Regulation Of Endothelial Barrier Function Research Articles

Heterogeneity of endothelial VE-PTP downstream polarization, Tie2 activation, junctional claudin-5, and permeability in the aorta and vena cava

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

Evaluation of Glycolysis and Mitochondrial Function in Endothelial Cells Using the Seahorse Analyzer.

Endothelial bioenergetics have emerged as a key regulator of endothelial barrier function. Glycolytic parameters have been linked to barrier enhancement, and interruption with mitochondrial complexes was shown to disrupt endothelial barrier. Therefore, a new technology that has been introduced to assess bioenergetics and metabolism has also made it possible to determine roles of specific energy production pathways in endothelial health. The Seahorse extracellular flux analysis by Agilent technologies is a state of the art tool that has been more frequently used to evaluate bioenergetics of endothelial cells. This chapter includes details about different assays that can be used to study endothelial cells using the Seahorse analyzer and how interpretation of the results can provide novel insight about endothelial metabolism.

Angiopoietin-like 4 to prevent endothelial damage during septic shock

Regulation of endothelial barrier function is critical for vascular homeostasis but a comprehensive understanding of sepsis-induced endothelial injury involved in organ failure is lacking. We have previously demonstrated that recombinant human Angiopoietin-like-4 (ANGPTL4) protects vascular integrity in the heart, reduces cardiac edema and no-reflow acutely after acute myocardial infarction (AMI). We have also shown that ANGPTL4 has protective effects on the cerebrovascular and functional damage after ischaemic stroke that shares some pathophysiological factors with AMI. Given the strong association between vascular barrier integrity and endothelial injury markers for predicting higher mortality in shocks, we here sought to evaluate the cardiovascular impact of a novel pharmacological strategy that preserves microvascular integrity aimed at reducing endothelial damage and fluid extravasation, in order to improve perfusion and reduce organ failure and death. Septic shock was induced in adult mice using lipopolysaccharides (LPS). Vascular leakage (total weight/dry weight) was assessed in highly metabolic tissues such as liver, kidneys, lungs and heart by gravimetry in control mice vs mice injected with recombinant human Angiopoietin-like-4 (ANGPTL4). To go further, integrity of tight junctions was studied by using endothelial cells treated with LPS and treated or not with ANGPTL4. Following septic shock, our results demonstrate that injection of recombinant human Angiopoietin-like-4 (ANGPTL4) was able to protect vascular barrier integrity by decreasing fluid extravasation in cardiorespiratory system. In addition, we demonstrate that ANGPTL4 was able to preserve the integrity of endothelial cells junctions after LPS treatment. These results show that ANGPTL4 is a regulator of endothelial barrier integrity during septic shock. This discovery of the vascular protective effects of ANGPTL4, thus being crucial for preventing endothelial damage during septic shock, is one of the first examples of the validity of applying vascular permeability-limiting drugs in shock states. Its therapeutic potential in other types of shocks is also presently under investigation.

The vasculoprotective effects of resveratrol are mediated via Kruppel-like factor 2 dependent protection of endothelial barrier function

Resveratrol is a naturally occurring polyphenolic compound that is thought to have vasculoprotective properties. Its observed effects are proposed, in part, to be mediated through the induction of endothelial Krϋppel-like factor 2 (KLF2) expression. KLF2 is a nuclear transcription factor that is highly expressed within the vascular endothelium. Studies from our laboratory and others have shown that this protein mediates vascular function through its transactivation domain, and its targeted expression promotes vascular health, notably by acting as an important positive regulator of endothelial barrier function. In this study, we demonstrate that resveratrol possesses endothelial barrier protective effects dependent on the presence of KLF2, with several key endothelial tight junction proteins expressed in a KLF2-dependent manner. Collectively, our findings identify KLF2 as essential for resveratrol-mediated endothelial barrier protection, thus further implicating KLF2 as a critical vasculoprotective factor.

SNP rs3803264 polymorphisms in THSD1 and abnormally expressed mRNA are associated with hemorrhagic stroke.

