Pulmonary Microvascular Endothelial Cells Barrier Research Articles

Investigation of the Role of Aging on Pulmonary Microvascular Endothelial Cell Barrier Function During Mechanical Ventilation

Elderly individuals have substantially elevated morbidity and mortality following pulmonary insult that require mechanical ventilation (MV) to sustain respiratory function. It is not known if an altered response to MV contributes to the worsened outcomes in the elderly. MV is associated with injury to pulmonary microvascular endothelial cells (PMVEC), leading to a compromised vascular barrier, and fluid and protein leakage within the tissue. However, the precise impact that age has on the vascular endothelium during MV is unknown. We hypothesized that aging augments PMVEC barrier dysfunction during MV. To address this hypothesis, young and aged mice were ventilated at a tidal volume of 20 mL/kg for 3 hours. To assess microvascular permeability, lungs were lavaged and abundance of the serum protein, immunoglobulin M, within the lavage fluid was measured. Lung tissue was dissociated into single cells and RNA was collected for single-cell RNA sequencing (scRNAseq). Subsequent analysis was performed using the Seurat package in R to give insight into the role of specific cell types, with a particular emphasis on microvascular endothelial cells. To directly assess molecular mechanisms directly in the cells, PMVEC were isolated from a separate cohort of non-ventilated young and aged mice and cultured in vitro. Cells were grown to confluency, then assessed for barrier integrity by Evans blue-labelled albumin leak across monolayers and immunofluorescence staining of cell junctional adherens and tight junction proteins, VE-cadherin and claudin-5. Compared to young, aged-ventilated animals had a significant increase in immunoglobulin M in their lavage fluid. Analysis of scRNAseq data revealed more robust activation and inflammation in the endothelial cells from aged animals following MV. Endothelial monolayers from aged animals in vitro exhibited a significant increase in albumin flux, which was associated with poor VE-cadherin and claudin-5 junction formation. In conclusion, PMVEC from aged mice exhibit compromised barrier function, leading to augmented permeability following MV. The age-dependent loss in barrier function appears to be due to an impaired ability to re-establish functional cell-cell junctions. These findings suggest that the aged vascular barrier is more susceptible to injury from MV, due to impaired PMVEC cell-cell junction integrity. Our results may highlight molecular pathways involved in predisposing aged individuals to worsened outcomes during MV, which can help in developing targeted therapeutics. Funded by Lawson Health Research Institute, CIHR, the Ontario Graduate Scholarship, and Western University. This is the full abstract presented at the American Physiology Summit 2024 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.

Abstract 14438: Loss of the ROR2 Tyrosine Kinase Receptor is Associated With Dysfunctional Angiogenesis in Pulmonary Hypertension via Inappropriate Focal Adhesion Activation and Disruption of the Endothelial Barrier

Background: Dysfunctional angiogenesis is a hallmark of pulmonary arterial hypertension (PAH), but its mechanisms are unclear. We recently demonstrated a key role for Wnt7a in lung angiogenesis. Wnt7a acts through ROR2, a Wnt pathway tyrosine kinase receptor. We elucidate how ROR2 controls angiogenic functions in pulmonary microvascular endothelial cells (PMVECs). Hypothesis: Loss of ROR2 activity is associated with PAH. Methods: Healthy and PAH PMVECs were obtained from explanted lungs. PMVECs were transfected with ROR2 siRNA/constructs, followed by functional and molecular studies. Focal adhesion (FA) activation and force generation was measured using FRET-based total internal reflection microscopy (TIRF). Mice with endothelial-specific conditional ROR2 KO (ROR2 ECKO) were phenotyped in normoxia and hypoxia. Results: ROR2 expression was found predominantly in the endothelium of healthy lung vessels but significantly reduced within PAH lesions. ROR2 knockdown (siROR2) reduced vascular sprouting comparable to PAH PMVECs. Interestingly, siROR2 and ROR2-deficient PAH PMVECs displayed increased fibronectin adhesion and hyperpermeability, which led us to look at focal adhesions (FA) and cell-cell junctions. Confocal and TIRF microscopy of siROR2 and ROR2-deficient PAH PMVECs revealed an increase in the FA number and force that inversely correlated with reduced VE-cadherin at cell junctions. However, transfection of a ROR2 construct in PAH PMVECs resulted in FAs activity normalization and junctional integrity accompanied by recovery of adhesion and permeability. Proteomic studies demonstrated that ROR2 interacts with Integrin b1 in focal adhesions, and its loss results in Integrin b1 activation, FA clustering, and high mechanical forces. In animal studies, we found that, compared to controls, ROR2 ECKO developed more severe pulmonary hypertension, right ventricular remodeling, microvascular reduction, and muscularization in hypoxia, which correlated with higher levels of integrin b1 activation and reduced VE-cadherin expression. Conclusions: ROR2 promotes PMVEC adhesion and barrier formation, and its loss is associated with dysfunctional angiogenesis. Restoring ROR2 could be a novel therapeutic strategy in PAH.

