of lipopolysaccharide stimulation. Results In vivo, EPO remarkably attenuated pulmonary interstitial and alveolar epithelial edema caused by renal IR injury. In vitro, the proliferation capacity of human umbilical vein endothelial cells was significantly increased under EPO stimulation, which correlated with changes in Jak-STAT3 signaling. Conclusion Our data indicated that EPO is able to ameliorate acute lung tissue damage induced by renal IR, and at least in part, via the Jak-STAT3 pathway. Highlights • Erythropoietin remarkably attenuated pulmonary interstitial and alveolar epithelial edema. • The proliferation capacity of human umbilical vein endothelial cells was significantly increased under erythropoietin stimulation. • Erythropoietin is able to ameliorate acute lung tissue damage induced by renal ischemia and reperfusion and, at least in part, via the Jak-STAT3 pathway. [ABSTRACT FROM AUTHOR]

Graphical abstract The suppression of Akt1 activity by LPS in pulmonary endothelial cells results in increased FoxO1/3a activation, in turn, leading to increased stromelysin1 expression/activity and reduced expression of tight junction proteins, particularly claudin5. Pharmacologically targeting FoxO or stromelysin activity inhibits LPS-induced pulmonary vascular injury and inflammation. Abstract Enhanced vascular permeability is associated with inflammation and edema in alveoli during the exudative phase of acute respiratory distress syndrome (ARDS). Mechanisms leading to the endothelial contribution on the early exudative stage of ARDS are not precise. We hypothesized that modulation of endothelial stromelysin1 expression and activity by Akt1-forkhead box-O transcription factors 1/3a (FoxO1/3a) pathway could play a significant role in regulating pulmonary edema during the initial stages of acute lung injury (ALI). We utilized lipopolysaccharide (LPS)-induced mouse ALI model in vivo and endothelial barrier resistance measurements in vitro to determine the specific role of the endothelial Akt1-FoxO1/3a-stromelysin1 pathway in ALI. LPS treatment of human pulmonary endothelial cells resulted in increased stromelysin1 and reduced tight junction claudin5 involving FoxO1/3a, associated with decreased trans-endothelial barrier resistance as determined by electric cell-substrate impedance sensing technology. In vivo , LPS-induced lung edema was significantly higher in endothelial Akt1 knockdown (EC-Akt1–/–) compared to wild-type mice, which was reversed upon treatment with FoxO inhibitor (AS1842856), stromelysin1 inhibitor (UK356618) or with shRNA-mediated FoxO1/3a depletion in the mouse lungs. Overall, our study provides the hope that targeting FoxO and styromelysin1 could be beneficial in the treatment of ALI. [ABSTRACT FROM AUTHOR]