Thrombospondin Type 1 Domain Containing Protein 1 (THSD1) has been suggested to be a new regulator of endothelial barrier function in the angiogenesis process, preserving vascular integrity. We sought to characterize the association of THSD1 genetic variants and mRNA expression with the risk of hemorrhagic stroke (HS) with population-based evidence. A case-control study was conducted with 843 HS cases and 1,400 healthy controls. A cohort study enrolled 4,080 participants free of stroke at baseline in 2009 and followed up to 2022. A synonymous variant, the main tag SNP rs3803264 of the THSD1 gene, was genotyped in all subjects, and peripheral leukocyte THSD1 mRNA expression was detected using RT-qPCR in 57 HS cases and 119 controls. In the case-control study, rs3803264 AG/GG variations are associated with a decreased risk of HS with odd ratio (OR) and 95% confidence interval (CI) of the dominant model of 0.788 (0.648-0.958), p = 0.017. In addition, rs3803264 and dyslipidemia had a multiplicative interaction [OR (95% CI) = 1.389 (1.032, 1.869), p = 0.030]. In the cohort study, a similar association strength of rs3803264 dominant model and the risk of HS was observed with the incidence rate ratio (IRR) of 0.734 and p-value of 0.383. Furthermore, the risk of HS showed a non-linear as THSD1 mRNA expression increased (p for non-linearity <0.001). For the subjects without hypertension, we observed THSD1 mRNA expression had a negative correlation with systolic blood pressure (SBP; ρ = -0.334, p = 0.022). SNP rs3803264 polymorphisms in THSD1 are associated with the decreased risk of HS and interacted with dyslipidemia, and a non-linear association was observed between THSD1 mRNA expression and the risk of HS.

Circulating dipeptidyl peptidase 3 and bio-adrenomedullin levels are associated with impaired outcomes in critically ill COVID-19 patients: a prospective international multicentre study.

IntroductionDipeptidyl-peptidase-3 (DPP3) is a protease involved in the degradation of several cardiovascular mediators. Adrenomedullin (bio-ADM) is a peptide essential for regulation of endothelial barrier function. In different shock-pathologies, both biomarkers are associated with disease-severity, organ dysfunction and mortality. Associations with outcome in critically ill COVID-19 patients are unknown.ObjectivesTo investigate associations of bio-ADM and cDPP3 with short-term outcome in critically ill COVID-19 patients (n=80).MethodsMulticenter prospective cohort study. Primary endpoint was 28-day mortality. Secondary endpoints included different severities of acute kidney injury (AKI).ResultscDPP3 levels were mainly associated with 28-day mortality; AUROC's of 0.69 (0.56–0.82, p=0.023), 0.77 (0.64–0.90, p<0.001) and 0.81 (0.65–0.96, p<0.001) at admission, day 3 and day 7, respectively. In contrast, bio-ADM levels were mainly associated with AKI: AUROC's of 0.64 (0.51–0.77, p=0.048), 0.75 (0.64–0.86, p<0.001) and 0.83 (0.74–0.93, p<0.001) for day 1, 3 and 7, respectively. Interestingly, patients with high levels of both cDPP3 and bio-ADM at day 7 had an additionally increased risk of 28-day-mortality; HR 11.8 (2.5–55.3, p<0.001).ConclusionscDPP3 and bio-ADM responses were associated with short-term mortality and AKI in critically ill COVID-19 patients, respectively. These findings suggest that treatment with specific antibodies modulating cDPP3 or bio-ADM related pathways may improve outcome of COVID-19.

Lymphangion‐Chip Experimental Platform Supports Co‐culture and Signaling Between Lymphatic Endothelial and Mural Cells

The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions that drive lymph flow. Even though the signaling between two main cellular components of a lymphangion, Lymphatic Endothelial Cells (LECs) and Lymphatic Muscle Cells (LMCs), is responsible for crucial lymphatic functions, there are no in vitro models that have included both cell types in a physiologically relevant environment. Here, a fabrication technique (Gravitational Lumen Patterning or GLP) is developed to create Lymphangion‐Chip. This organ‐on‐chip consists of co‐culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, lymphatic muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This model further demonstrated a robust sensitivity of this relative alignment of the two cells with respect to the presence or absence of co‐culture and mechanical forces (shear stress), thus suggesting that LECs and LMCs are biologically and functionally active within the chip. This system successfully characterizes the dynamics of cell size, density, growth, alignment, and intercellular gap due to co‐culture and shear. The time‐dependent decrease in the subendothelial gap strongly suggests proactive LEC‐LMC signaling as LECs and LMCs grow and proliferate within the device. Further exposure to pro‐inflammatory cytokines reveals that the device could produce the regulation of endothelial barrier function through the lymphatic muscle cells. Finally, measurement of intracellular calcium of LECs and LMCs while exposed to step and oscillatory flow profiles with various shear amplitudes revealed the bidirectional mechanism of calcium signaling within lymphatic vessel. Therefore, this organ‐chip technology allows researchers to include essential lymphatic vascular components in a tunable 3D microphysiological system that is suitable for use in preclinical research of lymphatic and blood mechanobiology, inflammation, and translational outcomes.

