Interendothelial Gap Formation Research Articles

CUMP elicits interendothelial gap formation during Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa utilizesa type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote interendothelial gap formation, impair motility, and arrest cell growth. The disruptive activities of cAMP and cGMP during the P. aeruginosa infection are established; however, little is known about the function of cUMP. Here, we tested the hypothesis that cUMP contributes to endothelial cell barrier disruption during P. aeruginosa infection. Using a membrane permeable cUMP analog, cUMP-AM, we revealed that during infection with catalytically inactive ExoY, cUMP promotes interendothelial gap formation in cultured pulmonary microvascular endothelial cells (PMVECs) and contributes to increased filtration coefficient in the isolated perfused lung. These findings indicate that cUMP contributes to endothelial permeability during P. aeruginosa lung infection.NEW & NOTEWORTHY During pneumonia, bacteria utilizea virulence arsenal to communicate with host cells. The Pseudomonas aeruginosa T3SS directly introduces virulence molecules into the host cell cytoplasm. These molecules are enzymes that trigger interkingdom communication. One of the exoenzymes is a nucleotidyl cyclase that produces noncanonical cyclic nucleotides like cUMP. Little is known about how cUMP acts in the cell. Here we found that cUMP instigates pulmonary edema during Pseudomonas aeruginosa infection of the lung.

Abstract 1104: Investigation Into The Potential Role Of Perceived Loneliness And Endothelial Dysfunction In A Community-based African American Cohort At-risk For Cardiovascular Disease: A Translational Approach

Background: Chronic stress is a significant contributor for cardiovascular disease (CVD) and existing health disparities. Loss of endothelial barrier integrity is a hallmark of CVD, but little is known how chronic stress impacts endothelial barrier integrity. This study aimed to investigate the signaling pathways of how loneliness-related chronic stress could impact endothelial barrier integrity. Methods: We examined the relationship among validated questionnaires of loneliness-related chronic stress and various hormonal and cytokines in a community-based cohort of African American adults at risk for CVD from resource-limited neighborhoods . Then, we conducted ex vivo as well as in vitro experiments to probe these effects and investigate the mechanism of action. Results: We determined that a 1-SD increase in loneliness was associated with a 0.32-increase (p=0.04) in the product of epinephrine (E, a catecholamine increased in individuals experiencing chronic stress) and TNFa (T, a cytokine increased in individuals experiencing loneliness), suggesting a synergism between epinephrine and TNFa. In ex vivo experiments, the product of E*T was associated with a loss in endothelial VE-cadherin expression (p=.03). In subsequent in vitro experiments to investigate this effect we treated human aortic endothelial cells with E, T, or their combination; together, these biomarkers decreased endothelial VE-cadherin expression (E: 0.94 ± 0.11, T: 0.79 ± 0.05 vs E+T: 0.58 ± 0.06-fold change of control, p<0.01) and dampened endothelial barrier integrity by increasing inter-endothelial gap formation (E: 1.59 ± 0.16 and T: 1.65 ± 0.17 vs E+T: 2.25 ± 0.13-fold change of control, p<0.001). Furthermore, we determined that the impact of E+T on endothelial cells is JAK/Stat signaling pathway dependent, establishing a putative mechanism. Conclusions: We found that a combination of epinephrine and TNFa decreased endothelial barrier integrity, suggesting an additive effect potentially accelerating CVD development and progression in individuals experiencing loneliness-related chronic stress. We provide a potential mechanism by which chronic stress could impact endothelial barrier integrity and function - ultimately worsening CVD health disparities.

Development of an In Vitro Model to Study Mechanisms of Ultrasound-Targeted Microbubble Cavitation–Mediated Blood–Brain Barrier Opening

Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood-brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability. This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment. This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model. Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability.

Skeletal muscle-derived FSTL1 starting up angiogenesis by regulating endothelial junction via activating Src pathway can be upregulated by hydrogen sulfide.

Hydrogen sulfide (H2S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H2S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H2S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H2S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H2S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H2S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway.NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H2S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

Donepezil is emerging as a potential therapeutic treatment in improving endothelial barrier function to prevent and treat coronary artery disease in Diabetes Mellitus.