Glutamine (GLN) is the most abundant free amino acid in the body, and is considered as a conditionally essential amino acid under stress conditions, acting as an important modulator of the immune response. We here investigated the role of exogenous GLN treatment on leukocyte migration after the onset of endotoxemia and the intracellular mechanisms of GLN actions on neutrophils. Two in vivo models of endotoxemia caused by lipopolysaccharide of Escherichia coli (LPS) injection were carried out in male outbred Balb/C mice 2-3 months old, as follow: (1) LPS (50 μg/kg) was intravenously injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 2 h later to investigate the role of GLN on the acute lung inflammation; (2) LPS (1 mg/kg) was intraperitoneally injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 18 h later to measure the effects of GLN on local and later phases of inflammation in the peritoneum. Results showed that GLN administration reduced the number of neutrophils in the inflamed lungs, partially recovery of the reduced number of leukocytes in the blood; reduced adhesion molecules on lung endothelium and on circulating neutrophils. Moreover, GLN treatment diminished the number of neutrophils, levels of chemotactic cytokine CXCL2 in the inflamed peritoneum, and neutrophils collected from the peritoneum of GLN-treated mice presented lower levels of Rho, Rac, and JNK. Together, our data show novel mechanisms involved in the actions of GLN on neutrophils migration. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Apple polyphenol relieves mean pulmonary arterial pressure, pulmonary vessel resistance in hypoxia-induced pulmonary hypertension model of rat. • After apple polyphenol administration, both endothelium intact and removed pulmonary vessel rings constricted by KCl or phenylephrine are relaxed. • Apple polyphenol up-regulates Ca2+ efflux and K+ influx in pulmonary arterial smooth muscle cell. • Apple polyphenol reduces pulmonary artery endothelium cell injury and apoptosis under hypoxia, and up-regulates NO level by enhancing endothelial nitric oxide synthase. Abstract Aim This study aims to test the effect of apple polyphenol (APP) on hypoxia-induced pulmonary arterial hypertension (PAH) and explore its possible underlying mechanisms. Methods and results Rats were treated with control, APP, hypoxia (8 h/d), hypoxia + APP. Mean pulmonary arterial pressure (mPAP) and pulmonary vessel resistance (PVR) were examined. Phenylephrine (PE)-pretreated pulmonary vessel rings were prepared for observation of APP administration. eNOS, sGC inhibitors (L-NAME, MB), Ca2+ channel blockers (NiCl 2 , Calhex231), K+ channel blockers (4-AP, 5-HD, TEA, BaCl 2) were applied to pulmonary vessel rings and pulmonary arterial smooth muscle cell (PASMC). Flow cytometry analysis and CCK-8 assay were applied to detect apoptosis of pulmonary artery endothelium cell (PAEC). Caspase-3, NO, eNOS, iNOS were detected in PAEC. APP reversed mPAP and PVR elevation in vivo. Contraction of pulmonary vessel rings with/without endothelium induced by hypoxia were inhibited by APP. APP effect was hindered by L-NAME or MB, and could be reduced by K+channel blockers. Further, APP was found to decrease cytosolic Ca2+ in PASMC and protect PAEC from apoptosis. In PAEC, Caspase-3, iNOS were decreased and NO, eNOS were increased after APP administration. Conclusions APP reverses pulmonary vasoconstriction through enzyme expression and cation channel activities, thus has effects of PASMC relaxation and PAEC protection. [ABSTRACT FROM AUTHOR]

Purpose: Fetal lung maturity (FLM) testing has been performed to help direct delivery timing in complex obstetrical conditions. We explored current practice patterns of FLM testing attempting to identify factors affecting its use.Materials and Methods: We distributed a 31-question survey to obstetricians and perinatologists that examined practice characteristics potentially affecting FLM usage. Logistic regression measured associations between these factors and test utilization. Weighted averages were calculated for conditions in which respondents considered FLM testing helpful.Results: Three hundred four surveys were completed. The response rate for respondents actively practicing obstetrics was 52%. The majority of respondents utilize FLM testing; however, 80% reported a decline in use over the past five years with 64% citing "published guidelines" as the reason. Respondents found FLM testing most applicable for poorly dated pregnancies. After an immature FLM test, 44% of respondents administer antenatal corticosteroids in the late-preterm period. None of the factors surveyed were significantly associated with FLM testing use.Conclusion: The majority of respondents use FLM testing although significant factors contributing to its use were not identified. We discover a high frequency of antenatal corticosteroid administration beyond 34 weeks gestational age in response to immature FLM indices that may be an area for future study. [ABSTRACT FROM AUTHOR]

Purpose: Fetal lung maturity (FLM) testing has been performed to help direct delivery timing in complex obstetrical conditions. We explored current practice patterns of FLM testing attempting to identify factors affecting its use.Materials and Methods: We distributed a 31-question survey to obstetricians and perinatologists that examined practice characteristics potentially affecting FLM usage. Logistic regression measured associations between these factors and test utilization. Weighted averages were calculated for conditions in which respondents considered FLM testing helpful.Results: Three hundred four surveys were completed. The response rate for respondents actively practicing obstetrics was 52%. The majority of respondents utilize FLM testing; however, 80% reported a decline in use over the past five years with 64% citing "published guidelines" as the reason. Respondents found FLM testing most applicable for poorly dated pregnancies. After an immature FLM test, 44% of respondents administer antenatal corticosteroids in the late-preterm period. None of the factors surveyed were significantly associated with FLM testing use.Conclusion: The majority of respondents use FLM testing although significant factors contributing to its use were not identified. We discover a high frequency of antenatal corticosteroid administration beyond 34 weeks gestational age in response to immature FLM indices that may be an area for future study. [ABSTRACT FROM AUTHOR]

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (βK82D) mutation in sickle cell Hb (βE6V/βK82D) and in a recombinant crosslinked Hb (rHb0.1/βK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of βCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the βK82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing βK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying βK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing βK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of βK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that βK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.