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

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

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.

Imbalance of Pulmonary Microvascular Endothelial Cell-Expression of Metalloproteinases and Their Endogenous Inhibitors Promotes Septic Barrier Dysfunction.

Sepsis is a life-threatening disease characterized by excessive inflammation leading to organ dysfunction. During sepsis, pulmonary microvascular endothelial cells (PMVEC) lose barrier function associated with inter-PMVEC junction disruption. Matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases (ADAM), which are regulated by tissue inhibitors of metalloproteinases (TIMPs), can cleave cell-cell junctional proteins, suggesting a role in PMVEC barrier dysfunction. We hypothesize that septic PMVEC barrier dysfunction is due to a disruption in the balance between PMVEC-specific metalloproteinases and TIMPs leading to increased metalloproteinase activity. The effects of sepsis on TIMPs and metalloproteinases were assessed ex vivo in PMVEC from healthy (sham) and septic (cecal ligation and perforation) mice, as well as in vitro in isolated PMVEC stimulated with cytomix, lipopolysaccharide (LPS), and cytomix + LPS vs. PBS. PMVEC had high basal Timp expression and lower metalloproteinase expression, and septic stimulation shifted expression in favour of metalloproteinases. Septic stimulation increased MMP13 and ADAM17 activity associated with a loss of inter-PMVEC junctional proteins and barrier dysfunction, which was rescued by treatment with metalloproteinase inhibitors. Collectively, our studies support a role for metalloproteinase-TIMP imbalance in septic PMVEC barrier dysfunction, and suggest that inhibition of specific metalloproteinases may be a therapeutic avenue for septic patients.

Tetramethylpyrazine ameliorates acute lung injury by regulating the Rac1/LIMK1 signaling pathway.

Acute lung injury (ALI) is a respiratory disorder characterized by severe inflammation of the alveoli and lung parenchyma. Tetramethylpyrazine (TMP), the main active compound in Ligusticum chuanxiong Hort (LC), can protect against lipopolysaccharide (LPS)-induced ALI. Our study aimed to investigate how TMP protects the endothelial cell barrier in pulmonary capillaries. We administered TMP intraperitoneally at different doses and found that acute lung injury in mice was improved, but not in a dose-dependent manner. TMP toxicity was tested in vitro. We observed that LPS-induced cytoskeletal remodeling was inhibited by TMP. Murine ALI was induced as follows: For the 1st hit, LPS (2mg/kg) was injected intraperitoneally; after 16h, for the 2nd hit, LPS (4mg/kg) was instilled intratracheally. The mice in treatment groups had TMP or dexamethasone administered intraperitoneally 30min prior to the 1st hit and 30min past the 2nd hit. Mice were euthanized 24h after the last injecting. We measured protein and mRNA levels using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase real-time PCR (RT-qPCR), respectively. The ultrastructural analysis was performed with transmission electron microscopy (TEM) and the cytoskeleton was observed by immunofluorescence. Immunohistochemistry and Western blotting were used to detect protein expression in the Rac1/LIMK1/ZO-1/occludin signal pathway. The results showed that TMP treatment decreased inflammatory cell infiltration and alleviated LPS-induced damage in lung tissue. Also, TMP significantly inhibited the Rac1/LIMK1/ZO-1/occludin signaling pathway. Our findings show that using TMP during sepsis can protect the pulmonary microvascular endothelial cell barrier and suppress inflammation. Therefore, TMP may have a promising therapeutic role in preventing acute lung injury from sepsis.

