Human Pulmonary Endothelial Cells Research Articles

ATP13A3 variants promote pulmonary arterial hypertension by disrupting polyamine transport

Abstract Aims Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated variants may contribute to disease pathogenesis. Methods and Results We studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. Conclusion We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.

Human Placental Mesenchymal Stem Cells-Exosomes Alleviate Endothelial Barrier Dysfunction via Cytoskeletal Remodeling through hsa-miR-148a-3p/ROCK1 Pathway.

Endothelial barrier disruption of human pulmonary vascular endothelial cells (HPVECs) is an important pathogenic factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Mesenchymal stem cells-exosome (MSCs-Exo) represents an ideal carrier for cell-free therapy. The therapeutic implication and underlying mechanism of human placental MSCs-Exo (HPMSCs-Exo) in ALI/ARDS need to be further explored. HPMSCs-Exo was extracted from HPMSCs and characterized. Then, the therapeutic effects of exosomes were evaluated in ALI mice and HPVECs. RNA-sequencing was applied to reveal the miRNA profile of HPMSCs-Exo and differentially expressed genes (DEGs) in HPMSCs-Exo-pretreated HPVECs. The targets of miRNAs were predicted by bioinformatics methods and correlated to DEGs. Finally, the role of hsa-miR-148a-3p/ROCK1 pathway in HPVECs has been further discussed. The results showed that HPMSCs-Exo could downregulate Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), upregulate the expression of zonula occludens-1 (ZO-1) and F-actin, promote HPVECs migration and tube formation, reduce cytoskeletal disorders and cell permeability, and thus improve ALI/ARDS. RNA-sequencing revealed the DEGs were mainly enriched in cell junction, angiogenesis, inflammation, and energy metabolism. HPMSCs-Exo contains multiple miRNAs which are associated with cytoskeletal function; the expression abundance of hsa-miR-148a-3p is the highest. Bioinformatic analysis identified ROCK1 as a target of hsa-miR-148a-3p. The overexpression of hsa-miR-148a-3p in HPMSCs-Exo promoted the migration and tube formation of HPVECs and reduced ROCK1 expression. However, the overexpression of ROCK1 on HPVECs reduced the therapeutic effect of HPMSCs-Exo. HPMSCs-Exo represents a protective regimen against endothelial barrier disruption of HPVECs in ALI/ARDS, and the hsa-miR-148a-3p/ROCK1 pathway plays an important role in this therapeutics implication.

Growth hormone-releasing hormone antagonists counteract interferon-γ – induced barrier dysfunction in bovine and human endothelial cells

GHRH regulates the secretion of GH from the anterior pituitary gland. An emerging body of evidence suggests that the activities of that neuropeptide are not limited to the GH/IGF-I axis, but they expand towards the mediation of inflammatory processes. GHRHAnt were developed to oppose the activities of GHRH in malignancies, and have been associated with strong anti-inflammatory and anti-oxidative effects in a diverse variety of tissues, including the lungs. In the present study we report that GHRHAnt oppose interferon-γ – induced paracellular hyperpermeability and reactive oxygen species generation in bovine and human pulmonary endothelial cells; and suppress interferon-γ – triggered STAT3, cofilin and ERK1/2 activation. Our observations substantiate previous findings on the protective effects of GHRHAnt in endothelial inflammation and barrier break-down.

E2F1 Mediates SOX17 Deficiency-Induced Pulmonary Hypertension.

Rare genetic variants and genetic variation at loci in an enhancer in SOX17 (SRY-box transcription factor 17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutations in SOX17 in PAH pathogenesis has not been reported. SOX17 expression was evaluated in the lungs and pulmonary endothelial cells (ECs) of patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion were generated to determine the role of SOX17 deficiency in the pathogenesis of PAH. Human pulmonary ECs were cultured to understand the role of SOX17 deficiency. Single-cell RNA sequencing, RNA-sequencing analysis, and luciferase assay were performed to understand the underlying molecular mechanisms of SOX17 deficiency-induced PAH. E2F1 (E2F transcription factor 1) inhibitor HLM006474 was used in EC-specific Sox17 mice. SOX17 expression was downregulated in the lung and pulmonary ECs from patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion induced spontaneously mild pulmonary hypertension. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced pulmonary hypertension in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and antiapoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP (bone morphogenetic protein) signaling. E2F1 signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction. Pharmacological inhibition of E2F1 in Sox17 EC-deficient mice attenuated pulmonary hypertension development. Our study demonstrated that endothelial SOX17 deficiency induces pulmonary hypertension through E2F1. Thus, targeting E2F1 signaling represents a promising approach in patients with PAH.

