Endothelial Cell Barrier Research Articles

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.

Blood-brain barrier shuttle peptides enhance AAV transduction in the brain after systemic administration

The adeno-associated virus (AAV) vector has been used in preclinical and clinical trials of gene therapy for central nervous system (CNS) diseases. One of the biggest challenges of effectively delivering AAV to the brain is to surmount the blood-brain barrier (BBB). Herein, we identified several potential BBB shuttle peptides that significantly enhanced AAV8 transduction in the brain after a systemic administration, the best of which was the THR peptide. The enhancement of AAV8 brain transduction by THR is dose-dependent, and neurons are the primary THR targets. Mechanism studies revealed that THR directly bound to the AAV8 virion, increasing its ability to cross the endothelial cell barrier. Further experiments showed that binding of THR to the AAV virion did not interfere with AAV8 infection biology, and that THR competitively blocked transferrin from binding to AAV8. Taken together, our results demonstrate, for the first time, that BBB shuttle peptides are able to directly interact with AAV and increase the ability of the AAV vectors to cross the BBB for transduction enhancement in the brain. These results will shed important light on the potential applications of BBB shuttle peptides for enhancing brain transduction with systemic administration of AAV vectors.

Microfluidic platform for the real time measurement and observation of endothelial barrier function under shear stress.

Electric cell-substrate impedance sensing (ECIS) is a quickly advancing field to measure the barrier function of endothelial cells. Most ECIS systems that are commercially available use gold electrodes, which are opaque and do not allow for real-time imaging of cellular responses. In addition, most ECIS systems have a traditional tissue culture Petri-dish set up. This conventional set-up does not allow the introduction of physiologically relevant shear stress, which is crucial for the endothelial cell barrier function. Here, we created a new ECIS micro-bioreactor (MBR) that incorporates a clear electrode made of indium tin oxide in a microfluidic device. Using this device, we demonstrate the ability to monitor the barrier function along culture of cells under varying flow rates. We show that while two cell types align in the direction of flow in responses to high shear stress, they differ in the barrier function. Additionally, we observe a change in the barrier function in response to chemical perturbation. Following exposure to EDTA that disrupts cell-to-cell junctions, we could not observe distinct morphological changes but measured a loss of impedance that could be recovered with EDTA washout. High magnification imaging further demonstrates the loss and recovery of the barrier structure. Overall, we establish an ECIS MBR capable of real-time monitoring of the barrier function and cell morphology under shear stress and allowing high-resolution analysis of the barrier structure.

Abstract B41: Tumor endothelial cells as a targetable gateway that modulates access of drugs to cancer cells