The dipeptidyl peptidase-4 inhibitor linagliptin ameliorates LPS-induced acute lung injury by maintenance of pulmonary microvascular barrier via activating the Epac1/AKT pathway

Pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction is a main pathophysiological feature of sepsis-related acute lung injury (ALI). This study aimed to investigate whether the dipeptidyl peptidase (DPP)-4 inhibitor linagliptin could protect against LPS-induced PMVECs barrier disruption and its underlying molecular mechanisms. A classical ALI animal model and LPS-treated PMVECs were applied and all were treated with or without linagliptin. Cellular experiments demonstrated that linagliptin could mitigate LPS-induced PMVECs hyperpermeability and intercellular junction (VE-cadherin, β-catenin, and ZO-1) disruption in a dose-dependent manner. Correspondingly, it was observed that linagliptin pretreatment distinctly relieved LPS-induced lung injury, oxidative stress, and pulmonary edema in vivo. Furthermore, we found that the inhibition of oxidative stress by linagliptin may be achieved by reversing impaired mitochondrial function. Mechanistically, linagliptin administration promoted the activation of the Epac1 pathway and its downstream AKT pathway, while inhibition of the Epac1/Akt signaling pathway significantly alleviated the above-mentioned protective effect of linagliptin on the PMVECs barrier. Taken together, these data suggest that linagliptin can effectively reserve PMVECs barrier dysfunction and inhibit oxidative stress to protect against ALI via activating the Epac1/AKT signaling pathway, and thus may become a potential clinical therapeutic strategy for ALI.

Protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells.

To investigate the protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells (PMVECs). Malondialdehyde and superoxide dismutase (SOD) were used to determine the optimal modeling of lung hypoxic injury cells. CCK-8 method was used to detect cell viability for determining the effective dose of areca nut polyphenols. Rat PMVECs were divided into control group, hypoxia model group and areca nut polyphenols group. BCA method was used to detect the protein concentration of each group, and the oxidative stress level in PMVECs was measured. Western blotting was used to detect the expression of inflammatory and apoptosis-related proteins. Immunofluorescence staining was used to detect the expression of occludin and zonula occludens (ZO) 1. Transwell chamber was used to detect transendothelial electrical resistance, and rhodamine fluorescent dye was used to detect PMVECs barrier permeability. The hypobaric hypoxia-induced cell injury model was established by culturing PMVECs for 48 h at 1% oxygen concentration. The 20 μg/mL areca nut polyphenols significantly reversed the survival rate and the oxidative stress of PMVECs in hypoxia model group (all P<0.05). Areca nut polyphenols had significant inhibitory effect on the up-regulation of inflammation-related proteins, including nuclear factor-κB (NF-κB) and nuclear factor-E2-related factor (Nrf) 2 in hypoxia model group (all P<0.05). And areca nut polyphenols could reduce hypoxia-induced PMVECs apoptosis by down-regulating the expressions of apoptosis-related proteins, including cysteine aspartic acid specific protease (caspase) 3, Bcl-2 associated X protein (Bax) in PMVECs (all P<0.05). In addition, areca nut polyphenols effectively improves the transendothelial electrical resistance and barrier permeability of PMVECs through elevating the expression of occludin and ZO-1 (all P<0.05). Areca nut polyphenols can inhibit the hypoxic damage of PMVECs by reducing oxidative stress and apoptosis down-regulating the expression of inflammatory proteins and reducing membrane permeability.