Cavin-2 loss exacerbates hypoxia-induced pulmonary hypertension with excessive eNOS phosphorylation and protein nitration

Pulmonary hypertension (PH) is associated with a poor prognosis even in recent years. Caveolin-1 (CAV1), a caveolae-associated protein, is a causal gene in PH. Cavin-2, one of the other caveolae-associated proteins, forms protein complexes with CAV1 and influences each other's functions. However, the role of Cavin-2 in PH has not been thoroughly investigated. To clarify the role of Cavin-2 in PH, we exposed Cavin-2-deficient (Cavin-2 KO) mice to hypoxia. A part of the analyses was confirmed in human pulmonary endothelial cells (HPAECs). After 4-week 10% O2 hypoxic exposure, we performed physiological, histological, and immunoblotting analyses. Right ventricular (RV) systolic pressure elevation and RV hypertrophy were exacerbated in Cavin-2 KO mice with hypoxia-induced PH (Cavin-2 KO PH mice). The vascular wall thickness of pulmonary arterioles was aggravated in Cavin-2 KO PH mice. Cavin-2 loss reduced CAV1 and induced sustained endothelial nitric oxide synthase (eNOS) hyperphosphorylation in the Cavin-2 KO PH lungs and HPAECs. NOx production associated with eNOS phosphorylation was also increased in the Cavin-2 KO PH lung and HPAECs. Furthermore, the nitration of proteins, including protein kinase G (PKG), was raised in the Cavin-2 KO PH lungs. In conclusion, we revealed that Cavin-2 loss exacerbated hypoxia-induced PH. Our results suggest that Cavin-2 loss leads to sustained eNOS hyperphosphorylation in pulmonary artery endothelial cells via CAV1 reduction, resulting in Nox overproduction-mediated nitration of proteins, including PKG, in smooth muscle cells.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

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

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

EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1, TRF2, and RAD50.

Telomeres are nucleoprotein structures located at the end of each chromosome, which function in terminal protection and genomic stability. Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo. As relatively long-lived mammals based on body size, bats display unique telomeric patterns, including the up-regulation of genes involved in alternative lengthening of telomeres (ALT), DNA repair, and DNA replication. At present, however, the relevant molecular mechanisms remain unclear. In this study, we performed cross-species comparison and identified EPAS1, a well-defined oxygen response gene, as a key telomeric protector in bat fibroblasts. Bat fibroblasts showed high expression of EPAS1, which enhanced the transcription of shelterin components TRF1 and TRF2, as well as DNA repair factor RAD50, conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion. Based on a human single-cell transcriptome atlas, we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation. Using in vitro-cultured human pulmonary endothelial cells, we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans. In addition, the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence. In conclusion, we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging, drawing insights from the longevity of bats.

Blood myeloid cells differentiate to lung resident cells and respond to pathogen stimuli in a 3D human tissue-engineered lung model.