Abstract Current treatments have shown limited success in reversing DM induced vascular endothelial barrier dysfunction. Vascular endothelium forms the inner most lining of the blood vessels, regulates variety of biological processes such as angiogenesis, wound healing and host defense mechanisms. During inflammatory conditions, such as diabetes mellitus and myocardial infarction endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins, such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications. Therefore, we hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. To investigate the signaling mechanism involved in the protection offered by Donepezil, we found that Donepezil prevented loss of VE-cadherin in endothelial junctions, and abrogated stress fiber formation in endothelial cell exposed to high-glucose solution. In conclusion, our study shows that Donepezil maintained endothelial barrier function and improved wound healing process in endothelial cells exposed to high glucose. Chicago State University CTRE grant to Professor Nadeem Fazal, MD, PhD

S100A6 is a positive regulator of PPP5C-FKBP51-dependent regulation of endothelial calcium signaling.

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter-endothelial cell gaps and barrier disruption. The immunophilin FK506-binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC . Free PPP5C assumes an autoinhibitory state, which has low "basal" catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium-dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C-FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C-dependent manner and by FKBP51 in a S100A6-dependent manner. Further, calcium entry-induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C-FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry-induced disruption.

Reversible Cavitation-Induced Junctional Opening in an Artificial Endothelial Layer.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.

Endothelial dysfunction in cerebral aneurysms

Endothelial cell (EC) dysfunction is known to contribute to cerebral aneurysm (CA) pathogenesis. Evidence shows that damage or injury to the EC layer is the first event in CA formation. The mechanisms behind EC dysfunction in CA disease are interrelated and include hemodynamic stress, hazardous nitric oxide synthase (NOS) activity, oxidative stress, estrogen imbalance, and endothelial cell-to-cell junction compromise. Abnormal variations in hemodynamic stress incite pathological EC transformation and inflammatory zone formation, ultimately leading to destruction of the vascular wall and aneurysm dilation. Hemodynamic stress activates key molecular pathways that result in the upregulation of chemotactic cytokines and adhesion molecules, leading to inflammatory cell recruitment and infiltration. Concurrently, oxidative stress damages EC-to-EC junction proteins, resulting in interendothelial gap formation. This further promotes leukocyte traffic into the vessel wall and the release of matrix metalloproteinases, which propagates vascular remodeling and breakdown. Abnormal hemodynamic stress and inflammation also trigger adverse changes in NOS activity, altering proper EC mediation of vascular tone and the local inflammatory environment. Additionally, the vasoprotective hormone estrogen modulates gene expression that often suppresses these harmful processes. Crosstalk between these sophisticated pathways contributes to CA initiation, progression, and rupture. This review aims to outline the complex mechanisms of EC dysfunction in CA pathogenesis.

Metformin Prevents High Glucose Induced Disruption of Coronary Artery Endothelial Barrier Function and Accelerates Wound Healing Via Regulating Junctional Protein, VE‐Cadherin