LPS induces pulmonary microvascular endothelial cell barrier dysfunction by upregulating ceramide production

The specific role of ceramides in pulmonary microvascular endothelial cell (PMVEC) barrier dysfunction remains unclear. In the present study, pretreatment with pan-caspase inhibitors significantly reduced LPS-induced PMVEC apoptosis and helped to stimulate PMVEC barrier reconstruction after 12 h but had no effect on PMVEC barrier dysfunction in the first 8 h. Further studies showed that imipramine, an acid sphingomyelinase (ASMase) inhibitor, significantly inhibited LPS-induced barrier dysfunction, while an siRNA targeting serine palmityl transferase subunit 1 (SPTLC1) and the pharmacological inhibitor myriocin did not inhibit early acute barrier dysfunction but significantly inhibited PMVEC apoptosis and apoptosis-dependent delayed barrier dysfunction. In addition, LPS was shown to activate RhoA by inducing transient receptor potential channel 6 (TRPC6) overexpression and calcium influx through the ASMase/ceramide pathway, and activation of RhoA further induced the cytoskeletal rearrangement of PMVECs and destruction of intercellular junctions, ultimately leading to early acute PMVEC barrier dysfunction. However, regarding apoptosis-dependent delayed barrier dysfunction, the ceramide-induced de novo synthesis pathway in paracellular cells induced the apoptosis of PMVECs, in which Txnip overexpression inhibited Trx activity and subsequently activated ASK1 in the context of LPS-induced PMVEC apoptosis, acting upstream of the ceramide-induced activation of p38 MAPK and JNK. At the same time, in rats with LPS- or exogenous C8 ceramide-induced ALI, ceramide was demonstrated to play an important role in lung injury by inducing the Txnip/TRX/ASK1/P38 and JNK pathways. Thus, the Txnip/TRX/ASK1/p38 and JNK pathways might be involved in ceramide-mediated PMVEC apoptosis in LPS-induced ALI.

Tumor necrosis factor-α requires Ezrin to regulate the cytoskeleton and cause pulmonary microvascular endothelial barrier damage

Acute respiratory distress syndrome (ARDS) is a rapidly progressive disease with unknown pathogenesis. Damage of pulmonary microvascular endothelial cells (PMVECs) caused by inflammatory storm caused by cytokines such as TNF-α is the potential pathogenesis of ARDS. In this study, we examined the role of ezrin and Rac1 in TNF-α-related pathways, which regulates the permeability of PMVECs. Primary rat pulmonary microvascular endothelial cells (RPMVECs) were isolated and cultured. RPMVECs were treated with rat TNF-α (0, 1, 10, 100 ng/ml), and the cell activity of each group was measured using a CCK8 kit. The integrity of endothelial barrier was measured by transendothelial resistance (TEER) and FITC-BSA flux across RPMVECs membranes. Pulldown assay and Western blot was used to detect the activity of RAS-associated C3 botulinum toxin substrate 1 (Rac1) and Ezrin phosphorylation. Short hairpin RNA (shRNA) targeting ezrin and Rac1 was utilized to evaluate the effect of RPMVECs permeability and related pathway. The effects of ezrin and Rac1 on cytoskeleton were confirmed by immunofluorescence. Our results revealed that active Rac1 was essential for protecting the RPMVEC barrier stimulated by TNF-α, while active ezrin could partially destroy the PMVEC barrier by reducing Rac1 activity and regulating the subcellular structure of the cytoskeleton. These findings may be used to create new therapeutic strategies for targeting Rac1 in the treatment of ARDS.

The Role of TIMP3 in Microvascular Endothelial Cell‐Extracellular Matrix Interaction and Regulation of Microvascular Barrier Function