Abstract Introduction: Based upon our demonstration that the human endothelial cell (EC) lining of the tumor vasculature of human prostate adenocarcinoma is preserved in primary xenografts of intact surgical remnant tissue, this study sought to determine the consequences of targeted perturbation of this key component of the tumor microenvironment on access of chemotherapeutic agents to prostate cancer cells (CaP). Methods: Primary xenografts of intact CaP tissue were employed to investigate multiple aspects of the response of the human ECs to iatrogenic modulation, including changes in gene expression, changes in cell-cell interaction, and in the function of human tissue/tumor microvascular EC-mediated, blood–tissue barrier. SCID mice were castrated and implanted with a device for sustained delivery of testosterone (T) to establish “humanized” mice. Freshly procured surgical specimens of human CaP or benign human prostate were transplanted onto the “humanized” mice within 3-7 days. Xenografts were allowed 30 days to engraft before the integrity of the human microvascular EC barrier was perturbed by removal of the T delivery device, T-deprivation (T-D). T-D induced apoptosis of the AR-expressing human ECs was maximal on Day 3, resulting in loss of 40-60% of human ECs. Significantly, the microvascular compartment re-endothelialized by Day 15 after T-D, in spite of the absence of T, providing a unique model for analysis of the role of the repaired human EC compartment in “acquired resistance.” After various times/treatments, xenografts were harvested, enzymatically disaggregated, and EC and CaP cells isolated using cell type-specific surface marker antibody-conjugated magnetic beads. Results: Enhanced access to the xenograft tissue of imaging markers in circulation during the interval of the compromised EC barrier, and after re-endothelialization, was demonstrated by: MRI (contrast dyes), MS (nanoparticles), immunohistochemistry (IHC) (lectins), and photo-acoustic imaging (hemoglobin). RNA-Seq of EC and epithelial/CaP populations characterized similarities/differences in gene expression profiles between cells recovered from fresh tissue versus established xenografts, and the effects of T-D in xenografts. Of ~2000 genes identified in the literature as AR-regulated/interacting, few were affected in either ECs or epithelial cells by T-D, with the notable exception of KLK2, KLK3, KLK4, MSMB, and ORM2. Expression profiles of membrane transporters and pumps for cellular uptake and efflux of chemotherapeutic agents were different between fresh tissue and established xenograft. Cisplatin (Cis-Pt) was investigated because it is excluded effectively by the membrane pumps/transporters in human prostate ECs, and it effectively kills slowly/nonproliferating cancer cells. IHC analysis using an antibody specific for Cis-Pt-induced DNA dimers demonstrated significantly greater levels of Pt-DNA adducts in xenografts treated during the T-D induced “window.” Quantitative analysis by Cytof technology of adduction in ECs and CaP cells isolated from xenografts is under way. Conclusions: Transient destruction of the EC barrier by T-D provides a therapeutically important “window” during which chemotherapeutic agents that are effectively excluded by the intact prostate EC barrier flood the CaP tissue microenvironment, providing therapeutically significant levels at reduced systemic dosages. Current analyses are focused on characterization of genes that respond to T-D, particularly differential characterization of gene expression profiles in the tumor ECs that re-endothelialize CaP xenografts after T-D. This model provides a unique tool for analysis of the roles of targeted microvascular damage in development of organ-specific therapies, and of the evolution of the EC compartment in response to iatrogenic intervention in “acquired resistance.” Citation Format: Michael J. Greene, Gary J. Smith, Yue Wu. Tumor endothelial cells as a targetable gateway that modulates access of drugs to cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B41.

Vascular endothelial injury in severe fever with thrombocytopenia syndrome caused by the novel bunyavirus

Severe fever with thrombocytopenia syndrome virus (SFTSV) infection typically causes acute fever, thrombocytopenia and leucopenia, presenting with a high case fatality rate. The pathogenesis of SFTSV infection, however, is not well described. It was hypothesized that endothelial dysfunction might play part in the disease process. In current study, we retrospectively analyzed the clinical manifestations among a large group of confirmed SFTS cases and found evidence of plasma leakage and vascular endothelial injury. Then we established a SFTSV infection cell model and determined the infectivity and stimulation of SFTSV on vascular endothelial cells in vitro. The hyperpermeability of endothelial cells directly induced by SFTSV was confirmed by electrical resistance and dextran diffusion assay. The virus induced alterations of cell junctions and cytoskeleton was also revealed. It's suggested that vascular endothelial cell injury and barrier function damage were induced after SFTSV infection, which is a vital but neglected pathogenesis of SFTS.

Abstract 461: Enhanced Glycolysis in Endothelial Cells Protects Against Atherosclerosis

Background: Increased aerobic glycolysis in endothelial cells (ECs) of atheroprone areas of blood vessels is hypothesized to drive increased inflammation and lesion burden but the direct links are not yet established. The goal of this study was to determine how metabolic adaptation in ECs impacts atherosclerosis. Methods: The immunostaining, Real-time PCR, Western blots, Seahorse assayes are used to analyze the expression and activity of PRKA/AMPKs and metabolic glycolysis in the arterial endothelium of mice and cultured endothelial cells under disturbed flows. Staining of Edu, TUNNEL and Evans blue were utilized to evaluate endothelial turnover and endothelium integrity. Oil Red O staining and histological cellular characterization were performed to evaluate atherosclerosis. Results: ECs exposed to disturbed flow in vivo and in vitro exhibit increased levels of PRKA/AMPKs (protein kinase AMP-activated; AMP-activated protein kinases) and glycolysis. Selective deletion of endothelial Prkaa1 (protein kinase AMP-activated catalytic subunit alpha1) reduced glycolysis, compromised EC proliferation and EC monolayer integrity, and accelerated the formation of atherosclerotic lesions in hyperlipidemic mice. Rescue of impaired glycolysis in Prkaa1-deficient ECs through Slc2a1 transfection enhanced EC viability and integrity of the EC barrier and reversed susceptibility to atherosclerosis. These results suggest that increased glycolysis in the endothelium of atheroprone arteries is a protective mechanism. Conclusions: Endothelial glycolysis plays a vital role in EC proliferation, repair of the EC monolayer, and protects mice from atherosclerosis. Thus, approaches such as activating AMPK activity or increasing endothelial glycolysis are preventive and therapeutic for arterial diseases.