Introduction: Respiratory infections remain a leading global health concern. Models that recapitulate the cellular complexity of the lower airway of humans will provide important information about how the immune response reflects the interactions between diverse cell types during infection. We developed a 3D human tissue-engineered lung model (3D-HTLM) composed of primary human pulmonary epithelial and endothelial cells with added blood myeloid cells that allows assessment of the innate immune response to respiratory infection. Methods: The 3D-HTLM consists of small airway epithelial cells grown at air-liquid interface layered on fibroblasts within a collagen matrix atop a permeable membrane with pulmonary microvascular endothelial cells layered underneath. After the epithelial and endothelial layers had reached confluency, an enriched blood monocyte population, containing mostly CD14+ monocytes (Mo) with minor subsets of CD1c+ classical dendritic cells (cDC2s), monocyte-derived dendritic cells (Mo-DCs), and CD16+ non-classical monocytes, was added to the endothelial side of the model. Results: Immunofluorescence imaging showed the myeloid cells migrate through and reside within each layer of the model. The myeloid cell subsets adapted to the lung environment in the 3D-HTLM, with increased proportions of the recovered cells expressing lung tissue resident markers CD206, CD169, and CD163 compared with blood myeloid cells, including a population with features of alveolar macrophages. Myeloid subsets recovered from the 3D-HTLM displayed increased expression of HLA-DR and the co-stimulatory markers CD86, CD40, and PDL1. Upon stimulation of the 3D-HTLM with the toll-like receptor 4 (TLR4) agonist bacterial lipopolysaccharide (LPS), the CD31+ endothelial cells increased expression of ICAM-1 and the production of IL-10 and TNFα was dependent on the presence of myeloid cells. Challenge with respiratory syncytial virus (RSV) led to increased expression of macrophage activation and antiviral pathway genes by cells in the 3D-HTLM. Discussion: The 3D-HTLM provides a lower airway environment that promotes differentiation of blood myeloid cells into lung tissue resident cells and enables the study of respiratory infection in a physiological cellular context.

Mechanisms of pulmonary endothelial permeability and inflammation caused by extracellular histone subunits H3 and H4.

Extracellular DNA-binding proteins such as histones are danger-associated molecular pattern released by the injured tissues in trauma and sepsis settings, which trigger host immune response and vascular dysfunction. Molecular events leading to histone-induced endothelial cell (EC) dysfunction remain poorly understood. This study performed comparative analysis of H1, H2A, H2B, H3, and H4 histone subunits effects on human pulmonary EC permeability and inflammatory response. Analysis of transendothelial electrical resistance and EC monolayer permeability for macromolecues revealed that H3 and H4, but not H1, H2A, or H2B caused dose-dependent EC permeability accompanied by disassembly of adherens junctions. At higher doses, H3 and H4 activated nuclear factor kappa B inflammatory cascade leading to upregulation EC adhesion molecules ICAM1, VCAM1, E-selectin, and release of inflammatory cytokines. Inhibitory receptor analysis showed that toll-like receptor (TLR) 4 but not TLR1/2 or receptor for advanced glycation end inhibition significantly attenuated deleterious effects of H3 and H4 histones. Inhibitor of Rho-kinase was without effect, while inhibition of Src kinase caused partial preservation of cell-cell junctions, H3/H4-induced permeability and inflammation. Deleterious effects of H3/H4 were blocked by heparin. Activation of Epac-Rap1 signaling restored EC barrier properties after histone challenge. Intravenous injection of histones in mice caused elevation of inflammatory markers and increased vascular leak. Post-treatment with pharmacological Epac/Rap1 activator suppressed injurious effects of histones in vitro and in vivo. These results identify H3 and H4 as key histone subunits exhibiting deleterious effects on pulmonary vascular endothelium via TLR4-dependent mechanism. In conclusion, elevation of circulating histones may represent a serious risk of exacerbated acute lung injury (ALI) and multiple organ injury during severe trauma and infection.

Polymorphonuclear neutrophil activation by Src phosphorylation contributes to HLA-A2 antibody-induced transfusion-related acute lung injury

Human leukocyte antigen (HLA)-A2 antibody contributes to the pathogenesis of transfusion-related acute lung injury (TRALI) via polymorphonuclear neutrophil (PMN) activation, but the signaling pathways involved this process remain largely undefined. In this study, we sought to study the signaling pathways involved in the pathogenesis of HLA-A2-induced TRALI. Lipopolysaccharide (LPS), and the plasma from the HLA-A2 antibody-positive donors were utilized to establish a rat model of TRALI. Human pulmonary endothelial cells (HPMECs) were in vitro co-cultured with HLA-A2 antibody-treated PMNs and then treated with LPS to induce a cytotoxicity model. The effects of HLA-A2 antibody on HPMEC injury were evaluated in this model. Besides, dasatinib was used to block the Src phosphorylation to explore whether Src involved in the TRALI or HPMEC injury induced by HLA-A2 antibody. The HLA-A2 antibody plus LPS induced TRALI and stimulated PMN activation in rats. HLA-A2 antibody-induced TRALI could be attenuated via depletion of PMN. HLA-A2 antibody activated NF-κB and NLRP3 inflammasome. In addition, HLA-A2 antibody aggravated the HPMEC injuries and the release of PMN surfaces makers, but dasatinib treatment reversed this effect, indicating that HLA-A2 antibody activated PMNs and exacerbated TRALI by stimulating phosphorylation of Src followed by activation of NF-κB and NLRP3 inflammasome, which was validated in vivo. In summary, HLA-A2 induced PMNs by activating NF-κB/NLRP3 inflammasome via phosphorylated-Src elevation, thereby exacerbating TRALI. This study highlights promising target for the treatment of antibody-mediated TRALI.