We have previously shown that Gs‐coupled adenosine receptors (A2a) are primarily involved in adenosine‐induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine‐induced Rac1 activation and EC barrier enhancement is mediated by Gs‐dependent stimulation of cAMP‐dependent Epac1‐mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively‐active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine‐induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine‐induced increase in TER. We next examined the role of cAMP‐activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine‐induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine‐induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine‐induced increases in TER and Tiam1 depletion had no effect on adenosine‐induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine‐induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP‐dependent Epac1/Vav2/Rap1‐mediated signaling. [ABSTRACT FROM AUTHOR]

A hallmark of acute respiratory distress syndrome (ARDS) is pulmonary vascular permeability. In these settings, loss of barrier integrity is mediated by cell-contact disassembly and actin remodeling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability in ARDS. The sweet taste receptor T1R3 is a G protein-coupled receptor, activated following exposure to sweet molecules, to trigger a gustducin-dependent signal cascade. In recent years, extraoral locations for T1R3 have been identified; however, no studies have focused on T1R3 within the vasculature. We hypothesize that activation of T1R3, in the pulmonary vasculature, plays a role in regulating endothelial barrier function in settings of ARDS. Our study demonstrated expression of T1R3 within the pulmonary vasculature, with a drop in expression levels following exposure to barrier-disruptive agents. Exposure of lung microvascular endothelial cells to the intensely sweet molecule sucralose attenuated LPS- and thrombin-induced endothelial barrier dysfunction. Likewise, sucralose exposure attenuated bacteria-induced lung edema formation in vivo. Inhibition of sweet taste signaling, through zinc sulfate, T1R3, or G-protein siRNA, blunted the protective effects of sucralose on the endothelium. Sucralose significantly reduced LPS-induced increased expression or phosphorylation of the key signaling molecules Src, p21-activated kinase (PAK), myosin light chain-2 (MLC2), heat shock protein 27 (HSP27), and p110α phosphatidylinositol 3-kinase (p110αPI3K). Activation of T1R3 by sucralose protects the pulmonary endothelium from edemagenic agent-induced barrier disruption, potentially through abrogation of Src/PAK/p110αPI3K-mediated cell-contact disassembly and Src/MLC2/HSP27-mediated actin remodeling. Identification of sweet taste sensing in the pulmonary vasculature may represent a novel therapeutic target to protect the endothelium in settings of ARDS. [ABSTRACT FROM AUTHOR]

Diesel exhaust particles (DEP) and their ultrafine fraction (UFP) are known to induce cardiovascular effects in exposed subjects. The mechanisms leading to these outcomes are still under investigation, but the activation of respiratory endothelium is likely to be involved. Particles translocation through the air-blood barrier and the release of mediators from the exposed epithelium have been suggested to participate in the process. Here we used a conditioned media in vitro model to investigate the role of epithelial-released mediators in the endothelial cells activation. Diesel UFP were sampled from a Euro 4 vehicle run over a chassis dyno and lung epithelial BEAS-2B cells were exposed for 20 h (dose 5 μg/cm 2 ). The exposure media were collected and used for endothelial HPMEC-ST1.6R cells treatment for 24 h. The processes related to oxidative stress and inflammation were investigated in the epithelial cells, accordingly to the present knowledge on DEP toxicity. The release of IL-6 and VEGF was significantly augmented in diesel exposed cells. In endothelial cells, VCAM-1 and ICAM-1 adhesion molecules levels were increased after exposure to the conditioned media. By interfering with IL-6 binding to its endothelial receptor, we demonstrate the role of this interleukin in inducing the endothelial response. [ABSTRACT FROM AUTHOR]