Abstract 213: Human Genetic Variations in Neuroimmune Guidance Cues and their Role in Premature Atherosclerosis

Introduction: Neuroimmune guidance cues (NGCs), a family of proteins originally known for controlling neuronal migration, have been shown to be involved in atherosclerosis in mice models by regulating adhesion of monocytes to the vascular endothelium. The NGC family consists of multiple receptors and ligands enabling an interplay between inflammatory cells and the vasculature endothelium. We set out to investigate the role of NGCs in atherosclerosis in humans. Methods and results: We sequenced a total of 77 NGC genes in a unique cohort of 89 patients with premature atherosclerotic (PAS), in whom no clear CVD risk factor was present. A total of 178 rare variants were identified and we classified the pathogenicity of these rare (MAF&lt;0.05) variants by high Combined Annotation Dependent Depletion (CADD) scores. Based on literature and cosegregating pedigrees of the 88 possible deleterious variants we chose two variants in Netrin1 (NTN1) and EphrinB2 (EFNB2) to explore and validate their causality in human atherosclerosis. The expression of both these genes is regulated by flow and inflammatory stimuli. Under atherosclerotic conditions endothelial cells upregulate EFNB2 while NTN1 is downregulated. In addition, using immunohistochemical staining on human aorta’s, we found that NTN1, which is normally expressed on human endothelial cells, is less abundant in increasing stages of atherosclerosis. For EFNB2, a cell surface transmembrane ligand for Eph receptors, we performed several functional assays using HUVEC cells that overexpress EFNB2 , showing that EFNB2 is affecting endothelial cell barrier function, as well as proliferation. Conclusion: In patients with PAS rare missense variants in NGC genes were identified. Further functional- and expression assays showed an effect of NTN1 and EFNB2 on different aspects of the human atherosclerotic process. The results obtained from these studies substantiate the role of NGCs in human atherosclerotic disease and may further identify NGCs as novel potential therapeutic targets in our strive against cardiovascular disease.

Application of Heparin/Collagen-REDV Selective Active Interface on ePTFE Films to Enhance Endothelialization and Anticoagulation.

Expanded polytetrafluoroethylene (ePTFE) prosthetic valves have been widely used in clinical applications in Asian countries. However, these valves still have limits with regard to thrombosis, neointimal hyperplasia, restenosis, and valvar vegetation. The achievement of in situ endothelialization on implant materials is a promising way to overcome those limits. Here, heparin/collagen multilayers were fabricated on ePTFE films via a layer-by-layer (LBL) self-assembly technique, and then, the endothelial cell (EC) adhesive peptide sequence Arg-Glu-Asp-Val (REDV) was immobilized on the multilayers. After modification with the heparin/collagen multilayers with or without REDV peptide, less platelet activation and aggregation were observed, the blood coagulation time was increased, and the hemolysis rate was decreased comparedto that on pristine ePTFE films. The REDV-functionalized ePTFE films positively impacted early EC adhesion, later cell proliferation and cell activity. The EC barrier was confirmed to be successfully achieved on the functionalized ePTFE film surface in vitro. The successful assembly of the REDV-functionalized heparin/collagen multilayer on ePTFE films improved the blood compatibility, anticoagulant properties, and cell compatibility of the films in vitro, and thus, represents a candidate approach for applications requiring quick in situ endothelialization in vivo.

Chemical Composition of Scrophularia lucida and the Effects on Tumor Invasiveness in Vitro.