Circulating extracellular histones exacerbate acute lung injury by augmenting pulmonary endothelial dysfunction via TLR4-dependent mechanism.

Extracellular histones released into the circulation following trauma, sepsis, and ARDS may act as potent damage-associated molecular pattern signals leading to multiple organ failure. Endothelial cell (EC) dysfunction caused by extracellular histones has been demonstrated in vitro and in vivo; however, precise mechanistic details of histone-induced EC dysfunction and exacerbation of ongoing inflammation remain poorly understood. This study investigated the role of extracellular histones in exacerbating preexisting endothelial dysfunction and acute lung injury. Histone subunits H3 and H4, but not H1, H2A, or H2B, induced permeability in human pulmonary EC. H3 and H4 at concentrations above 30 µg/mL caused EC inflammation reflected by activation of the NF-κB pathway, transcriptional activation, and release of cytokines and chemokines including IL-6 and IL-8, and increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1. Pharmacological inhibitors targeting Toll-like receptor TLR4 but not TLR2/6, blocked histone-induced EC dysfunction. H3 and H4 also strongly augmented EC permeability and inflammation caused by Gram-negative and Gram-positive bacterial particles, endotoxin, and TNFα. Heparin blocked histone-induced augmentation of EC inflammation caused by endotoxin and TNFα. Injection of histone in mouse models of lung injury caused by bacterial wall lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus (HKSA) augmented ALI parameters: increased protein content, cell count, and inflammatory cytokine secretion in bronchoalveolar lavage fluid. Important clinical significance of these findings is in the demonstration that even a modest increase in extracellular histone levels can act as a severe exacerbating factor in conjunction with other EC barrier disruptive or proinflammatory agents.

The suppressive effects of Mer inhibition on inflammatory responses in the pathogenesis of LPS-induced ALI/ARDS.

The pathogenesis of sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) has not yet been fully elucidated. Growth arrest-specific 6 (Gas6) has marked effects on hemostasis and reduces inflammation through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Here, we found that plasma concentrations of Gas6 and soluble Mer were greater in patients with severe sepsis or septic ALI/ARDS compared with those in normal healthy donors. To determine whether the Gas6-Mer axis was critical in the pathogenesis of ALI/ARDS, we investigated the effects of intravenous administration of the selective Mer inhibitor UNC2250 on lipopolysaccharide (LPS)-induced ALI in mouse models subjected to inhalation of LPS. UNC2250 markedly inhibited the infiltration into the lungs of neutrophils and monocytes with increased amounts of Gas6 and Mer proteins, severe lung damage, and increased amounts of reactive oxygen species (ROS) in LPS-induced ALI in mice. In human pulmonary aortic endothelial cells, LPS induced decreases in the amounts of endothelial nitric oxide synthase, thrombomodulin, and vascular endothelial-cadherin, which was blocked by treatment with UNC2250. UNC2250 also inhibited the LPS-dependent increases in cell proliferation and enhanced apoptosis in HL-60 cells, a human neutrophil-like cell line, and RAW264.7 cells, a mouse monocyte/macrophage cell line. These data provide insights into the potential multiple beneficial effects of the Mer inhibitor UNC2250 as a therapeutic reagent to treat inflammatory responses in ALI/ARDS.

Ischemia-Reperfusion Injury in a Simulated Lung Transplant Setting Differentially Regulates Transcriptomic Profiles between Human Lung Endothelial and Epithelial Cells.