BackgroundDuring sepsis, disruption of the pulmonary microvascular endothelial cell (PMVEC) barrier is associated with lung dysfunction and death. PMVEC‐PMVEC interactions through adherens and tight junctions are crucial for maintaining barrier function, as disruption of these junctions causes protein leak. Multiple mechanisms support PMVEC junctional and barrier function, including PMVEC binding to the extracellular matrix (ECM). Data suggest that decreased PMVEC‐ECM interactions under pro‐inflammatory conditions, indicated by altered focal adhesion kinase (FAK) phosphorylation, may be associated with increased leak. Tissue inhibitor of metalloproteinases 3 (TIMP3) is shown to regulate barrier function as PMVEC from Timp3−/− mice have increased leak; however, the specific mechanisms are unknown. Interestingly, previous studies in the developing lung found that TIMP3 can mediate PMVEC‐ECM interactions. Based on this, I hypothesized that TIMP3 maintains PMVEC barrier function by promoting PMVEC‐ECM interactions.MethodPMVEC from wild type (WT) and Timp3−/− mice were cultured on different matrices and stimulated with pro‐inflammatory cytokines (sepsis) vs control PBS. PMVEC‐ECM interactions were assessed using adhesion assays, immunocytochemistry, and the XPERT‐permeability assay.ResultsExamination of PMVEC adhesion to fibronectin, laminin, vitronectin, and collagen I indicated preferential adhesion of WT PMVEC to fibronectin with lowest adhesion to collagen. While Timp3−/− PMVEC appeared to also preferentially adhere to fibronectin, overall adhesion to the ECM vs. WT PMVEC was reduced. Assessment of phosphorylated FAK (pFAK) through immunocytochemistry revealed reduced abundance of pFAK in both cell types under cytomix‐treatment vs. PBS. Additionally, pFAK organisation and localised distribution around the periphery of cell borders appeared to be reduced in Timp3−/− vs. WT PMVEC. The XPERT‐permeability assay revealed differences in total inter‐PMVEC avidin fluorescence and specifically avidin leak diffusion distance between PMVEC and the ECM in WT vs. Timp3−/− cells, especially under cytomix‐treatment.SignificanceThese findings demonstrate that sepsis disrupts the interaction of WT PMVEC with ECM, which is further enhanced in Timp3−/− cells. The results suggest that TIMP3 may play an important role in regulating PMVEC‐ECM interactions, and could help elucidate therapeutic targets to maintain or rescue PMVEC barrier integrity in sepsis.Support or Funding InformationHeart and Stroke Foundation of Ontario, Ontario Thoracic Society Grant‐in‐Aid, NSERC Discovery Grant

Inhibition of Metalloproteinase Activity Promotes PMVEC Barrier Function Under Septic Conditions

BackgroundSepsis is a life‐threatening human disease with significant mortality characterized by excessive inflammation, which can lead to endothelial dysfunction, microvascular injury and organ damage. During sepsis, endothelial cells, especially the pulmonary microvascular endothelial cells (PMVEC), become injured leading to loss of barrier function and accumulation of protein‐rich edematous fluid. Metalloproteinases, including the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families, are capable of cleaving cell surface proteins, such as cell‐cell junctional proteins, suggesting a potential role in septic PMVEC barrier dysfunction. Metalloproteinase activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). Recent work in our lab found that PMVEC lacking TIMP3, a critical regulator of both MMP and ADAM activity, had increased permeability compared to wild type (WT) PMVEC.HypothesisWe hypothesize that PMVEC‐derived metalloproteinase activity will be increased under septic conditions and that vascular permeability will be reduced with the application of synthetic metalloproteinase inhibitors.Materials and MethodsPMVEC isolated from WT and Timp3−/− mice were stimulated with PBS (control) or cytomix (equimolar tumour necrosis factor α, interferon γ, and interleukin 1β) + lipopolysaccharide (LPS; septic) for 4 hours. Metalloproteinase activity was then assessed in the conditioned media and cell lysate, and trans‐PMVEC macromolecular flux was assessed using Evans blue‐labelled albumin. Additionally, PMVEC surface localization of VE‐cadherin (adherens junction) and claudin‐5 (tight junction) was assessed by immunofluorescence on WT and Timp3−/− PMVEC. To confirm the role of metalloproteinases, WT and Timp3−/− PMVEC were treated with synthetic metalloproteinase inhibitors.ResultsAnalysis of metalloproteinase activity revealed increased MMP13 and ADAM17 activity in septic PMVEC and Timp3−/− PMVEC leading to a loss of inter‐PMVEC junctional proteins and subsequent PMVEC barrier dysfunction. Importantly, the application of synthetic metalloproteinase inhibitors such as BB94, CL82198 and TAPI2 reduced the permeability and disruption of VE‐cadherin and claudin 5 under septic conditions.Discussion and conclusionIncreased metalloproteinase activity under septic conditions is associated with disrupted inter‐PMVEC junctional protein localization and loss of barrier function suggesting metalloproteinases are critical mediators of septic PMVEC barrier dysfunction. Further, PMVEC‐derived TIMP3 appears to be an important regulator of PMVEC‐derived metalloproteinase activity as loss of TIMP3 is associated with increased metalloproteinase activity and subsequent PMVEC barrier dysfunction. Thus, these studies suggest that inhibition of metalloproteinase activity may promote PMVEC barrier function by reducing inter‐junctional protein degradation during sepsis and thereby reducing septic vascular permeability.Support or Funding InformationOTSHeart and Stroke OGS