Abstract 378: Lymphangion-chip: It Recreates Intercellular Signaling Between Lymphatic Vascular Endothelial And Mural Cells In Health And Disease

The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions. Even though the signaling between two main cellular components of a lymphangion, Lymphatic Endothelial Cells (LECs) and Lymphatic Muscle Cells (LMCs), is responsible for crucial functions, there are no in vitro models that have included both cell types in a physiologically relevant environment. Here, a fabrication technique is developed to create a Lymphangion-Chip. This organ-on-chip consists of co-culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, the muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This model further demonstrated a robust sensitivity of this relative alignment of the two cells as well as dynamics of cell size, density, growth, and intercellular gap with respect to the presence or absence of co-culture and mechanical forces (shear). The time-dependent decrease in the subendothelial gap strongly suggests proactive LEC-LMC signaling as LECs and LMCs grow and proliferate within the device. Further exposure to pro-inflammatory cytokines reveals that the device could produce the regulation of endothelial barrier function through the muscle cells. Finally, measurement of intracellular calcium of LECs and LMCs while exposed to step and oscillatory flow profiles with various shear amplitudes revealed the bidirectional mechanism of calcium signaling within the platform. Therefore, this technology allows researchers to include essential lymphatic vascular components in a tunable 3D microphysiological system that is suitable for use in preclinical research of lymphatic mechanobiology, inflammation, and translational outcomes.

The value of bioactive adrenomedullin and dipeptidyl peptidase 3 to predict short-term unfavourable outcomes after cardiac surgery: A prospective cohort study.

Adrenomedullin (ADM) is a key regulator of endothelial barrier function and vascular tone. Dipeptidyl peptidase 3 (DPP3) is a protease involved in the degradation of several cardiovascular mediators. Increased levels of bioactive ADM (bio-ADM) and circulating DPP3 (cDPP3) were found to predict short-term outcome in cardiogenic shock patients. To investigate the unknown temporal profiles of bio-ADM and cDPP3 and their association with short-term outcome following cardiac surgery. Prospective observational study of 203 adult cardiac surgery patients admitted to the intensive care unit (ICU) postoperatively. Plasma bio-ADM and cDPP3 levels were measured at ICU admission (day 1) and on days 2 and 3. Biomarker prediction of prolonged vasopressor dependency (>3 days), acute kidney injury (AKI) and prolonged ICU length of stay (ICU-LOS) (>3 days). bio-ADM and cDPP3 levels displayed distinct temporal profiles following cardiac surgery. cDPP3 levels were highest on day 1 and strongly correlated with surgical complexity and duration but subsequently normalised on day 2 in most patients. In contrast, bio-ADM levels on day 1 were within the normal range but subsequently increased. Day 2 bio-ADM levels were strongly associated with study outcomes: the area under the receiver-operating curves (AUROC) were 0.82 (95% CI, 0.72 to 0.92) for prolonged vasopressor dependency, 0.87 (0.81 to 0.92) for AKI and 0.82 (0.75 to 0.90) for prolonged ICU-LOS (all P < 0.0001). cDPP3 levels on day 2 also predicted these outcomes, albeit to a lesser extent, with AUROCs of 0.73 (95% CI, 0.64 to 0.81) for prolonged vasopressor dependency, 0.69 (0.61 to 0.77) for AKI and 0.70 (0.62 to 0.79) for prolonged ICU-LOS (all P < 0.0001). Following cardiac surgery, increased bio-ADM levels are strongly associated with unfavourable short-term outcomes, whereas cDPP3 levels are mainly related to surgery complexity and duration. On the basis of these findings, ADM-modulating therapies may have beneficial effects in cardiac surgery patients whereas DPP3-targeted therapies should be reserved for patient categories with higher baseline disease severity.

The Cx43 Carboxyl-Terminal Mimetic Peptide αCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner.

The Cx43 carboxyl-terminus (CT) mimetic peptide, αCT1, originally designed to bind to Zonula Occludens 1 (ZO1) and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing (ECIS), a TRITC-dextran Transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell–cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular chiral orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell–cell border localization of endothelial VE-cadherin, the tight junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1 binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema.