Current understanding of mechanisms of ischemia-reperfusion-induced lung injury during lung preservation and transplantation is mainly based on clinical observations and animal studies. Herein, we used cell and systems biology approaches to explore these mechanisms at transcriptomics levels, especially by focusing on the differences between human lung endothelial and epithelial cells, which are crucial for maintaining essential lung structure and function. Human pulmonary microvascular endothelial cells and human lung epithelial cells were cultured to confluent, subjected to different cold ischemic times (CIT) to mimic static cold storage with preservation solution, and then subjected to warm reperfusion with a serum containing culture medium to simulate lung transplantation. Cell morphology, viability, and transcriptomic profiles were studied. Ischemia-reperfusion injury induced a CIT time-dependent cell death, which was associated with dramatic changes in gene expression. Under normal control conditions, endothelial cells showed gene clusters enriched in the vascular process and inflammation, while epithelial cells showed gene clusters enriched in protein biosynthesis and metabolism. CIT 6 h alone or after reperfusion had little effect on these phenotypic characteristics. After CIT 18 h, protein-biosynthesis-related gene clusters disappeared in epithelial cells; after reperfusion, metabolism-related gene clusters in epithelial cells and multiple gene clusters in the endothelial cells also disappeared. Human pulmonary endothelial and epithelial cells have distinct phenotypic transcriptomic signatures. Severe cellular injury reduces these gene expression signatures in a cell-type-dependent manner. Therapeutics that preserve these transcriptomic signatures may represent new treatment to prevent acute lung injury during lung transplantation.

EICOSAPENTAENOIC ACID REDUCES EXPRESSION OF PLATELET ACTIVATION AND AGGREGATION PROTEINS IN PULMONARY AND VASCULAR ENDOTHELIUM DURING INFLAMMATION

BACKGROUND Atherothrombotic cardiovascular (CV) disease leads to platelet aggregation and thrombus formation. Icosapent ethyl is an ethyl ester of eicosapentaenoic acid (EPA), and was shown to reduce ischemic events in high-risk patients (REDUCE-IT). IPE is approved by Health Canada to reduce CV risk among patients with elevated TG levels as an add-on to maximally tolerated statin therapy. In this study, the effects of EPA on expression of proteins associated with platelet activation and aggregation were measured in cytokine (IL-6) treated pulmonary and vascular endothelial cells (ECs). METHODS AND RESULTS Human pulmonary ECs (PECs) and umbilical vein ECs (HUVECs) were treated with vehicle or EPA (40/10 µM in PECs/HUVECs) and IL-6 (12 ng/mL) for 24 h. Proteomic analysis was performed using liquid chromatography/mass spectroscopy that detected modulation of >1,000 proteins. Proteins which showed significantly (p < 0.05) greater than 1-fold change between treatment groups were further assessed. EPA significantly downregulated a total of 36 proteins involved in platelet activation, signaling, aggregation, in EC following cytokine exposure. Platelet endothelial cell adhesion molecule (PECAM) was downregulated in both HUVECs and PECs (1.2-fold) by EPA. In PECs, EPA significantly modulated 26 additional proteins related to platelet activation, including amyloid-beta precursor protein (1.1-fold decrease) and thrombin receptor (PAR-1,1.3-fold decrease). In HUVECs, the expression of nine other proteins were modulated related to platelet activation, including superoxide dismutase (1.6-fold increase). CONCLUSION EPA significantly reduced expression of multiple EC proteins required for platelet activation and aggregation, including PECAM in both pulmonary and vascular tissues. These findings indicate potential novel anti-platelet mechanisms for EPA that may account for reduced ischemic events in REDUCE-IT. Atherothrombotic cardiovascular (CV) disease leads to platelet aggregation and thrombus formation. Icosapent ethyl is an ethyl ester of eicosapentaenoic acid (EPA), and was shown to reduce ischemic events in high-risk patients (REDUCE-IT). IPE is approved by Health Canada to reduce CV risk among patients with elevated TG levels as an add-on to maximally tolerated statin therapy. In this study, the effects of EPA on expression of proteins associated with platelet activation and aggregation were measured in cytokine (IL-6) treated pulmonary and vascular endothelial cells (ECs). Human pulmonary ECs (PECs) and umbilical vein ECs (HUVECs) were treated with vehicle or EPA (40/10 µM in PECs/HUVECs) and IL-6 (12 ng/mL) for 24 h. Proteomic analysis was performed using liquid chromatography/mass spectroscopy that detected modulation of >1,000 proteins. Proteins which showed significantly (p < 0.05) greater than 1-fold change between treatment groups were further assessed. EPA significantly downregulated a total of 36 proteins involved in platelet activation, signaling, aggregation, in EC following cytokine exposure. Platelet endothelial cell adhesion molecule (PECAM) was downregulated in both HUVECs and PECs (1.2-fold) by EPA. In PECs, EPA significantly modulated 26 additional proteins related to platelet activation, including amyloid-beta precursor protein (1.1-fold decrease) and thrombin receptor (PAR-1,1.3-fold decrease). In HUVECs, the expression of nine other proteins were modulated related to platelet activation, including superoxide dismutase (1.6-fold increase). EPA significantly reduced expression of multiple EC proteins required for platelet activation and aggregation, including PECAM in both pulmonary and vascular tissues. These findings indicate potential novel anti-platelet mechanisms for EPA that may account for reduced ischemic events in REDUCE-IT.

