Pulmonary Microvascular Endothelial Cell Monolayers Research Articles

The calcineurin–NFATc pathway modulates the lipid mediators in BAL fluid extracellular vesicles, thereby regulating microvascular endothelial cell barrier function

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.

Bortezomib Increased Vascular Permeability by Decreasing Cell–Cell Junction Molecules in Human Pulmonary Microvascular Endothelial Cells

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell–cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and β-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and β-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell–cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

The impact of aging on pulmonary microvascular endothelial cell barrier function

Elderly individuals have substantially elevated morbidity and mortality during conditions of lung injury. However, the precise underlying mechanisms leading to this elevated mortality are ill-defined. Lung injury is associated with damage to pulmonary microvascular endothelial cells (PMVEC) leading to a compromised vascular barrier, fluid and protein leakage into the tissue, and subsequent respiratory dysfunction. Cell-cell junctions, including adherens junctions and tight junctions, are crucial for the maintenance of the endothelial barrier and have been shown to be dysregulated in various disease models. Pilot data from our lab demonstrated increased pulmonary vascular leak in aged mice during lung injury compared to young mice. We hypothesized that aging contributes to PMVEC barrier dysfunction due to impaired cell-cell junction integrity.To address our hypothesis, PMVEC isolated from young and aged mice were cultured in vitro until a confluent monolayer was formed. Endothelial barrier integrity was assessed by Evans blue-labelled albumin flux across the monolayers, as well as immunofluorescence staining of the adherens junction protein, vascular endothelial (VE)-cadherin, and tight junction protein, claudin-5. Localization of leak in relation to junctional proteins was performed using the fluorescently labelled macromolecule, NeutrAvidin. Proteomics analysis was conducted to identify differentially enriched pathways in young and aged PMVEC.Endothelial monolayers from aged mice exhibited a significant increase in albumin leak associated with augmented VE-cadherin and claudin-5 disruption vs. endothelial monolayers form young mice. Furthermore, NeutrAvidin staining localized to paracellular regions with VE-cadherin discontinuity was increased in PMVEC monolayers from aged mice vs. young mice, suggesting that the junctional disruption in aged PMVEC was directly associated with leak. Proteomics analysis revealed pathways known to be implicated in endothelial barrier dysfunction, including regulation of oxidoreduction and protein processing and trafficking, that were altered in PMVEC from aged mice.In summary, PMVEC from aged mice exhibit compromised barrier function, which is directly associated with impaired cell-cell junction integrity. We will next validate and target protein pathways identified in our proteomics analysis that were dysregulated in aged PMVEC to potentially rescue age-induced barrier dysfunction. These findings may highlight molecular pathways involved in predisposing aged individuals to worsened outcomes during lung injury, which can help in the development of therapeutics. Funded by Lawson Health Research Institute, CIHR, and Western University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel HIF-1-target gene isthmin1 contributes to hypoxia-induced hyperpermeability of pulmonary microvascular endothelial cells monolayers.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

Role of adenosine A2b receptors in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide

To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism. Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay. Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)% vs. (27.66±3.57)%, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β (ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)% vs. (27.66±3.57)%], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10-6 cm/s): 39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05). Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.

Mechanical signaling in a pulmonary microvascular endothelial cell monolayer

The mechanical microenvironment of an endothelial cell includes a stable protein scaffold on the basal side, flowing blood on the apical side and contractile cells on the lateral sides. Interaction with the protein scaffold and flowing blood modulates the ability of endothelial cells to migrate, align and maintain barrier function. Interaction with neighbors provides the endothelial monolayer unique “collective” properties. However, the nature of local mechanical signaling – i.e., the local functional consequence of a cell interacting with its contractile neighbors – remains unclear. Using an advancing sheet of pulmonary microvascular endothelial cells, here we examine the mechanical properties of an individual cell and its neighboring region. By combining Monolayer Stress Microscopy (MSM) with a novel analysis, we assessed several mechanical properties of an individual cell and its neighboring region. Across the monolayer, mechanical properties of the neighboring region defined multicellular “subdivisions” wherein constituent cells were exposed to a similar mechanical microenvironment. Adjacent subdivisions were separated by a narrow interface where adjoining cells were exposed to remarkably different mechanical microenvironments. Comparison of temporal fluctuations in mechanical properties of individual cells and those of their neighboring regions suggested three distinct intercellular mechanical signaling processes. These processes indicated that change in size, shape and speed of individual cells is associated with change in contractile forces in their neighboring regions. In summary, we present a novel approach to assess the mechanical interactions of individual cells with their contractile neighbors and identify potential functional consequences of such interactions.