Despite the great strides have been made in treating diabetes induced vascular endothelial barrier dysfunction, current treatments have shown limited success in reversing vascular pathologies. Increase in endothelial permeability via modification of endothelial junctional proteins, such as vascular endothelial cadherin (VE‐Cadherin), leads to the formation of minute inter‐endothelial gaps, and the infiltration of protein‐rich fluid in the interstitial space. If remained unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life‐threatening complications. Hyperglycemia‐induced endothelial dysfunction contributes vascular complications of diabetes. Moreover, wound healing impairment is also increasingly recognized to be a consequence of hyperglycemia‐induced dysfunction of endothelial in type 2 diabetes mellitus (T2DM). Metformin, an oral anti‐hyperglycemic agent, is the first‐line drug in the clinic for patients with T2DM. Clinical studies suggested that the beneficial effect of metformin on the incidence of diabetes complications was not only related to its action on blood glucose normalization. We hypothesized that the metformin prevents high glucose‐induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells (HCAE) via preventing the destabilization of VE‐cadherin. Endothelial permeability was evaluated by using Electric Cell‐substrate Impedance Sensing (ECIS) mechanism, a measure of trans‐endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. Our data show that 25mM glucose concentration increased endothelial permeability and abrogated the wound healing process. Metformin at the dose of 10mM significantly prevented alteration of endothelial barrier function and enhances wound healing process in the presence of high glucose concentration. To investigate the signaling mechanism involved in the protection offered by metformin, we found that metformin prevented myosin light chain kinase (MYLK) phosphorylation and increased in VE‐cadherin expression, suggesting abrogation of increase endothelial contractility and dismantling the junctional proteins in presence of high glucose concentration. In conclusion, our study shows that metformin maintained endothelial barrier function and improved the process of wound healing in endothelial cells exposed to high glucose. Thus, metformin is emerging as a potential therapeutics in improving coronary endothelial barrier function in diabetes mellitus.Support or Funding InformationThis work was supported by funding from CTRE SEED Grant Award to Mohammad Tauseef.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiopoietin-1 haploinsufficiency affects the endothelial barrier and causes hereditary angioedema.

Different mutations of the angiopoietin-1 gene (ANGPT1) have been associated with the occurrence of hereditary angioedema (HAE). The purpose of the study is to clarify whether the ANGPT1 A119S variant plays its role via haploinsufficiency or a dominant negative effect. The ability of ANGPT1 A119S variant to affect the endothelial barrier function was assessed by immunocytochemistry. Inter-endothelial gap formation molecules primarily responsible for cell-cell adhesions of HUVECs, vascular endothelial (VE)-cadherin and β-catenin, and reorganization of the F-actin cytoskeletal were evaluated. In invitro conditions mimicking the heterozygous state, the p.A119S variant significantly reduced the capability to bind its natural receptor (80.7% of normal), less than the homozygous condition (59.1%). After stimulation of VEGF or bradykinin, the addiction to equimolar amounts of wtANGPT1 and ANGPT1 p.A119S clearly reduced the expression of VE-cadherin on the endothelial cell surface (31% and 24% respectively). Likewise, cell surface expression of β-catenin was reduced and severe gap formation between adjacent HUVECs developed. In cultured cells, β-catenin expression was mostly observed along the cell surface. Treatment with equimolar amounts of wtANGPT1 and ANGPT1 p.A119S failed to restore the reorganization of the F-actin cytoskeletal elements. ANGPT1 p.A119S variant in homozygous condition further diminished VE-cadherin and β-catenin expression and failed to reduce stress fibre formation significantly affecting the endothelial barrier functionality. Present data show that in a heterozygous state the p.A119S substitution results in a pathogenic loss of function of the protein due to a mechanism of haploinsufficiency. The ANGPT1 reduced ability to counteract the increment of endothelial permeability produced by inducers, such as VEGF and bradykinin, stimulate vascular leakage and reorganization of the F-actin cytoskeletal elements. As a result, a partial impairment of the ANGPT1 functionality, like when dominant mutations occur, represents a pathophysiological cause of HAE.

RSPO3 impairs barrier function of human vascular endothelial monolayers and synergizes with pro-inflammatory IL-1

BackgroundEndothelial barrier dysfunction characterized by hyperpermeability of the vascular endothelium is a key factor in the pathogenesis of chronic inflammatory diseases and affects clinical outcomes. In states of chronic inflammation, mediators secreted by activated immune cells or vascular endothelium may affect the barrier function and permeability of the vascular endothelium. The matricellular R-spondin family member RSPO3 is produced by inflammatory-activated human monocytes and vascular endothelial cells, but its effects in the regulation of vascular endothelial barrier function remains elusive.MethodsThe present study investigates the effects of RSPO3 on the barrier function of adult human primary macro- and micro- vascular endothelial monolayers. Tight monolayers of primary endothelial cells from human coronary and pulmonary arteries, and cardiac, brain, and dermal microvascular beds were treated with RSPO3 either alone or in combination with pro-inflammatory mediator IL-1β. Endothelial barrier function was assessed non-invasively in real-time using Electric Cell-substrate Impedance Sensing.ResultsRSPO3 treatment critically affected barrier function by enhancing the permeability of all vascular endothelial monolayers investigated. To confer hyperpermeable phenotype in vascular endothelial monolayers, RSPO3 induced inter-endothelial gap formation by disrupting the β-catenin and VE-cadherin alignment at adherens junctions. RSPO3 synergistically enhanced the barrier impairing properties of the pro-inflammatory mediator IL-1β.ConclusionHere, we show that the matricellular protein RSPO3 is a mediator of endothelial hyperpermeability that can act in synergy with the inflammatory mediator IL-1β. This finding stimulates further studies to delineate the endothelial barrier impairing properties of RSPO3 and its synergistic interaction with IL-1β in chronic inflammatory diseases.