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

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

Association between Leflunomide and Pulmonary Hypertension.

<b>Rationale:</b> Pulmonary hypertension (PH) has been described in patients treated with leflunomide. <b>Objectives:</b> To assess the association between leflunomide and PH. <b>Methods:</b> We identified incident cases of PH in patients treated with leflunomide from the French PH Registry and through the pharmacoVIGIlAnce in Pulmonary ArTerial Hypertension (VIGIAPATH) program between September 1999 to December 2019. PH etiology, clinical, functional, radiologic, and hemodynamic characteristics were reviewed at baseline and follow-up. A pharmacovigilance disproportionality analysis using the World Health Organization's global database was conducted. We then investigated the effect of leflunomide on human pulmonary endothelial cells. Data are expressed as median (min-max). <b>Results:</b> Twenty-eight patients treated with leflunomide before PH diagnosis was identified. A total of 21 (75%) had another risk factor for PH and 2 had two risk factors. The median time between leflunomide initiation and PH diagnosis was 32 months (1-120). Right heart catheterization confirmed precapillary PH with a cardiac index of 2.37 L⋅min^-1 ⋅m^-2 (1.19-3.1) and elevated pulmonary vascular resistance at 9.63 Wood Units (3.6-22.1) without nitric oxide reversibility. Five patients (17.9%) had no other risk factor for PH besides exposure to leflunomide. No significant hemodynamic improvement was observed after leflunomide withdrawal. The pharmacovigilance disproportionality analysis using the World Health Organization's database revealed a significant overrepresentation of leflunomide among reported pulmonary arterial hypertension-adverse drug reactions. <i>In vitro</i> studies showed the dose-dependent toxicity of leflunomide on human pulmonary endothelial cells. <b>Conclusions:</b> PH associated with leflunomide is rare and usually associated with other risk factors. The pharmacovigilance analysis suggests an association reinforced by experimental data.

The Effect of ABO Immune Complexes on Endothelial Cell Integrity As Measured By Electric Cell-Substrate Impedance Sensing (ECIS) and Confocal Microscopy