Phosphorylated ERM Mediates Lipopolysaccharide Induced Pulmonary Microvascular Endothelial Cells Permeability Through Negatively Regulating Rac1 Activity

IntroductionThe endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. MethodsRat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1mg/L, 1mg/L, and 10mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway. ResultsLPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other. ConclusionPhosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.

Secreted Caspase‐1 Contracts Amyloid‐related Danger Molecules Released from Pulmonary Microvascular Endothelial Cell as a Novel Stress Response during Infection

Amyloid Beta (Aβ) are a group of peptides present in several oligomeric states that upon misfolding cause prionopathies. Emerging evidence indicates that Pseudomonas aeruginosa infection and associated host immune responses in pulmonary microvascular endothelial cells (PMVECs) elicit extracellular release of Aβ. These Aβ transmit cytotoxicity independent of the primary infection propagating PMVEC barrier disruption and exacerbating lung injury. Inflammasomes are protein recognition receptors that assemble upon sensing danger molecules including Aβ, resulting in activation and extracellular secretion of caspase‐1. Caspase‐1 propagates innate immune responses by activating interleukins and causing pyroptosis, a programmed cell death. Higher levels of circulating caspase‐1 have been positively correlated with disease severity in critically ill septic patients. Paradoxically, evidence in experimental models indicates that caspase‐1 activation is protective for barrier and multiorgan function, however, the underlying mechanisms are yet to be determined. We therefore hypothesized that secreted caspase‐1 degrades Aβ maintaining PMVEC monolayer integrity during Aβ‐induced injury. Infection of PMVECs with P. aeruginosa strain PA103 stimulated release of Aβ to the culture medium as assessed by Thioflavin T (ThT), a benzothiazole dye that increases in fluorescence upon binding to Aβ fibrils/aggregates. Strikingly, Aβ accumulated in bacteria‐free culture supernatant is cytotoxic when added to naïve PMVECs. Pretreatment of Aβ‐containing bacteria‐free culture supernatant with recombinant caspase‐1 reduced Aβ levels as measured by western blotting and ablated cytotoxicity. In addition, pretreatment of recombinant Aβ with recombinant caspase‐1 prevented Aβ fibril/aggregate formation in vitro. Moreover, CRISPR/Cas9 caspase‐1 mutant PMVECs were enriched with cytotoxic Aβ following P. aeruginosa infection and caspase‐1 mutant PMVECs were more susceptible to damage when treated with Aβ‐containing bacteria‐free culture supernatant. Together with bioinformatics evidence of a caspase‐1 recognition motif (FRHD7), our data strongly suggest caspase‐1 cleaves Aβ as a protective response. To support our hypothesis, we collected plasma from 45 critically ill patients recruited from the University of South Alabama ICU and 7 control subjects under an IRB‐approved study for analysis of circulating caspase‐1 and Aβ. All critically ill patients demonstrated higher levels of Aβ compared to healthy controls. Pulmonary septic patients displayed higher levels of Aβ. Caspase‐1 measured by ELISA was found to be highest in extra‐pulmonary septic patients. Importantly, there was an inverse correlation between caspase‐1 levels and ThT across all critically ill patients. In conclusion, our data indicate that P. aeruginosa infection‐induced release of Aβ from PMVECs elicits a feedforward cytotoxic effect that is inhibited by caspase‐1. Thus, caspase‐1 constitutes a novel protective stress response that contracts Aβ danger molecules generated during infection.Support or Funding InformationNIH R01 HL118334 to D.F.A. and J.P.A.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Distinct Induced Calcium Entry Pathways on Mitochondrial Respiration in Pulmonary Microvascular Endothelial Cells