A detannified methanolic extract of Scrophularia lucida L. attenuated the formation of cancer cell-induced circular chemorepellent induced defects (CCIDs) in the lymph endothelial cell barrier, which resemble entry ports for the intravasating tumor into the vasculature as a prerequisite for lymph node metastasis. Therefore, the composition of this extract was studied in an activity-guided approach. Since no data on the secondary metabolites of this plant were available, first phytochemical data were collected in the course of the fractionation of the extract. The study resulted in the identification of 14 substances, among them very rare iridoids, such as scrovalentinoside or koelzioside, and several flavonoids (e.g., nepitrin and homoplantaginin). One of the latter group, 2″-O-acetyl-homoplantaginin, is a new natural compound. In the most active fraction, the flavonoid hispidulin was identified as major component and the assay of the pure compound confirmed a contribution of hispidulin to the CCID-inhibitory effects of S. lucida. The activity of the two major iridoids in this assay was less compared to hispidulin.

HDAC7 in MLCP‐mediated endothelial barrier preservation

Acute lung injury (ALI) is an acute systemic inflammatory process characterized by pulmonary infiltrates hypoxemia and edema. ALI arises from a wide range of lung injuries such as toxins or inflammatory mediators. Despite the high level of mortality, to date no pharmacological therapies have been proven to improve ALI except from ventilation strategies. A deeper understanding of the pathogenic mechanisms involved remains a barrier to new and effective therapies that will reduce the vascular leak associated with ALI.A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier. Endothelial cells (EC) are a specialized cell type that line the lumen of the blood vessels serving every major organ system and provide a semi‐selective barrier between the blood and the interstitial space. Epigenetic mechanisms have emerged as a major regulator of enduring changes in cell behavior and the therapeutic potential of inhibiting histone deacetylases (HDACs) for the treatment of cardiovascular and inflammatory diseases is gaining increasing attention. In addition to histones, it has been shown that HDACs can deacetylate many non‐histone proteins to regulate cellular functions such as cytoskeletal acetylation and polymerization, and signal transduction. Further, it has been shown that HDAC inhibition is directly involved in EC barrier preservation, however, the molecular mechanisms of HDAC‐mediated EC barrier regulation remain largely unexplored.Our data indicate that the class IIa histone deacetylases comprising of HDACs 4, 5, 7, 9 are involved in the regulation of pulmonary vascular barrier in vitro and in lipopolysaccharide (LPS)‐induced murine model of ALI. These HDACs are known to function as transcriptional co‐repressors, however, they also are able to shuttle from nucleus to the cytoplasm thus becoming accessible for extra nuclear targets. We demonstrate that HDAC 7 is directly involved in LPS‐induced human lung microvascular EC (HLMVEC) barrier compromise. Further, barrier‐disruptive effects of LPS are accompanied by HDAC7 phosphorylation and its nuclear export. Conversely, agents that protect the EC barrier such as adenosine, decrease its phosphorylation. These data suggest the involvement of phosphatase activity in HDAC7‐mediated EC barrier regulation. Our results show that HDAC7 is a substrate for myosin light chain (MLC) phosphatase (MLCP) in HLMVEC. Conversely, down‐regulation of HDAC7 may affect MLCP activity. However, the physiological role of HDAC7/MLCP crosstalk in EC barrier regulation is currently unexplored. It is known that activation of MLCP leads to dephosphorylation of MLC thus opposing actomyosin contraction. Our newly published results suggest that MLCP activation may be involved in EC barrier preservation via the dephosphorylation of unconventional MLCP cytoskeletal targets such as moesin. Our results demonstrate a direct association between HDAC7 and moesin, suggesting a novel role of a HDAC7/MLCP/moesin signaling complex in the regulation of EC barrier. Collectively, these data demonstrate the involvement of HDAC7 activity in MLCP‐mediated EC barrier preservation and suggest, for the first time, the importance of HDAC7 nuclear export in pulmonary vascular injury induced by edemagenic agents such as LPS.Support or Funding InformationNIH; Contract grant number: HL101902This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ceramide Containing Microparticles from Aged Stored Platelets Recapitulate Aspects of Murine Transfusion Related Acute Lung Injury