Purpose: Poor sleep quality and duration are associated with systemic endothelial dysfunction. However, an association between poor sleep and pulmonary endothelial dysfunction has not been elucidated. We sought to determine if there is a relationship between sleep duration and fractional exhaled nitric oxide (FeNO) concentrations as a surrogate for pulmonary endothelial function. Methods: We used three National Health and Nutrition Examination Survey (NHANES) cycles (2007-2012). Linear regression models were built with and without adjustment for age, sex, race, BMI, asthma/bronchitis, CRP, smoking, folate, renal function, respiratory infections, and steroid use. To examine a non-linear relationship, we introduced a spline, with single knot at mean sleep duration (7 h). Results: Of 13,173 participants (50.8% male, 44.2% Caucasian), 78% slept 6-8 hours (h). FeNO was significantly higher in the group sleeping 6-8 h (17.3 ± 14.9 ppb) than in the other two groups (16.0 ± 13.0 ppb, 15.9 ± 12.7 ppb for <6 and >8 h respectively; P < 0.001). In unadjusted linear regression, FeNO increased by 1.1 ppb for each hour increase in sleep up to 7 h ( P < 0.001). Increased sleep duration beyond 7 h saw a 0.96 ppb decrease in FeNO ( P < 0.001). After adjustment for confounders, FeNO increased by 1.09 ppb for each hour of sleep up to 7 h ( P = 0.001) and decreased by 0.71 ppb for each hour after ( P = 0.02). Conclusion: Sleeping less or more than 7 h is associated with pulmonary endothelial dysfunction as measured by FeNO. Further study is needed to evaluate mechanism(s) of this association and validity of FeNO as a marker of endothelial function. [ABSTRACT FROM AUTHOR]

• Investigation of the impact of P-gp on pulmonary disposition of inhaled drugs. • PET measurement of lung pharmacokinetics of i.t. aerosolized model P-gp substrate. • Use of transporter gene knockout rats and pharmacological P-gp inhibition. • Overall effect of P-gp depends on opposing effects at pulmonary epithelium and endothelium. In the lungs, the membrane transporter P-glycoprotein (P-gp) is expressed in the apical (i.e. lumen-facing) membrane of airway epithelial cells and in the luminal (blood-facing) membrane of pulmonary capillary endothelial cells. To better understand the influence of P-gp on the pulmonary disposition of inhaled P-gp substrate drugs, we measured the intrapulmonary pharmacokinetics of the intratracheally (i.t.) aerosolized model P-gp substrate [11C]metoclopramide in presence and absence of P-gp activity by means of positron emission tomography (PET) imaging in rats. Data were compared to data previously acquired with the model P-gp substrates (R)-[11C]verapamil and [11C] N -desmethyl-loperamide, using the same experimental set-up. Groups of wild-type rats, either untreated or treated with the P-gp inhibitor tariquidar, and Abcb1a/b(-/-) rats underwent 90-min dynamic PET scans after i.t. aerosolization of [11C]metoclopramide. Lung exposure to [11C]metoclopramide was expressed as the area under the right lung concentration-time curve (AUC lung). AUC lung values were significantly higher in Abcb1a/b(-/-) rats (1.8-fold, p ≤ 0.0001) and in tariquidar-treated wild-type rats (1.6-fold, p ≤ 0.01) than in untreated wild-type rats. This differed from previously obtained results with (R)-[11C]verapamil and [11C] N -desmethyl-loperamide, which showed decreased exposure in the rat lung in absence of P-gp activity. Our results suggest that transepithelial transfer of [11C]metoclopramide was not or only to a small extent affected by P-gp activity, presumably due to the compound's high passive permeability. The increased lung retention of [11C]metoclopramide may be due to decreased P-gp-mediated clearance into the blood in absence of P-gp activity in capillary endothelial cells. The overall effect of P-gp on the lung exposure to inhaled P-gp substrate drugs may, thus, be determined by a balance of opposing effects at the pulmonary epithelium and endothelium. [Display omitted] [ABSTRACT FROM AUTHOR]