Pulmonary microvascular endothelial cells (PMVECs) are distributed in a unique vascular bed exposed to the entire cardiac output and thus are influenced by circulating molecules that regulate calcium influx. Despite recognition of mitochondria's ability to buffer calcium, disparate results assessing the role of mitochondrial calcium and oxidative respiration are reported. The impact of distinct calcium entry pathways on mitochondrial respiration in PMVEC is unknown. We hypothesize that calcium entry pathways originating in spatially distinct cellular regions lead to unique changes in PMVEC mitochondrial respiration. Calcium entry dynamic patterns were assessed using an Andor spinning disk confocal microscope in confluent PMVEC monolayers loaded with the cytosolic calcium sensor CAL520. Thapsigargin (1 μM), an alkaloid that inhibits the sarcoendoplasmic reticulum calcium ATPase and elicits store‐depletion‐induced calcium entry, resulted in a rapid, global increase in cytosolic calcium. In contrast, 4α‐phorbol 12,13‐didecanoate (4αPDD, 5 μM), an agonist of the transient receptor potential vanilloid 4 channel (TRPV4), resulted in a slower increase in cytosolic calcium when compared to thapsigargin and elicited repetitive, short‐lived dynamic calcium events that spread into local cellular microdomains. In parallel studies, mitochondrial respiration was assessed by measuring oxygen consumption rate (OCR) using the Seahorse XF24 analyzer. 40,000 PMVECs were seeded per well 24 hours prior to running the assay. Experiments followed a standardized sequential protocol to measure different mitochondrial states. At t=21 min, after obtaining three basal measurements, cells were exposed to substrates for mitochondrial complex I (pyruvate 1 mM) and complex II (succinate 1 mM), then either thapsigargin (1 μM, n=20) or 4αPDD (5 μM, n=20) were added to activate distinct calcium entry pathways. At t=38 min, the complex V inhibitor oligomycin (1 μM) was added. At t=55 min, the mitochondrial respiration uncoupler FCCP (1 μM) was added. At t=73 min, the complex I inhibitor rotenone (1 μM) and the complex III inhibitor antimycin A (1 μM) were added. Control groups (n=20) were exposed to a similar protocol with addition of vehicle instead of thapsigargin or 4αPDD. In the control group, under basal conditions OCR measured 195 pmol/min. Addition of oligomycin decreased OCR to 68 pmol/min, while addition of uncoupler FCCP induced maximal respiration of 389 pmol/min. Addition of rotenone and antimycin A ablated mitochondrial respiration as expected. In controls, the respiratory ratio, defined as the ratio of basal to oligomycin OCR, was 3.05 ± 0.08 (mean ± SEM). Addition of thapsigargin significantly decreased the respiratory ratio (2.27 ± 0.01), while in contrast, the ratio increased after exposure to 4αPDD (8.02 ± 0.89) (p&lt;0.001, one‐way ANOVA). We conclude that the cytosolic calcium dynamics elicited by activation of store‐operated channels or TRPV4 in PMVECs differentially impact mitochondrial oxidative respiration.Support or Funding InformationNIH PO1 HL066299/Project 3 to M.I.T., NIH R01 HL118334 to D.F.A., and NIH S10OD02149 to USA microscopy core facility.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphorylated ERM Mediates Lipopolysaccharide Induced Pulmonary Microvascular Endothelial Cells Permeability Through Negatively Regulating Rac1 Activity

IntroductionThe endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. MethodsRat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1mg/L, 1mg/L, and 10mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway. ResultsLPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other. ConclusionPhosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.

Differential Mechanisms of Septic Human Pulmonary Microvascular Endothelial Cell Barrier Dysfunction Depending on the Presence of Neutrophils.