Background: ABO non-identical transfusions have been associated with increased risk of hemolytic transfusion reactions. We previously reported that the ABO immune complexes (ABO-IC) formed in vitro as a result of ABO non-identical exposure affect platelet function, red blood cell integrity, and clot kinetics (Vox Sang. 2016;110:219-26). We developed an in vitro model as a means to investigate the interaction between ABO-IC and endothelial cells (EC).Methods: The ABO-IC were prepared by combining equal volumes of concentrated O plasma (high titers of anti-A and anti-B) with A plasma (high concentration of A antigens). Anti-A titers were measured before and after mixing to confirm the presence of ABO-IC. ABO-known human pulmonary vessel EC were then seeded onto gold microarray plates to prepare for electric cell-substrate impedance sensing (ECIS). The trans-endothelial electrical resistance (TER) was recorded for 48 hours after the addition of media, PBS, thrombin, O, A, B, and/or AB plasmas added 1:1 with media, as well as various concentrations of ABO-IC. Statistics were performed using the chi-square test to compare TER values at various timepoints to baseline. Human pulmonary EC were cultured on glass-bottomed petri dishes for confocal microscopy. The cells were stained with fluorescent green calcein AM dye and assessed for viability at 100x magnification at baseline and again 15 minutes following the addition of ABO-IC.Results: Substantial decreases in TER were observed over the course of 2 hours in O and A EC when treated with non-identical plasma, and sustained over 10 hours (p<0.05 for all). Significant decreases in TER were also observed in cells treated with various concentrations of ABO-IC, over 2 hours and were found to be dose-dependent and independent of ABO type (p<0.05 for all). Unlike the ABO-IC, exposure to thrombin revealed a marked decrease in TER after 2 hours, although the final TER measurement revealed complete recovery (Table 1a-c). Incubation with ABO-identical plasma showed slight decreases in TER over time that were not statistically significant when compared to incubation with cell media (Figure 1a-b). Physical damage to the EC membrane were observed via confocal microscopy after 15 minutes of exposure to ABO-IC (Figures 2a-b).Discussion: The exposure of ABO-IC to human pulmonary vessel EC revealed significantly decreased monolayer integrity via ECIS and confocal microscopy. The decreases seen in the TER of EC incubated with non-identical plasma and ABO-IC indicate the potential for capillary leak as a possible reaction to ABO non-identical transfusion. We have shown that the substantial decrease in TER when exposed to plasma is ABO type-dependent, and that the damage caused to the cells happens within a few hours and is irreversible over 48 hours. The confocal images reveal a significant change in the morphology of the EC membrane within 15 minutes of exposure to ABO-IC. These preliminary data suggest that anti-A and ABO-IC may significantly affect EC viability and function. This in vitro model provides potential novel mechanisms for the detrimental effects of ABO non-identical transfusion on EC. Further investigation of biomarkers for cell viability and integrity in response to exposure to ABO-IC is underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vitamin D Repletion Mitigates Oxidative Stress Induced Pulmonary Artery Remodeling in Sickle Cell Disease

The lung is a primary target of end organ pathology in patients with sickle cell disease (SCD). Pulmonary artery remodeling, a common feature at autopsy in SCD, is characteristic of the pulmonary hypertension of SCD. In an autopsy cohort of 20 patients with SCD, we found that pulmonary artery remodeling was a pathology of the young patient. Patients with severe remodeling had less than a 15% probability of surviving to 25 years of age, whereas those with milder remodeling had an 80% probability of living beyond 50. Therefore, we wanted to understand the mechanisms underpinning pulmonary artery remodeling.Using benchtop ion trap mass spectrometry on extracted pulmonary proteins from the autopsy study, we screened for oxidative “fingerprints” (protein modifications) that were specific for SCD. We found that the lungs from patients with SCD carried a profound oxidative burden. Oxidative modifications arising from the hydroxyl (∙OH), superoxide (∙O2-) peroxynitrite (ONOO-),and eosinophil peroxidase were all significantly elevated compared to samples prepared from identical lung blocks from unaffected controls at autopsy. However, of the oxidative modifications detected, only the signature from peroxynitrite, 3-nitro tyrosine, (3-NT) could differentiate those SCD patients with profound pulmonary artery remodeling from those with milder forms of this pathology. After adjusting for age, the 3-NT content was increased 16 fold in the severely remodeled pulmonary tissues. Using immunohistochemistry, we found that 3NT was exquisitely localized to remodeling vessels and areas of smooth muscle proliferation. We assayed plasma proteins from patients with SCD at steady state and found a consistently elevated 3-NT content when compared to control plasma. The 3-NT containing proteins in living patients correlated strongly with NT-proBNP, but inversely with plasma free hemoglobin at steady state.Peroxynitrite, a strong smooth muscle mitogen, is formed via the reaction of NO with ∙O2-, therefore we reasoned that the presence of the superoxide radical was transforming NO into ONOO-. Extracellular (EC) superoxides are neutralized by EC-superoxide dismutases (SODs) to preserve the vaso-regulatory effects of NO. EC-SOD is tethered to the outer membrane of cells to allow for diffusion of NO without superoxide attack. Using immunohistochemistry, electron microscopy, and linear-ion trap mass spectrometry with comparative proteomics on paraffin-free lung tissue we noted, that pulmonary EC-SOD was 60-fold depleted in the remodeling pulmonary vessels and endothelium in patients with SCD at autopsy. The plasma of patients at steady state, compared to unaffected controls, however, contained increased, but inactive EC-SOD indicating an SCD-specific release of EC-SOD from the outer membrane of endothelial surfaces and into the plasma compartment.Using an in vitro culture of human pulmonary endothelial cells, we quantified EC-SOD on the cell surface using ELISA. Plasma from patients with SCD significantly reduced the amount of EC-SOD detectable on the endothelial cells. Plasma from healthy control subjects was without effect.We, and many other groups have demonstrated that patients with SCD have compromised flow mediated dilation (FMD) - an NO mediated process. We found that both plasma EC-SOD and 3-NT content of plasma proteins inversely correlated with FMD in patients with SCD. However, high dose vitamin D repletion (100,000 units ergocalciferol weekly) improved FMD within 8 weeks and reduced both plasma EC-SOD and 3NT content of plasma proteins, thus restoring NO function. After an 8 week washout period of the ergocalciferol, FMD values, plasma EC-SOD, and plasma 3-NT content returned to baseline, pathological steady state levels.Furthermore, plasma from the 8 week ergocalciferol treated patients lost the ability to deplete EC-SOD from the endothelial cell surface in vitro, whereas after the washout, plasma from the same subject effectively removed EC-SOD from the endothelial cells.Taken together these data suggest a new mechanism of pulmonary hypertension in patients with SCD, one in which NO in the presence of superoxide radical promotes pulmonary artery remodeling and predicts an early death. Vitamin D, through an as yet undetermined mechanism, appears to restore NO function, and re-localize EC-SOD to the appropriate vascular compartment. DisclosuresKutlar:Sancilio & Co (OMEG-411-02): Other: Chair of Data and Safety Monitoring Board; BlueBird Bio: Other: Member of Data Monitoring Committee; Reprixys Pharmaceuticals Corporation (formerly known as Selexys Pharmaceuticals Corporation, which is not affiliated with Selexis S.A.): Research Funding; Novartis: Research Funding.