BACKGROUNDTransfusion‐related acute lung injury (TRALI) presents as chest infiltrates and hypoxia after 0.02–1% of transfusions with an associated mortality of 5–15%. Aged platelet products elicit TRALI via a mechanism invovling formation of the sphingolipid ceramide (CER) by acid sphingomyelinase (ASM), which causes lung injury in vivo and impairs pulmonary endothelial cell (EC) barrier integrity in vitro. Concomitantly, aged platelets form abundant microparticles (PMPs) which can shuttle nucleic acids, proteins, and ‐ importantly ‐ lipids, to target cells.OBJECTIVETo test the hypothesis that aged (5 days) but not fresh (1 day) platelets will generate PMP that mediate TRALI in a CER‐dependent manner.METHODSPMPs from male C57BL/6 wild type (WT) or Smpd1−/− (ASM knockout) platelets stored for 1 (fresh) or 5 (aged) days duration were isolated by differential centrifugation and filtration before enumeration (flow cytometry) and CER analysis (mass spectroscopy). Cultured human microvascular pulmonary ECs (passage 6–8) were incubated with PBS (sham) or 5×105 fresh or aged PMPs for 4 hours and transendothelial electrical resistance (TEER) was measured every 30 minutes. For TRALI induction, BALB/c male mice primed with 2 mg/kg intraperitoneal LPS two hours prior to intravenous (IV) infusion of 10 ml/kg PMPs (≈ 5×106) or saline (sham) and were assessed (6 hours later) by measuring lung tissue wet‐to‐dry (W/D) ratios, bronchoalveolar lavage fluid protein (BAL‐p) and lung tissue myeloperoxidase activity (MPO).RESULTSCompared with fresh WT PMP, aged WT PMPs were more numerous and CER‐enriched. In contrast, aged ASM KO PMPs were less numerous compared with fresh ASM KO PMP but contained similar CER content as WT aged PMPs. Aged (CER rich) but not fresh (CER poor) PMPs lead to reductions in TEER (&lt; 2 hours) in vitro in keeping with EC loss of barrier integrity which persisted for &gt; 4 hours. Reductions in TEER seen over 4 hours with aged PMPs was prevented with pre‐treatment of WT PMPs with ceramidase (0.75 μg/mL) prior to incubation with ECs. LPS primed mice receiving IV aged WT PMPs exhibited increased W/D (p &lt; 0.05), MPO and BAL‐p (p&lt;0.05) relative to mice receiving LPS priming in addition to saline, fresh PMPs or ASM KO aged PMPs.CONCLUSIONSCeramide formation in aged platelets contributes to PMP formation and impairs human EC barrier integrity resulting in TRALI. Removal or modification of aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationThis work was jointly funded by Canadian Blood Services and the Canadian Institute of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microfluidics: A high-throughput system for the assessment of the endotheliopathy of trauma and the effect of timing of plasma administration on ameliorating shock-associated endothelial dysfunction.

Early resuscitation after trauma-hemorrhagic shock with plasma rather than crystalloid may ameliorate systemic endothelial cell (EC) injury and dysfunction (endotheliopathy of trauma). We postulated that endothelial-lined microfluidic networks would be a useful platform to study the EC activation/injury under flow conditions to mimic trauma-hemorrhagic shock. We then used the microfluidic system to further characterize the protective effects and optimal timing of plasma infusion on the development of "endotheliopathy of trauma" in our model. Human umbilical vein ECs were added to microfluidic flow channels, and after overnight perfusion, the cells were subsequently treated with epinephrine and exposed to hypoxia reoxygenation. Media alone or 5% human plasma was perfused either immediately following treatment (early plasma) or after a 3-hour delay (late plasma). Glycocalyx injury was indexed by fluorescent microscopy and shedding of syndecan 1 and hyaluronic acid. Endothelial markers of activation/injury were also measured and included soluble thrombomodulin, tissue plasminogen activator, plasminogen activator inhibitor 1, and angiopoietins 1 and 2. Sheddase activity was indexed by ADAM metallopeptidase domain 17. Endothelial cell and glycocalyx barrier function studies using microfluidic devices are a more realistic model of the glycocalyx endothelial vascular barrier than studies performed on ECs using static (no flow) conditions. Conditions that mimic the internal milieu following hemorrhagic shock result in glycocalyx degradation and an inflammatory prothrombotic response by the endothelium. "Early" use of plasma in the microfluidic channel perfusate mitigated against these effects. Later perfusion with plasma had no protective effect. A temporal effect to plasma administration was noted in our biomimetic model of the endothelial vascular barrier following shock. This suggests a protective role to "early" plasma administration in the severely injured patient.