Sepsis is characterized by injury of pulmonary microvascular endothelial cells (PMVEC) leading to barrier dysfunction. Multiple mechanisms promote septic PMVEC barrier dysfunction, including interaction with circulating leukocytes and PMVEC apoptotic death. Our previous work demonstrated a strong correlation between septic neutrophil (PMN)-dependent PMVEC apoptosis and pulmonary microvascular albumin leak in septic mice in vivo; however, this remains uncertain in human PMVEC. Thus, we hypothesize that human PMVEC apoptosis is required for loss of PMVEC barrier function under septic conditions in vitro. To assess this hypothesis, human PMVECs cultured alone or in coculture with PMN were stimulated with PBS or cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) in the absence or presence of a pan-caspase inhibitor, Q-VD, or specific caspase inhibitors. PMVEC barrier function was assessed by transendothelial electrical resistance (TEER), as well as fluoroisothiocyanate-labeled dextran and Evans blue-labeled albumin flux across PMVEC monolayers. PMVEC apoptosis was identified by (1) loss of cell membrane polarity (Annexin V), (2) caspase activation (FLICA), and (3) DNA fragmentation [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)]. Septic stimulation of human PMVECs cultured alone resulted in loss of barrier function (decreased TEER and increased macromolecular flux) associated with increased apoptosis (increased Annexin V, FLICA, and TUNEL staining). In addition, treatment of septic PMVEC cultured alone with Q-VD decreased PMVEC apoptosis and prevented septic PMVEC barrier dysfunction. In septic PMN–PMVEC cocultures, there was greater trans-PMVEC macromolecular flux (both dextran and albumin) vs. PMVEC cultured alone. PMN presence also augmented septic PMVEC caspase activation (FLICA staining) vs. PMVEC cultured alone but did not affect septic PMVEC apoptosis. Importantly, pan-caspase inhibition (Q-VD treatment) completely attenuated septic PMN-dependent PMVEC barrier dysfunction. Moreover, inhibition of caspase 3, 8, or 9 in PMN–PMVEC cocultures also reduced septic PMVEC barrier dysfunction whereas inhibition of caspase 1 had no effect. Our data demonstrate that human PMVEC barrier dysfunction under septic conditions in vitro (cytomix stimulation) is clearly caspase-dependent, but the mechanism differs depending on the presence of PMN. In isolated PMVEC, apoptosis contributes to septic barrier dysfunction, whereas PMN presence enhances caspase-dependent septic PMVEC barrier dysfunction independently of PMVEC apoptosis.

Caspase‐Dependent Septic Pulmonary Microvascular Endothelial Cell Barrier Dysfunction is Associated with Vascular Endothelial‐Cadherin Disruption

BackgroundSepsis often causes dysfunction of pulmonary microvascular endothelial cells (PMVEC) leading to severe pulmonary edema. We previously reported in a murine model of sepsis that lung injury, including PMVEC barrier dysfunction, was strongly caspase‐dependent, and associated with PMVEC apoptosis. The mechanism through which caspases mediate PMVEC barrier function, however, was not determined. The classic role of caspases is the initiation and execution of apoptosis; however, caspases have important biological functions other than apoptosis. Indeed, caspases can cleave multiple targets, including proteins associated with cell‐cell junctions.ObjectiveTo identify the mechanisms through which caspases regulate septic PMVEC barrier dysfunction.HypothesisPMVEC barrier dysfunction in sepsis is mediated by caspase‐dependent disruption of inter‐PMVEC junctions.MethodsHuman PMVEC cultured alone or in co‐culture with neutrophils (PMN) were stimulated with cytomix (equimolar tumour necrosis factor α, interleukin 1β and interferon γ to mimic septic conditions). PMVEC barrier function was assessed by flux of Evans blue labelled‐albumin in a transwell assay. Specific microscopic localization of leak was determined by imaging paracellular fluorescein isothiocyanate labelled‐avidin leak across PMVEC monolayers in biotin‐coated wells. PMVEC apoptosis was examined using 3 markers: caspase activation (FLICA staining), loss of cell membrane polarity (Annexin V staining), and DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labelling [TUNEL] staining). PMVEC cell‐cell junctions were examined using immunofluorescence with antibodies against vascular endothelial (VE)‐cadherin.ResultsWe found that co‐culture of PMVEC with PMN significantly disrupts PMVEC barrier function under both basal and septic conditions vs. PMVEC cultured alone. Compared to cytomix‐treated PMVEC alone, the presence of PMN also significantly enhanced septic PMVEC caspase activation (FLICA staining), but this increase did not appear to be associated with any change in PMVEC apoptosis (Annexin V or TUNEL staining) vs. PMVEC cultured alone. We also found that microscopic paracellular leak co‐localized around cells positive for caspase activity, and was highly associated with disruption of circumferential PMVEC surface VE‐cadherin staining (97.1%). Moreover, PMVEC treatment with Q‐VD, a pan‐caspase inhibitor, rescued the septic barrier dysfunction, microscopic leak, and VE‐cadherin disruption.ConclusionOur data demonstrates that neutrophil presence enhances caspase‐dependent septic PMVEC barrier dysfunction independently of PMVEC apoptosis partly through disruption of VE‐cadherin.Support or Funding InformationThis work was supported by research funding from the Ontario Thoracic Society (S. Gill), the Program of Experimental Medicine (S. Gill and S. Mehta), and the Heart &amp; Stroke Foundation of Ontario/Canada grants #G‐16‐00014621 (S. Mehta and S. Gill).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. Controlled, in vitro, ex vivo, and in vivo laboratory study. Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Cell-free hemoglobin promotes primary graft dysfunction through oxidative lung endothelial injury.