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.

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.

Activation of A2b adenosine receptor decreases lipopolysaccharide-induced pulmonary microvascular permeability

To explore the role of the A2b adenosine receptor (Adora2b) in lipopolysaccharide (LPS)-induced injury of human pulmonary microvascular endothelial cells (HPMECs), and its mechanism. HPMECs were cultured in vitro. The LPS dose-effect experiment, time-effect experiment and the Adora2b agonist/antagonist intervention experiment were performed respectively. (1) Dose-effect and time-effect experiments: HPMECs were stimulated with 1, 10, 100, 1 000 μg/L LPS for 24 hours, or 100 μg/L LPS for 4, 8, 12, 16, 24 hours. Cell viability was measured by cell counting kit-8 (CCK8). The protein and mRNA expressions of Adora2b were determined by Western Blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively. (2) Adora2b agonist/antagonist intervention experiment: serum-starved HPMECs 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 incubated with 100 μg/L of LPS for 24 hours. The HPMECs without treatment were served as blank control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. The monolayer permeability of HPMECs was determined by fluorescein isothiocyanate (FITC)-dextran. Cell cycle was analyzed by flow cytometry. The mRNA expressions of VE-cadherin, occludin, vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1) were determined by RT-PCR. (1) Dose-effect and time-effect experiments: LPS induced the decreased cell viability of HPMECs in dose and time-dependent manner. Compared with the control group, the protein expression of Adora2b was sharply up-regulated after 100 μg/L or 1 000 μg/L LPS stimulation. Meanwhile, LPS was shown to cause a dose and time-dependent induction of Adora2b transcript level. (2) Adora2b agonist/antagonist intervention experiments: compared with the control group, the monolayer permeability of HPMECs was rapidly enhanced after LPS treatment, and lower cell viability and proliferation, as well as the expression of cell junction and angiogenic factors were downregulated. Compared with LPS group, 0.1, 1, 10 μmol/L BAY 60-6583 pretreatment could decrease the endothelial cell barrier leakage in a dose-dependent manner [Pd: (203.06±15.24)%, (164.15±17.82)%, (125.69±10.38)% vs. (218.53±12.05)%], and promote cell proliferation of HPMECs [the proportion of S and G2 phases: (24.36±1.40)%, (32.37±0.95)%, (40.05±2.99)% vs. (18.83±0.73)%]. Pretreatment of 10 μmol/L BAY60-6583 also upregulated the mRNA expressions of cell junction and angiogenic factors [VE-cadherin (2-ΔΔCt): 2.17±0.23 vs. 0.56±0.10, occludin (2-ΔΔCt): 5.32±0.28 vs. 0.48±0.11, VEGF (2-ΔΔCt): 4.44±0.34 vs. 0.58±0.09, ANGPT-1 (2-ΔΔCt): 5.98±0.73 vs. 0.66±0.10, all P < 0.01]. PSB1115 pretreatment aggravated injury of microvascular endothelial cells after LPS incubation, with lower cell viability, slower proliferation and less expression of VEGF and ANGPT1. There was no influence of BAY 60-6583 or PSB1115 single treatment on cell viability, cell cycle and the expression of angiogenic factors in HPMECs. In vitro studies of cultured HPMECs exposed to LPS are identified as dose and time-dependent induction of Adora2b transcript and corresponding protein induction. Activation of Adora2b attenuates LPS-induced pulmonary microvascular endothelial cell barrier enhancement by regulating intercellular junction and promoting angiogenesis, suggesting Adora2b as potential therapeutic target in the treatment of LPS-induced forms of acute lung injury.