The Role of S100A6 in the PP5C‐FKBP51‐Mediated Inhibition of Endothelial Isoc

BackgroundDuring inflammation, mediators increase calcium influx into endothelial cells through the store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current, Isoc, promotes endothelial barrier disruption through the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased endothelial permeability, a vascular event contributing to pathophysiological states including inflammation and acute respiratory distress syndrome (ARDS). Identifying mechanisms of Isoc inhibition will aid in development of therapeutic strategies against endothelial permeability‐associated inflammation and ARDS. However, mechanisms of Isoc inhibition are poorly understood. We have shown that the immunophilin FKBP51 inhibits Isoc and contributes to the inhibition of calcium entry‐induced inter‐endothelial cell gap formation. We also determined that protein phosphatase 5 (PP5C) is required for the FKBP51‐mediated inhibition of Isoc and inter‐endothelial cell gap formation. However, PP5C has very low activity in the basal state. To exert its effect PP5C needs an activator. S100 proteins are potent activators of PP5C. S100 proteins are calcium‐dependent proteins and specifically, S100A6 can interact with tetratricopeptide repeat domain containing proteins such as PP5C and FKBP51. Further, S100A6 shows calcium‐induced translocation from cytosol to the plasma membrane where the ISOC channel is located. However, it is unknown whether S100A6 contributes to the PP5C‐FKBP51‐mediated inhibition of Isoc. The goal of this study was to determine whether S100A6 is critical in increasing the phosphatase activity of PP5C in a calcium‐dependent manner in endothelial cells to promote the PP5C‐FKBP51 axis‐mediated inhibition of Isoc.MethodsWe utilized quantitative RT‐PCR and immunoblot to determine whether S100 proteins are expressed in pulmonary endothelial cells. We performed co‐immunoprecipitation experiments to determine if S100A6 associates with the ISOC channel and PP5C protein in pulmonary microvascular endothelial cells (PMVECs) in presence and absence of calcium (2mM). To determine whether S100A6 activates PP5C in PMVECs we analyzed the phosphorylation status of tau protein with or without calcium (2mM), where decreased tau phosphorylation suggests increased PP5C activity. Whole cell patch clamp electrophysiology was used to measure Isoc in cells.ResultsWe showed that S100A6 is highly expressed in pulmonary endothelial cells. S100A6 co‐precipitated with the TRPC4 subunit of the ISOC channel as well as with PP5C in a calcium‐dependent manner. Upon S100A6 knock‐down in PMVECs, phosphorylation of tau increased suggesting that PP5C activity was deceased. In FKBP51 over‐expressed cells, Isoc was inhibited, but this effect was reduced by S100A6 knock‐down.ConclusionTogether these findings suggest that S100A6 activates PP5C leading to the FKBP51‐PP5C‐mediated inhibition of Isoc.Support or Funding InformationSupported by NIH R01HL107778.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myosin Light Chain Kinase‐210 Induces ER‐PM Junctions and STIM1 Puncta Formation to Augment Store‐Operated Ca2+ Entry