HSPA12A improves endothelial integrity to attenuate lung injury during endotoxemia through activating ERKs and Akt-dependent signaling

Acute lung injury (ALI) is a critical manifestation of sepsis/septic shock. Disruption of endothelial barrier function is critical for ALI pathogenesis; however, the regulation of endothelial barrier integrity remains largely unclear. Heat shock protein A12A (HSPA12A) is an atypical member of HSP70 family. We have recently demonstrated that hepatocyte HSPA12A attenuated the bacteria endotoxin (lipopolysaccharide, LPS)-induced liver injury. However, the role of HSPA12A in endothelial barrier function and ALI is unknown. Here in this study, HSPA12A showed upregulation in lungs of mice during bacteria endotoxin (lipopolysaccharide, LPS)-induced lung injury in vivo and in primary human umbilical vein endothelial cells (HUVECs) during LPS-induced barrier disruption in vitro. Knockout of HSPA12A in mice exacerbated LPS-induced ALI. Intriguingly, overexpression of HSPA12A in HUVECs attenuated the LPS-induced endothelial hyperpermeability. In line with this, HSPA12A overexpression increased VE-cadherin and decreased VEGF expression following LPS treatment in HUVECs. Also, knockout of HSPA12A enhanced the LPS-evoked pulmonary endothelial cell apoptosis in mice whereas overexpression of HSPA12A inhibited the LPS-induced death of HUVECs. The levels of ERKs and Akt phosphorylation in HUVECs were promoted by HSPA12A overexpression when cells exposed to LPS. Importantly, inhibition of either ERKs or Akt diminished the HSPA12A-induced protection from LPS-induced endothelial hyperpermeability and death. Taken together, these findings indicated that HSPA12A is a novel regulator of endothelial barrier function through both ERKs and Akt-mediated signaling. HSPA12A might represent a viable strategy for the pulmonary protection against endotoxemia challenge.

Purinergic Regulation of Endothelial Barrier Function

Increased vascular permeability is a hallmark of several cardiovascular anomalies, including ischaemia/reperfusion injury and inflammation. During both ischaemia/reperfusion and inflammation, massive amounts of various nucleotides, particularly adenosine 5′-triphosphate (ATP) and adenosine, are released that can induce a plethora of signalling pathways via activation of several purinergic receptors and may affect endothelial barrier properties. The nature of the effects on endothelial barrier function may depend on the prevalence and type of purinergic receptors activated in a particular tissue. In this review, we discuss the influence of the activation of various purinergic receptors and downstream signalling pathways on vascular permeability during pathological conditions.

Prostaglandin D2 strengthens human endothelial barrier by activation of E-type receptor 4

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D2 (PGD2) is a potent inflammatory lipid mediator with vasoactive properties. Previous studies suggest that PGD2 is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD2 is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD2 and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD2 and BW245c. Stimulation with PGE2 or PGD2 reduced AKT phosphorylation which could be reversed by prior blockade of EP4 receptors. These data demonstrate a novel mechanism by which PGD2 may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function.

The Good and Bad Faces of Oxidized Phospholipids: Friends or Foes of Vascular Endothelium?

A diverse group of bioactive lipids are generated from the oxidation of phospholipids during various pathological conditions such as lung injury, sepsis, and autoimmune diseases. An elevated level of circulating oxidized phospholipids with potent inflammatory activities has been recognized as a hallmark of various diseases including atherosclerosis. On the other hand, the oxidation of a principal membrane phospholipid 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine (PAPC) results in the formation of a heterogeneous mixture of full length and fragmented oxidized species with both beneficial and detrimental effects on pulmonary endothelium. Among these, the extensive studies from this research group have established that full length oxidized species present in oxidized PAPC (OxPAPC) have pronounced endothelial barrier protective and anti‐inflammatory properties. This has been documented in various cellular and animal models of lung injury and inflammation using a wide range of chemical and mechanical stimuli. Moreover, OxPAPC stimulation of endothelial cells generates anti‐inflammatory molecules such as lipoxin that mediate the recovery of inflamed lungs. This review briefly summarizes the current knowledge on OxPAPC‐mediated positive regulation of endothelial barrier function and its potential as a promising therapeutic target for lung injury and inflammation.Practical Applications: This review briefly summarizes the current knowledge on OxPAPC‐mediated positive regulation of endothelial barrier function and its potential as a promising therapeutic target for lung injury and inflammation.Products of phospholipid oxidation may exhibit disruptive or protective effects on vascular endothelium depending on the extent of oxidation. Full length oxidized phospholipids stimulated endothelial barrier recovery and resolution of acute lung injury or sepsis by triggering multiple mechanisms enhancing endothelial barrier properties and diminishing inflammation.