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.

Interleukin 22 attenuated angiotensin II induced acute lung injury through inhibiting the apoptosis of pulmonary microvascular endothelial cells

Apoptosis of pulmonary microvascular endothelial cells (PMVECs) was considered to be closely related to the pathogenesis of acute lung injury (ALI). We aim to investigate whether IL-22 plays protective roles in lung injury through inhibiting the apoptosis of PMVECs. ALI model was induced through subcutaneous infusion of angiotensin II (Ang II). Lung injury and infiltration of inflammatory cells were evaluated by determining the PaO2/FiO2, calculation of dry to weight ratio in lung, and immunohistochemisty analysis. Apoptosis of PMVECs was determined using TUNEL assay and flow cytometry, respectively. Immunofluorescence and Western blot analysis were used to determine the expression and localization of STAT3, as well as the nucleus transmission of STAT3 from cytoplasm after IL22 treatment. Pathological findings showed ALI was induced 1 week after AngII infusion. IL22 inhibited the AngII-induced ALI, attenuated the edema in lung and the infiltration of inflammatory cells. Also, it contributed to the apoptosis of PMVECs induced by AngII. Meanwhile, significant increase was noticed in the expression of STAT3, phosphorylation of Y705-STAT3, and migration from cytoplasm to the nucleus after IL-22 treatment (P < 0.05). The activation of STAT3 by IL22 showed significant attenuation after AG490 treatment. Our data indicated that IL22 showed protective effects on lung injury through inhibiting the AngII-induced PMVECs apoptosis and PMVEC barrier injury by activating the JAK2/STAT3 signaling pathway.

Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions.

Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix]) of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

Caspase-1 Activation Protects Lung Endothelial Barrier Function during Infection-Induced Stress.

Dysregulated activation of the inflammasome-caspase-1-IL-1β axis elicits damaging hyperinflammation during critical illnesses, such as pneumonia and sepsis. However, in critical illness models of Salmonella infection, burn, or shock, caspase-1 inhibition worsens outcomes. These paradoxical effects suggest that caspase-1 drives novel protective responses. Whether the protective effects of caspase-1 activation involve canonical immune cell and/or nonimmune cell responses is unknown. The objective of this study was to test the hypothesis that, in addition to its recognized proinflammatory function, caspase-1 initiates protective stress responses in nonimmune cells. In vivo, lung epithelial and endothelial barrier function and inflammation were assessed in mice infected with Pseudomonas aeruginosa in the presence or absence of a caspase-1 inhibitor. Lung endothelial barrier function was assessed ex vivo in isolated, perfused rat lungs infected with P. aeruginosa in the presence or absence of a caspase-1 inhibitor. Endothelial barrier function during P. aeruginosa infection was assessed in vitro in cultured rat wild-type pulmonary microvascular endothelial cells (PMVECs) or recombinant PMVECs engineered to decrease caspase-1 expression. We demonstrated in vivo that caspase-1 inhibition in P. aeruginosa-infected mice ameliorated hyperinflammation, but, counterintuitively, increased pulmonary edema. Ex vivo, caspase-1 inhibition increased pulmonary permeability in P. aeruginosa-infected isolated rat lungs. To uncouple caspase-1 from its canonical inflammatory role, we used cultured rat PMVECs in vitro and discovered that genetic knockdown of caspase-1 accelerated P. aeruginosa-induced barrier disruption. In conclusion, caspase-1 is a sentinel stress-response regulator that initiates proinflammatory responses and also initiates novel response(s) to protect PMVEC barrier function during pneumonia.

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.