Primary graft dysfunction (PGD) is acute lung injury within 72 hours of lung transplantation. We hypothesized that cell-free hemoglobin (CFH) contributes to PGD by increasing lung microvascular permeability and tested this in patients, ex vivo human lungs, and cultured human lung microvascular endothelial cells. In a nested case control study of 40 patients with severe PGD at 72 hours and 80 matched controls without PGD, elevated preoperative CFH was independently associated with increased PGD risk (odds ratio [OR] 2.75, 95%CI, 1.23-6.16, P = 0.014). The effect of CFH on PGD was magnified by reperfusion fraction of inspired oxygen (FiO2) ≥ 0.40 (OR 3.41, P = 0.031). Isolated perfused human lungs exposed to intravascular CFH (100 mg/dl) developed increased vascular permeability as measured by lung weight (CFH 14.4% vs. control 0.65%, P = 0.047) and extravasation of Evans blue-labeled albumin dye (EBD) into the airspace (P = 0.027). CFH (1 mg/dl) also increased paracellular permeability of human pulmonary microvascular endothelial cell monolayers (hPMVECs). Hyperoxia (FiO2 = 0.95) increased human lung and hPMVEC permeability compared with normoxia (FiO2 = 0.21). Treatment with acetaminophen (15 μg/ml), a specific hemoprotein reductant, prevented CFH-dependent permeability in human lungs (P = 0.046) and hPMVECs (P = 0.037). In summary, CFH may mediate PGD through oxidative effects on microvascular permeability, which are augmented by hyperoxia and abrogated by acetaminophen.

Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis.

Specific therapies targeting cellular and molecular events of sepsis induced Acute Lung Injury (ALI) pathogenesis are lacking. We have reported a pivotal role for Nuclear Factors of Activated T cells (NFATc3) in regulating macrophage phenotype during sepsis induced ALI and subsequent studies demonstrate that NFATc3 transcriptionally regulates macrophage CCR2 and TNFα gene expression. Mouse pulmonary microvascular endothelial cell monolayer maintained a tighter barrier function when co-cultured with LPS stimulated NFATc3 deficient macrophages whereas wild type macrophages caused leaky monolayer barrier. More importantly, NFATc3 deficient mice showed decreased neutrophilic lung inflammation, improved alveolar capillary barrier function, arterial oxygen saturation and survival benefit in lethal CLP sepsis mouse models. In addition, survival of wild type mice subjected to the lethal CLP sepsis was not improved with broad-spectrum antibiotics, whereas the survival of NFATc3 deficient mice was improved to 40–60% when treated with imipenem. Passive adoptive transfer of NFATc3 deficient macrophages conferred protection against LPS induced ALI in wild type mice. Furthermore, CP9-ZIZIT, a highly potent, cell-permeable peptide inhibitor of Calcineurin inhibited NFATc3 activation. CP9-ZIZIT effectively reduced sepsis induced inflammatory cytokines and pulmonary edema in mice. Thus, this study demonstrates that inhibition of NFATc3 activation by CP9-ZIZIT provides a potential therapeutic option for attenuating sepsis induced ALI/pulmonary edema.

Eosinophil transendothelial migration induced by the bronchoalveolar lavage fluid of acute eosinophilic pneumonia.