Endothelial Barrier Restoration after Inflammation‐Induced Hyperpermeability

Inflammation is characterized by an increase in endothelial barrier permeability (hyperpermeability) to macromolecules. Major efforts have focused on understanding the mechanisms involved in the hyperpermeability response of endothelial cells; however, less attention has been given to the restoration of vascular barrier integrity following hyperpermeability. Because many negative effects of hyperpermeability are due to its persistence beyond what is required for preserving organ function, we are investigating mechanisms that terminate hyperpermeability and thereby restore microvascular barrier properties. Understanding the mechanisms involved in the restoration of endothelial barrier integrity will have direct clinical impact because sustained inflammation is a hallmark in clinical disorders such as trauma and ischemia‐reperfusion. We demonstrated that eNOS translocation from plasma membrane to cytosol is required for the onset of hyperpermeability. We also demonstrated that stimulation of Epac1 (exchange protein activated by cAMP) contributes to restoration of basal permeability after ischemia‐reperfusion. In this study, we tested the hypothesis that 1) hyperpermeability induces delayed increase in cAMP concentration; and 2) cAMP stimulates Epac1 and VASP (vasodilator‐stimulated phosphoprotein) to promote translocation of eNOS back to the plasma membrane, and restores endothelial barrier properties. We use human microvascular (HMVEC) and mouse myocardial endothelial cells (Myend) to study the restoration of endothelial barrier after hyperpermeability induced by platelet‐activating factor (PAF). Myend VASP knockout (KO) endothelial cells (EC) have significantly greater baseline permeability to macromolecules as well as hyperpermeability response to PAF compared to wild type Myend cells. Stimulation of Epac1 in HMVEC inactivates their hyperpermeability response to PAF. However, Epac1 stimulation fails to inactivate PAF‐induced hyperpermeability in VASP KO Myend EC. PAF activates VASP by phosphorylation at Serine 157 and Serine 239. This phosphorylation is sensitive to eNOS inhibition indicating that PAF stimulates production of eNOS‐derived NO and VASP phosphorylation in a sequential manner. In addition, stimulation of Epac1 after the onset of PAF‐induced hyperpermeability translocates eNOS back from the cytosol to the plasma membrane in HMVEC and in control Myend. eNOS is preferentially located in the cytosol in Myend VASP KO cells. Lack of VASP prevents movement of eNOS associated with stimulation of Epac1. We conclude that VASP is a required protein for movement of eNOS and restoration of EC barrier.Support or Funding InformationRutgers School of Gradute StudiesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alpha‐2c Adrenergic Receptor Promotes the Malignant Phenotype of Colon Cancer Cells