Myosin light chain kinase‐Long (MYLK‐L or MYLK‐210), a Ca2+‐calmodulin dependent kinase containing a unique 922‐amino acid N‐terminal domain is a predominant isoform of MYLK expressed in endothelial cells (ECs). N‐terminus of MYLK‐210 has been shown to contain several single nucleotide polymorphisms (SNPs) in an actin binding region which is associated with severity of acute lung injury (ALI). However, significance of SNPs in regulating MYLK‐210 function remains unclear. The classical function of MYLK‐210 is to catalyze the phosphorylation of myosin light chain to induce EC contraction, leading to inter‐endothelial gap formation and increase permeability, a hall mark of ALI. Intriguingly, we showed that MYLK‐210, through its actin‐binding motif, regulates store‐operated Ca2+ entry (SOCE) but not receptor‐operated Ca2+ entry (ROCE). We showed that MYLK‐210 null endothelial cells (ECs) or MYLK‐210 depleted ECs displayed diminished Ca2+ entry in response to SOCE‐activator thapsigargin and thrombin. We generated MYLK‐210 constructs lacking C‐terminus (N‐MYLK) or N‐terminus (C‐MYLK) to investigate the role of specific domains of MYLK‐210 in inducing SOCE activity. We found that N‐MYLK but not C‐MYLK rescued SOCE activity in MYLK‐210 null ECs, indicating thereby that MYLK regulates SOCE in a kinase independent manner. Additionally, SOCE activation failed to increase lung vascular hyper‐permeability in MYLK‐210 null mice. We identified unique actin binding motif, DVRGLL within N terminus of MYLK‐L which enables MYLK‐210 to induce SOCE. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta which translocate to plasma membrane that trigger SOCE via plasmalemmal Ca2+selective channels such as transient receptor potential channel 1 (TRPC1) and Orai1. Thus, we addressed if MYLK‐L controlled STIM1 function by regulating puncta formation. We showed that STIM1 formed puncta in control ECs but these puncta were markedly reduced in MYLK depleted ECs. Formation of endoplasmic reticulum (ER)‐plasma membrane (PM) junction is required for STIM1 activation of SOCE. Using MAPPER (membrane‐attached peripheral ER) that selectively labels ER–PM junctions, we found that MYLK induced ER‐PM junction, but these junctions were markedly reduced in the density in MYLK‐knockdown cells. Our study thereby defines for the first time the clinical validity of SNPs within actin binding region of MYLK in inducing ALI through organizing ER‐PM junctions, STIM1 activity, SOCE and lung vascular hyperpermeability.Support or Funding InformationNational Institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective role of FKBP51 in calcium entry-induced endothelial barrier disruption.

Pulmonary artery endothelial cells (PAECs) express a cation current, ISOC (store-operated calcium entry current), which when activated permits calcium entry leading to inter-endothelial cell gap formation. The large molecular weight immunophilin FKBP51 inhibits ISOC but not other calcium entry pathways in PAECs. However, it is unknown whether FKBP51-mediated inhibition of ISOC is sufficient to protect the endothelial barrier from calcium entry-induced disruption. The major objective of this study was to determine whether FKBP51-mediated inhibition of ISOC leads to decreased calcium entry-induced inter-endothelial gap formation and thus preservation of the endothelial barrier. Here, we measured the effects of thapsigargin-induced ISOC on the endothelial barrier in control and FKBP51 overexpressing PAECs. FKBP51 overexpression decreased actin stress fiber and inter-endothelial cell gap formation in addition to attenuating the decrease in resistance observed with control cells using electric cell-substrate impedance sensing. Finally, the thapsigargin-induced increase in dextran flux was abolished in FKBP51 overexpressing PAECs. We then measured endothelial permeability in perfused lungs of FKBP51 knockout (FKBP51–/–) mice and observed increased calcium entry-induced permeability compared to wild-type mice. To begin to dissect the mechanism underlying the FKBP51-mediated inhibition of ISOC, a second goal of this study was to determine the role of the microtubule network. We observed that FKBP51 overexpressing PAECs exhibited increased microtubule polymerization that is critical for inhibition of ISOC by FKBP51. Overall, we have identified FKBP51 as a novel regulator of endothelial barrier integrity, and these findings are significant as they reveal a protective mechanism for endothelium against calcium entry-induced disruption.