Membranous Expression of Integrin α5β1 is Elevated in Endothelial Cells of Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is an incurable pathology characterized by remodeling of small pulmonary arteries leading to increased pulmonary vascular resistance and right ventricular heart failure. The endothelial dysfunction and endothelial-to-mesenchymal transition (EndoMT) observed in PAH contribute to the formation of plexiform lesions. Fibronectin is a commonly used molecular marker of EndoMT and is abundantly present in the plexiform lesions observed in human PAH. We have previously observed that pulmonary artery endothelial cells (PAECs) from the lesion forming Sugen/hypoxia rat model of PAH demonstrate increased proliferation and angiogenesis compared to PAECs from normoxic control animals. As the fibronectin receptor integrin α5β1 promotes endothelial proliferation and angiogenesis, we aimed to determine whether the expression of integrin α5β1 was elevated in PAH PAECs. Sugen/hypoxia rat PAECs (PAH PAECs) or normoxic control cells were received from the University of South Alabama. Initially, we observed an increase in mRNA expression of integrins α5, αV and β1 in PAH PAECs compared to control cells using a PCR screening array for molecular markers of epithelial-to-mesenchymal transition. Increased protein expression of the integrin α5, αV and β1 monomers were also observed in membrane fractions of PAH PAECs via western blot, compared to the cell membranes of control PAECs following a cell fractionation assay. To confirm the presence of the integrin α5β1 heterodimer at the membranes of PAH PAECs, co-immunoprecipitation of integrins α5 and β1 was performed from the membrane fractions of PAH and control PAECs. Following immunoprecipitation, western blot analysis demonstrated a strong coprecipitation of integrin α5 and β1 in membrane fractions of PAH PAECs, compared to weak coprecipitation in control PAECs. To confirm the increased membranous expression of integrin α5β1 in human tissue, human pulmonary endothelial cells from patients with idiopathic PAH (IPAH), familial PAH (FPAH) and control cells from failed donors (FD) were analyzed with an integrin-mediated cell adhesion array for cell surface expression of alpha and beta integrins. Human cells were provided by PHBI under the Pulmonary Hypertension Breakthrough Initiative (PHBI)*. Monomeric α5- and β1-mediated adhesion and heterodimeric α5β1-mediated adhesion were increased in IPAH and FPAH cells compared to FD control pulmonary endothelial cells. Collectively, these data strongly indicate that protein levels of the integrin α5β1 fibronectin receptor are increased at the cell membranes of pulmonary endothelial cells in PAH.