Cancer remains one of the leading causes of death worldwide, with colorectal cancer (CRC) being the third most common type. Accumulating evidence shows that stress plays a pivotal role in the malignant phenotype of CRC, by promoting hallmarks of CRC including cellular proliferation, migration, invasion, and angiogenesis. During stressful situations, the sympathetic nervous system is activated, causing an elevation in levels of catecholamines, including epinephrine which elicits its effects mainly by activating adrenoceptors. Interestingly, however, the role of alpha2C adrenergic receptor (a2c‐AR) in CRC malignancy remains obscure. Here, we proposed that activation of a2c‐AR potentiates the malignant phenotype of human CRC cells. Treatment of human colon cancer cell lines, SW480 and HT‐29, with increasing concentrations of a2‐AR specific agonist (UK 14,304; 10, 30 or 100 nM) for different time points (24, 48, or 72 hours) did not cause a significant change in proliferation. However, using monolayer scratch assay, a significant increase in migration was observed after treatment with UK 14,304 (100 nM) for 8 and 12 hours in both cell lines. This was further supported by a transwell migration assay where UK 14,304 increased cell migration. Pretreatment with a specific a2c‐AR antagonist (JP 1302; 100 nM) abolished the UK 14,304‐induced cell migration. This increase in migratory capacity was concomitant with increased Rho activation evident by translocation of Rho from cytoplasm to the membrane as well as via a Rhotekin‐binding activation assay. Moreover, treatment with UK 14,304 evoked actin reorganization, an element known to be a prerequisite for cell migration. The induced Rho activation and actin cytoskeletal rearrangement could be mediated by increased oxidative stress since treatment with UK 14,304 increased ROS production. Importantly, ROS is known to induce migration of these cells. Furthermore, adhesion of colon cancer cells to TNF‐alpha‐activated HUVECs was promoted by UK 14,304. This UK14,304‐induced adhesion was followed by a dramatic increase in transendothelial migration of SW480 through HUVECs, clearly indicating that a2c‐AR activation may promote intra‐ or extravasation. Because breaking the endothelial cell barrier requires matrix digestion, we measured the levels of matrix metalloproteinases (MMPs). Importantly, stimulation of a2c‐ARs by UK14,304 increased the production of MMP2 and MMP9, and JP 1302 abolished this UK14,304‐induced production. Taken together, our data suggests that a2c‐AR potentiates the migratory and invasive capacities and hence the malignant phenotype of human colon cancer cells. Therefore, a2c‐AR may represent a novel target in the treatment of CRC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration.

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which alternative signaling entities can be created; yet, how receptor heterodimers affect receptor pharmacology remains unknown. Previous observations suggested a biochemical antagonism between GPCRs, CXCR4 and CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on the membrane of cancer cells, inhibiting their metastatic potential in vitro However, the reduced signaling entities responsible for the observed functional outputs remain elusive. This study now delineates the signaling mechanism whereby heterodimeric association between CXCR4 and CB2, induced by simultaneous agonist treatment, results in decreased CXCR4-mediated cell migration, invasion, and adhesion through inhibition of the Gα13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Gα13 (GNA13), and subsequently, the small GTPase RhoA, which is required for directional cell migration and the metastatic potential of cancer cells. These studies in prostate cancer cells demonstrate decreased protein expression levels of Gα13 and RhoA upon simultaneous CXCR4/CB2 agonist stimulation. Furthermore, the agonist-induced heterodimer abrogated RhoA-mediated cytoskeletal rearrangement resulting in the attenuation of cell migration and invasion of an endothelial cell barrier. Finally, a reduction was observed in the expression of integrin α5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices in vitro Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease.Implications: This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migration. Mol Cancer Res; 16(4); 728-39. ©2018 AACR.

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.

Developmental differences in focal adhesion kinase expression modulate pulmonary endothelial barrier function in response to inflammation.

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

TGF-\u03b21 promotes cell barrier function upon maturation of corneal endothelial cells

Human corneal endothelial cells (HCECs) easily become fibroblastic-like when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor β (TGF-β) could be a key factor in this phenomenon; however, TGF-β is also known to maintain the endothelium in a quiescent state in vivo. This work aimed to compare the effects of TGF-β1 on the phenotype of HCECs during the proliferation and maturation phases. Our results show that addition of TGF-β1 during the active proliferation phase produced fibroblastic HCECs and loss of the cell junction markers ZO-1 and n-cadherin, independent from the presence of epidermal growth factor (EGF). By contrast, addition of TGF-β1 in maturation media containing few mitogens led to an endothelial phenotype and functional cell junctions as HCECs developed a high trans-endothelial resistance. Furthermore, addition of AG-1478, an epithelial growth factor receptor inhibitor, enhanced the gain of the endothelial phenotype and cell barrier function. Overall, these results show that TGF-β1 can be used to promote the formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs. A two-phase culture of HCECs using distinct proliferation and maturation media could also be key for developing ideal HCEC culture conditions.

Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: Molecular mechanisms and barrier-protective role.

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

Myosin light chain kinase ( MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y464 and Y471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.