Prolonged Morphine Exposure Induces Increased Firm Adhesion in an in Vitro Model of the Blood-Brain Barrier.

The blood–brain barrier (BBB) has been defined as a critically important protective barrier that is involved in providing essential biologic, physiologic, and immunologic separation between the central nervous system (CNS) and the periphery. Insults to the BBB can cause overall barrier damage or deregulation of the careful homeostasis maintained between the periphery and the CNS. These insults can, therefore, yield numerous phenotypes including increased overall permeability, interendothelial gap formation, alterations in cytokine and chemokine secretion, and accelerated cellular passage. The current studies expose the human brain microvascular endothelial cell line, hCMEC/D3, to prolonged morphine exposure and aim to uncover the mechanisms underlying alterations in barrier function in vitro. These studies show alterations in the mRNA and protein levels of the cellular adhesion molecules (CAMs) intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and activated leukocyte cell adhesion molecule that correlate with an increased firm adhesion of the CD3+ subpopulation of peripheral blood mononuclear cells (PBMCs). Overall, these studies suggest that prolonged morphine exposure may result in increased cell migration into the CNS, which may accelerate pathological processes in many diseases that involve the BBB.

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.

We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a high-molecular-weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU and the conditional expression of an activity-attenuated ExoU induced time-dependent pulmonary microvascular endothelial cell gap formation that was paralleled by the loss of intracellular tau and the concomitant appearance of high-molecular-weight extracellular tau. Transfer of the high-molecular-weight tau in filtered supernatant to naïve endothelial cells resulted in intracellular accumulation of tau clusters, which was accompanied by cell injury, interendothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant but did not retrieve higher-molecular-weight endothelial tau and did not rescue the injurious effects of tau. Enrichment and transfer of high-molecular-weight tau to naïve cells was sufficient to cause injury. Thus we provide the first evidence for a pathophysiological stimulus that induces release and transmissibility of high-molecular-weight endothelial tau characteristic of an endothelial proteinopathy.

Pseudomonas aeruginosa Exoenzymes U and Y Induce a Transmissible Endothelial Tauopathy

Here, we tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors ExoY and ExoU induce endothelial tau oligomerization and release, causing transmissible cytotoxicity characteristic of an infectious tauopathy. Both the bacterial delivery of ExoY and ExoU, and the conditional expression of an activity‐attenuated ExoU, induced time‐dependent pulmonary microvascular endothelial cell (PMVEC) gap formation that was paralleled by loss of intracellular tau and concomitant appearance of extracellular tau oligomers. Transfer of tau oligomers in filtered supernatant onto naïve endothelial cells resulted in the intracellular accumulation of oligomeric tau inclusions accompanied by cytotoxicity, as determined by inter‐endothelial gap formation and decreased endothelial network stability in Matrigel. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant, but did not retrieve higher molecular mass tau oligomers, and did not rescue the cytotoxic effects of tau. Since cytotoxic effects were not rescued, we combined an electrophoresis and electroelution approach to recover the high molecular mass tau oligomers, treat PMVECs with these oligomers, and recapitulate cytotoxicity. Thus, we provide the first evidence for a pathophysiological stimulus that induces endothelial tau oligomerization, release and transmissible cytotoxicity, characteristic of an endothelial tauopathy.

Dimethyl fumarate attenuates cerebral edema formation by protecting the blood–brain barrier integrity

Brain edema is a hallmark of various neuropathologies, but the underlying mechanisms are poorly understood. We aim to characterize how tissue hypoxia, together with oxidative stress and inflammation, leads to capillary dysfunction and breakdown of the blood–brain barrier (BBB). In a mouse stroke model we show that systemic treatment with dimethyl fumarate (DMF), an antioxidant drug clinically used for psoriasis and multiple sclerosis, significantly prevented edema formation in vivo. Indeed, DMF stabilized the BBB by preventing disruption of interendothelial tight junctions and gap formation, and decreased matrix metalloproteinase activity in brain tissue. In vitro, DMF directly sustained endothelial tight junctions, inhibited inflammatory cytokine expression, and attenuated leukocyte transmigration. We also demonstrate that these effects are mediated via activation of the redox sensitive transcription factor NF-E2 related factor 2 (Nrf2). DMF activated the Nrf2 pathway as shown by up-regulation of several Nrf2 target genes in the brain in vivo, as well as in cerebral endothelial cells and astrocytes in vitro, where DMF also increased protein abundance of nuclear Nrf2. Finally, Nrf2 knockdown in endothelial cells aggravated subcellular delocalization of tight junction proteins during ischemic conditions, and attenuated the protective effect exerted by DMF. Overall, our data suggest that DMF protects from cerebral edema formation during ischemic stroke by targeting interendothelial junctions in an Nrf2-dependent manner, and provide the basis for a completely new approach to treat brain edema.