Pharmacological inhibition of β-catenin prevents EndMT in vitro and vascular remodeling in vivo resulting from endothelial Akt1 suppression

Endothelial to mesenchymal transition (EndMT), where endothelial cells acquire mesenchymal characteristics has been implicated in several cardiopulmonary, vascular and fibrotic diseases. The most commonly studied molecular mechanisms involved in EndMT include TGFβ, Notch, interleukin, and interferon-γ signaling. As of today, the contributions of Akt1, an important mediator of TGFβ signaling and a key regulator of endothelial barrier function to EndMT remains unclear. By using the ShRNA based gene silencing approach and endothelial-specific inducible Akt1 knockdown (ECKOAkt1) mice, we studied the role of Akt1 in EndMT in vitro and pathological vascular remodeling in vivo. Stable, Akt1 silenced (ShAkt1) human microvascular endothelial cells (HMECs) indicated increased expression of mesenchymal markers such as N-cadherin and α-SMA, phosphorylation of Smad2/3, cellular stress via activation of p38 MAP Kinase and the loss of endothelial nitric oxide synthase (eNOS) accompanied by a change in the morphology of HMECs in vitro and co-localization of endothelial and mesenchymal markers promoting EndMT in vivo. EndMT as a result of Akt1 loss was associated with increased expression of TGFβ2, a potent inducer of EndMT and mesenchymal transcription factors Snail1, and FoxC2. We observed that hypoxia-induced lung vascular remodeling is exacerbated in ECKOAkt1 mice, which was reversed by pharmacological inhibition of β-catenin. Thus, we provide novel insights into the role of Akt1-mediated β-catenin signaling in EndMT and pathological vascular remodeling, and present β-catenin as a potential target for therapy for various cardiopulmonary diseases involving vascular remodeling.

A Novel Microscopic Assay Reveals Heterogeneous Regulation of Local Endothelial Barrier Function

Blood vessels are covered with endothelial cells on their inner surfaces, forming a selective and semipermeable barrier between the blood and the underlying tissue. Many pathological processes, such as inflammation or cancer metastasis, are accompanied by an increased vascular permeability. Progress in live cell imaging techniques has recently revealed that the structure of endothelial cell contacts is constantly reorganized and that endothelial junctions display high heterogeneities at a subcellular level even within one cell. Although it is assumed that this dynamic remodeling is associated with a local change in endothelial barrier function, a direct proof is missing mainly because of a lack of appropriate experimental techniques. Here, we describe a new assay to dynamically measure local endothelial barrier function with a lateral resolution of ∼15 μm and a temporal resolution of 1 min. In this setup, fluorescence-labeled molecules are added to the apical compartment of an endothelial monolayer, and the penetration of molecules from the apical to the basal compartment is recorded by total internal reflection fluorescence microscopy utilizing the generated evanescent field. With this technique, we found a remarkable heterogeneity in the local permeability for albumin within confluent endothelial cell layers. In regions with low permeability, stimulation with the proinflammatory agent histamine results in a transient increase in paracellular permeability. The effect showed a high variability along the contact of one individual cell, indicating a local regulation of endothelial barrier function. In regions with high basal permeability, histamine had no obvious effect. In contrast, the barrier-enhancing drug forskolin reduces the permeability for albumin and dextran uniformly along the cell junctions. Because this new approach can be readily combined with other live cell imaging techniques, it will contribute to a better understanding of the mechanisms underlying subcellular junctional reorganization during wound healing, inflammation, and angiogenesis.

E. coli High Throughput Assay Identifies Regulators of Endothelial Barriers

Regulation of endothelial barrier function is critical for vascular homeostasis, as dynamic and local control of vascular permeability permits macromolecular transport, immune surveillance, and deposition of a fibrin barrier to contain infection at sites of inflammation. Many of the signaling pathways promoting useful vascular permeability, however, are also triggered during disease, resulting in prolonged or uncontrolled vascular leak. Hyper-permeability triggered by inflammation or ischemia in the heart, brain, or lung promotes edema, exacerbates disease progression, and impairs recovery. Currently there is no clinical solution for the regulation of the endothelial barriers. A main reason for the lack of translational solutions is the technical difficulties (e.g. low throughput, and lengthy assays) to explore libraries of small compounds to begin drug development. Regulating endothelial barriers is crucial in the prevention of disease, disease progression, and the discovery and implementation of new therapies. Claudins, and occludin, are Tight Junction membrane proteins (TJ-MP) that seal the paracellular space creating the endothelial barriers. Our laboratory has devised a strategy to express TJ-MPs in the outer membrane of E. coli. TJ-MP expression drives cell-cell interactions above the unicellular behavior of E. coli. TJ-MPs force this aggregation in a manner that correlates with their strength in the native endothelial barriers. The aggregation of E. coli can be quantified using flow cytometry. Our preliminary results suggest that our assay has sensitivity to detect hyper-permeability (barrier opening) as well as hypo-permeability (barrier closing or strengthening).

FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB.

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference–based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

Effects of the Humanized Anti-Adrenomedullin Antibody Adrecizumab (HAM8101) on Vascular Barrier Function and Survival in Rodent Models of Systemic Inflammation and Sepsis.

Adrenomedullin (ADM) is an important regulator of endothelial barrier function during sepsis. Administration of a murine antibody targeted against the N-terminus of ADM (HAM1101) resulted in improved outcome in models of murine sepsis. We studied the effects of a humanized form of this antibody (HAM8101, also known as Adrecizumab) on vascular barrier dysfunction and survival in rodent models of systemic inflammation and sepsis. Rats (n=48) received different dosages of HAM8101 or placebo (n = 8 per group), directly followed by administration of lipopolysaccharide (5 mg/kg). Twenty-four hours later, Evans Blue dye was administered to assess vascular leakage in kidney and liver tissue. Furthermore, mice (n = 24) were administered different dosages of HAM8101 or placebo (n = 6 per group), immediately followed by cecal ligation and puncture (CLP). Eighteen hours later, albumin, vascular endothelial growth factor (VEGF), and angiopoietin-1 were analyzed in the kidney. Finally, effects of single and repeated dose administration of HAM1101, HAM8101 and placebo on survival were assessed in CLP-induced murine sepsis (n = 60, n = 10 per group). Dosages of 0.1 and 2.5 mg/kg HAM8101 attenuated renal albumin leakage in endotoxemic rats. Dosages of 0.1, 2.0, and 20 mg/kg HAM8101 reduced renal concentrations of albumin and the detrimental protein VEGF in septic mice, whereas concentrations of the protective protein angiopoietin-1 were augmented. Both single and repeated administration of both HAM1101 and HAM8101 resulted in improved survival during murine sepsis. Pretreatment with the humanized anti-ADM antibody HAM8101 improved vascular barrier function and survival in rodent models of systemic inflammation and sepsis.

Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury

BackgroundEndothelial barrier dysfunction is central to the pathogenesis of sepsis-associated acute lung injury (ALI). Microtubule (MT) dynamics in vascular endothelium are crucial for the regulation of endothelial barrier function. Unfractionated heparin (UFH) possesses various biological activities, such as anti-inflammatory activity and endothelial barrier protection during sepsis.MethodsHere, we investigated the effects and underlying mechanisms of UFH on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. C57BL/6 J mice were randomized into vehicle, UFH, LPS and LPS + UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS + UFH group received intravenous UFH 0.5 h prior to LPS injection. Human pulmonary microvascular endothelial cells (HPMECs) were cultured for analyzing the effects of UFH on LPS-induced and nocodazole-induced hyperpermeability, F-actin remodeling, and LPS-induced p38 MAPK activation.ResultsUFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes, and increased the lung W/D ratio and Evans blue accumulation in vivo. Both in vivo and in vitro studies showed that UFH pretreatment blocked the LPS-induced increase in guanine nucleotide exchange factor (GEF-H1) expression and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, and microtubule (MT) disassembly in LPS-induced ALI mouse model and human pulmonary microvascular endothelial cells (HPMECs). These results suggested that UFH ameliorated LPS-induced endothelial barrier dysfunction by inhibiting MT disassembly and GEF-H1 expression. In addition, UFH attenuated LPS-induced hyperpermeability of HPMECs and F-actin remodeling. In vitro, UFH pretreatment inhibited LPS-induced increase in monomeric tubulin expression and decrease in tubulin polymerization and acetylation. Meanwhile, UFH ameliorates nocodazole-induced MTs disassembly and endothelial barrier dysfunction.Additionally, UFH decreased p38 phosphorylation and activation, which was similar to the effect of the p38 MAPK inhibitor, SB203580.ConclusionsUFH exert its protective effects on pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization and is associated with the p38 MAPK pathway.