Acute eosinophilic pneumonia (AEP) is characterized by a massive pulmonary infiltration of eosinophils. Mechanisms regulating the selective accumulation of eosinophils in AEP have not been fully established. The objective of this study was to evaluate the mechanisms of eosinophil accumulation in alveolar spaces through examination of bronchoalveolar lavage fluid (BALF) from AEP patients (AEP-BALF). Eosinophils were isolated from the blood of healthy subjects and were placed on a human pulmonary microvascular endothelial cell monolayer cultured on Transwell filters (Coster, Cambridge, MA, USA). A saline control solution or BALF from patients with AEP, sarcoidosis or hypersensitivity pneumonitis was applied to the lower compartment, and the transendothelial migration of the eosinophils was evaluated. The concentrations of cytokines and chemokines in BALF were also measured. Transmigration of eosinophils across endothelial cells was only induced by the AEP-BALF. This transmigration was blocked by anti-β2 integrin mAb. The concentrations of eotaxin-2 and monocyte chemotactic protein (MCP)-4, which are CC chemokine receptor (CCR) 3 ligands, were elevated in the AEP-BALF, and anti-CCR3 mAb or anti-MCP-4 mAb inhibited the AEP-BALF-induced transmigration of eosinophils. Furthermore, the concentration of leukotriene (LT) B4 was increased in the AEP-BALF, and an LTB4 receptor antagonist partially suppressed the AEP-BALF-induced transmigration of eosinophils. These findings suggest that CCR3 ligands including eotaxin-2 and MCP-4, and LTB4 play a role in the accumulation of eosinophils in AEP.

α2-adrenoreceptor modulated FAK pathway induced by dexmedetomidine attenuates pulmonary microvascular hyper-permeability following kidney injury.

Renal ischemia-reperfusion (rI/R) could cause remote acute lung injury (ALI) and combination of these two organ injuries can remarkably increase the mortality. This study aims to determine whether dexmedetomidine, an α2-adrenoreceptor agonist sedative, can ameliorate pulmonary microvascular hyper-permeability following rI/R injury and explore the underlying mechanisms. In vivo, C57BL/6J mice received dexmedetomidine (25μg/kg, i.p.) in the absence or presence of α2-adrenergic antagonist atipamezole (250μg/kg, i.p.) or focal adhesion kinase (FAK) inhibitor (30mg/kg, i.p.) before bilateral renal pedicle clamping for 45 minutes followed by 24 hours reperfusion. The lung histopathological changes and the permeability of pulmonary microvascular were assessed respectively. In vitro, the cultured C57BL/6J mice pulmonary microvascular endothelial cells (PMVECs) were treated with serum from mice with rI/R with or without dexmedetomidine and atipamezole. Trans-endothelial permeability and phospho-tyrosine397FAK, F-actin, VE-cadherin and ZO-1 in monolayer PMVECs were measured respectively in the presence or absence of rI/R serum, dexmedetomidine and FAK inhibitor. In vivo, dexmedetomidine remarkably attenuated lung injury and pulmonary microvascular hyper-permeability caused by rI/R injury, which was abolished by atipamezole or FAK inhibitor co-administration. In vitro, the permeability of PMVECs monolayer following exposure to serum from rI/R mice was increased significantly, and decreased by dexmedetomidine. Dexmedetomidine increased phospho-tyrosine397FAK in a time- and dose-dependent manner, which was correlated with the changes in trans-endothelial permeability. Our data indicated that dexmedetomidine is able to ameliorate remote pulmonary microvascular hyper-permeability induced by rI/R, at least in part, via FAK modulation.

Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability.

BackgroundLipopolysaccharide (LPS) was shown to induce an increase in caveolin-1 (Cav-1) expression in endothelial cells; however, the mechanisms regarding this response and the consequences on caveolae-mediated transcellular transport have not been completely investigated. This study aims to investigate the role of LPS-induced Cav-1 phosphorylation in pulmonary microvascular permeability in pulmonary microvascular endothelial cells (PMVECs).MethodsRat PMVECs were isolated, cultured, and identified. Endocytosis experiments were employed to stain the nuclei by DAPI, and images were obtained with a fluorescence microscope. Permeability of endothelial cultures was measured to analyze the barrier function of endothelial monolayer. Western blot assay was used to examine the expression of Cav-1, pCav-1, triton-insoluble Cav-1, and triton-soluble Cav-1 protein.ResultsThe LPS treatment induced phosphorylation of Cav-1, but did not alter the total Cav-1 level till 60 min in both rat and human PMVECs. LPS treatment also increased the triton-insoluble Cav-1 level, which peaked 15 min after LPS treatment in both rat and human PMVECs. LPS treatment increases the intercellular cell adhesion molecule-1 expression. Src inhibitors, including PP2, PP1, Saracatinib, and Quercetin, partially inhibited LPS-induced phosphorylation of Cav-1. In addition, both PP2 and caveolae disruptor MβCD inhibited LPS-induced increase of triton-insoluble Cav-1. LPS induces permeability by activating interleukin-8 and vascular endothelial growth factor and targeting other adhesion markers, such as ZO-1 and occludin. LPS treatment also significantly increased the endocytosis of albumin, which could be blocked by PP2 or MβCD. Furthermore, LPS treatment for 15 min significantly elevated Evans Blue-labeled BSA transport in advance of a decrease in transendothelial electrical resistance of PMVEC monolayer at this time point. After LPS treatment for 30 min, transendothelial electrical resistance decreased significantly. Moreover, PP2 and MβCD blocked LPS-induced increase in Evans Blue-labeled BSA level.ConclusionOur study demonstrates that LPS-induced Cav-1 phosphorylation may lead to the increase of transcellular permeability prior to the increase of paracellular permeability in a Src-dependent manner. Thus, LPS-induced Cav-1 phosphorylation may be a therapeutic target for the treatment of inflammatory lung disease associated with elevated microvascular permeability.