Abstract 179: Rho Kinase Isoform Contribution to Endothelial Contractility

Background: Rho kinase (ROCK) is the major effector protein of RhoA and is known to mediate F-actin stress fibers. ROCK activates myosin motors. Actomyosin-generated contractile forces can be transmitted to cell-cell junctions, resulting in increased junctional tension, junctional disassembly and endothelial hyperpermeability. In apparent contrast, basal ROCK activity is required for maintenance of barrier integrity . We hypothesize that these dual roles of ROCK could be contributed to the two highly homologous expressed isoforms. Methods: Traction force microscopy was utilized to study the endothelium-generated contractile forces after depletion of ROCK1 and/or ROCK2 by SiRNA approach in the absence and presence of the vaso-active agent thrombin. Results: The traction force landscape showed that ROCK is a major contributor to endothelial contraction and permeability. Depletion of ROCK1 and ROCK2 resulted in a blockade of contractile response to thrombin and a marked reduction in endothelial hyperpermeability. Detailed force distribution maps revealed that the absence of ROCK1, but not of ROCK2, resulted in an increase in baseline contractile forces (siROCK1: 88.1 ± 4.1 Pa, siROCK2: 53.6 ± 3.4 Pa, Control: 56.2 ± 6.1 Pa), which was associated with the formation of numerous inter-endothelial gaps upon stimulation with thrombin. Knockdown of ROCK2 stabilized the endothelial barrier and largely prevented traction force enhancements. Exposure to thrombin resulted in all conditions in an increase of traction forces and hyperpermeability. Conclusion: Both ROCK isoforms have a distinct function in endothelial contractility and permeability. ROCK1 is predominantly contributing to barrier maintenance under baseline conditions, whereas ROCK2 mediates the thrombin-induced contractile force enhancements and subsequent barrier dysfunction. Funding: Dutch Heart Foundation 2011T072

Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability

Infection with dengue virus (DV) can result in dengue hemorrhagic fever and dengue shock syndrome, where patients suffer from bleeding and plasma leakage involving endothelial cells. Angiopoietins (Ang) 1 and 2 are important angiogenic factors that affect endothelial barrier integrity. In this study, DV was observed to induce endothelial leakage at multiplicity of infection of 10 in primary human umbilical vein endothelial cells (HUVEC) with interendothelial gap formation. Immunostaining of vascular endothelial cadherin (VE-cadherin) and zona occludin 1 (ZO-1) showed the absence of these endothelial junctional proteins at the cell-cell contact zones between adjacent cells. In addition, Ang1 that is required for protecting against endothelial hyperpermeability was found to be down-regulated during DV infection. Treatment with increasing concentrations of recombinant Ang1 was shown to prevent DV-induced endothelial hyperpermeability in a dose-dependent manner by preventing the down-regulation of VE-cadherin and ZO-1 at cell membrane. In contrast, the expression of Ang2, the natural antagonist of Ang1, was observed to be up-regulated during DV infection. Recombinant Ang2 added to HUVEC at non-toxic concentrations showed decreased in transendothelial electrical resistance reading and the down-regulation of VE-cadherin and ZO-1. These findings suggest that DV reduces the expression of Ang1 and enhances the expression of Ang2 in endothelial cells and that this imbalance of Ang 1 and Ang 2 may play a contributing role to the increased permeability of human primary endothelial cells during DV infection.