Neuraminidase Enhances Pseudomonas Aeruginosa Secretome Disruption of the Rat Pulmonary Microvascular Endothelial Cell Monolayer

Neuraminidase (NA), an enzyme that removes sialic acids and is produced by many pathogenic organisms, has previously been shown to cause endothelial barrier disruption. P. aeruginosa strains secrete a variety of proteins, including NA, into the extracellular environment, the collection of which may be termed the secretome. Many of these proteins are virulence factors and expression can vary across strains, as well as over the course of growth and in response to environmental factors. For example, strain PAO1 expresses LasB, an elastase which has been previously implicated in disruption of endothelial monolayers by cleaving VE‐cadherin, which is sialylated, but strain Pa103 does not. This study investigates the role of NA in P. aeruginosa secretome disruption of the pulmonary microvascular endothelial cell (PMVEC) monolayer. PMVEC monolayers were treated with secretome from various P. aeruginosa strains including the wildtype PAO1, a NA deleted strain, PAO1Δ2794, and the wildtype strain Pa103. Secretomes from the different strains caused gap formation in the PMVEC monolayer at differing times. Large gaps were observed at 18 hours after treatment with PAO1 secretome, while similar damage from the PAO1Δ2794 secretomes was not observed until 26 hours after treatment. Only minor damage was observed upon treatment with Pa103 secretome at any time point. The delay in damage observed between the wild‐type secretome treatment and the NA‐deleted secretome lead to the conclusion that NA enhances disruption of the PMVEC barrier by P. aeruginosa PAO1 secretome. Supported by R01HL107778.

Establishment of model of serum-caused damage to pulmonary microvascular endothelial cells of mice with renal ischemia-reperfusion injury

Objective To establish the model of serum-caused damage to pulmonary microvascular endothelial cells (PMVECs) of mice with renal ischemia-reperfusion (I/R) injury. Methods Mice PMVECs were cultured to measure the standard trans-endothelial electrical resistance (TER) in the monolayer of PMVECs. When PMVECs were cultured and arranged in compact monolayer and TER was achieved, they were divided into 4 groups (n = 3 each) using a random number table: serum of normal mice group (NS group) and different concentrations (5%, 10% and 20%) of serum of mice with renal I/R injury groups (IRS5 group, IRS10group and IRS20 group). The PMVECs were cultured for 1 h in the serum-free endothelial culture medium. The 0.8 and 0.2 ml culture medium containing 20% serum of normal mice were then added to the upper and lower chambers, respectively, in group NS. The 0.8 and 0.2 ml culture medium containing 5%, 10% and 20% serum of mice with renal I/R injury were then added to the upper and lower chambers in IRS5, IRS10 and IRS20 groups, respectively.100 μg/ml FITC-BSA 100 μl was added to the upper chamber in the four groups. At 3, 6, 9, 12, 15, 18, 21 and 24 h of incubation, the PMVEC monolayer permeability (apparent permeability coefficient, Pa) was detected. Results Compared with NS group, the Pa was significantly increased at 12 and 15 h of incubation in IRS5 group, and the Pa was increased at 6-24 h of incubation in IRS10 and IRS20 groups. Compared with IRS5 group, the Pa at 21 and 24 h in IRS10 group and at 9-24 h in IRS20 group were significantly increased. Conclusion Both 10% and 20% serum of mice with renal I/R injury can successfully establish the model of damage to PMVECs, and 20% serum causes a more severe damage. Key words: Reperfusion injury; Kidney; Serum; Endothelial cells