Disassembly Of Adherens Junctions Research Articles

Mechanisms of vascular inflammaging: TNF-alpha mediates endothelial CEACAM1 expression by NF-KB and beta-Catenin in a biphasic manner

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Research Foundation Introduction Chronic inflammation with progressive age, also called inflammaging, is a hallmark of the pathogenesis of cardiovascular diseases. Previously we have shown increased expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) with progressive age in the vasculature of mice and humans that leads to a chronic pro-inflammatory milieu via mutual upregulation with TNF-α. Furthermore, CEACAM1 critically promotes vascular aging processes like collagen deposition, endothelial permeability as well as enhanced oxidative stress. Therefore, CEACAM1 is a main mediator of vascular inflammaging that may facilitate progress into cardiovascular diseases. In this study we identified the mechanisms that underly the upregulation of endothelial CEACAM1 expression by TNF-α in detail. Methods Wildtype (WT) and TNF-α knockout (Tnf-/-) mice of different age were used to determine the impact of TNF-α on age-dependent CEACAM1 expression in vivo. Underlying mechanisms of TNF-α-dependent CEACAM1 upregulation were further analyzed in an endothelial cell culture model using pharmacological inhibition and siRNA-mediated knockdown of signaling pathways. Activation of signaling pathways was confirmed by determining the phosphorylation status of signaling components via Western Blot and analysis of nuclear translocation of NF-κB and β-Catenin using subcellular fractionation. Results Immunohistochemical analyses of aortic sections from WT and Tnf-/- mice of different age reveal that TNF-α critically contributes to the aging-related upregulation of endothelial CEACAM1 expression in vivo. Mechanistic analyses in vitro showed that TNF-α upregulated CEACAM1 expression in a time-dependent manner. In pharmacological experiments we identified an early NF-κB- and a delayed β-Catenin-mediated response. This finding was supported by time-matched nuclear translocation of NF-κB and β-Catenin. Furthermore, siRNA-mediated knockdown of the β-Catenin-targeted transcription factor TCF4 reduced TNF-α-mediated CEACAM1 upregulation. Co-immunoprecipitation analyses show that β-Catenin is released by TNF-α-mediated adherens junctions disassembly. Furthermore, TNF-α enhanced activating phosphorylation of Akt kinase at Ser473. Akt kinase in turn increased phosphorylation of β-Catenin at Ser552, a modification reported to augment its transcriptional activity. Additionally, Akt kinase facilitated β-Catenin signaling by inhibition of its degradation via phosphorylation of GSK3β at Ser9. Conclusion Taken together, this study identified the signaling mechanisms that underly the vascular upregulation of CEACAM1 with age. Our results directly link endothelial barrier impairment, an initial step in the pathogenesis of atherosclerosis, to CEACAM1 expression. Since CEACAM1 critically promotes vascular inflammaging and age-dependent vascular alterations this further emphasizes the potential of CEACAM1 as a novel target to prevent cardiovascular diseases.

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

Oxidative stress-induced MMP- and γ-secretase-dependent VE-cadherin processing is modulated by the proteasome and BMP9/10

Classical cadherins, including vascular endothelial (VE)-cadherin, are targeted by matrix metalloproteinases (MMPs) and γ-secretase during adherens junction (AJ) disassembly, a mechanism that might have relevance for endothelial cell (EC) integrity and vascular homeostasis. Here, we show that oxidative stress triggered by H2O2 exposure induced efficient VE-cadherin proteolysis by MMPs and γ-secretase in human umbilical endothelial cells (HUVECs). The cytoplasmic domain of VE-cadherin produced by γ-secretase, VE-Cad/CTF2—a fragment that has eluded identification so far—could readily be detected after H2O2 treatment. VE-Cad/CTF2, released into the cytosol, was tightly regulated by proteasomal degradation and was sequentially produced from an ADAM10/17-generated C-terminal fragment, VE-Cad/CTF1. Interestingly, BMP9 and BMP10, two circulating ligands critically involved in vascular maintenance, significantly reduced VE-Cad/CTF2 levels during H2O2 challenge, as well as mitigated H2O2-mediated actin cytoskeleton disassembly during VE-cadherin processing. Notably, BMP9/10 pretreatments efficiently reduced apoptosis induced by H2O2, favoring endothelial cell recovery. Thus, oxidative stress is a trigger of MMP- and γ-secretase-mediated endoproteolysis of VE-cadherin and AJ disassembly from the cytoskeleton in ECs, a mechanism that is negatively controlled by the EC quiescence factors, BMP9 and BMP10.

Dopamine ameliorates hyperglycemic memory-induced microvascular dysfunction in diabetic retinopathy.

Dopamine is a neurotransmitter that mediates visual function in the retina and diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of blindness; however, the role of dopamine in retinal vascular dysfunction in DR remains unclear. Here, we report a mechanism of hyperglycemic memory (HGM)-induced retinal microvascular dysfunction and the protective effect of dopamine against the HGM-induced retinal microvascular leakage and abnormalities. We found that HGM induced persistent oxidative stress, mitochondrial membrane potential collapse and fission, and adherens junction disassembly and subsequent vascular leakage after blood glucose normalization in the mouse retinas. These persistent hyperglycemic stresses were inhibited by dopamine treatment in human retinal endothelial cells and by intravitreal injection of levodopa in the retinas of HGM mice. Moreover, levodopa supplementation ameliorated HGM-induced pericyte degeneration, acellular capillary and pericyte ghost generation, and endothelial apoptosis in the mouse retinas. Our findings suggest that dopamine alleviates HGM-induced retinal microvascular leakage and abnormalities by inhibiting persistent oxidative stress and mitochondrial dysfunction.

Mechanisms of pulmonary endothelial permeability and inflammation caused by extracellular histone subunits H3 and H4.

Extracellular DNA-binding proteins such as histones are danger-associated molecular pattern released by the injured tissues in trauma and sepsis settings, which trigger host immune response and vascular dysfunction. Molecular events leading to histone-induced endothelial cell (EC) dysfunction remain poorly understood. This study performed comparative analysis of H1, H2A, H2B, H3, and H4 histone subunits effects on human pulmonary EC permeability and inflammatory response. Analysis of transendothelial electrical resistance and EC monolayer permeability for macromolecues revealed that H3 and H4, but not H1, H2A, or H2B caused dose-dependent EC permeability accompanied by disassembly of adherens junctions. At higher doses, H3 and H4 activated nuclear factor kappa B inflammatory cascade leading to upregulation EC adhesion molecules ICAM1, VCAM1, E-selectin, and release of inflammatory cytokines. Inhibitory receptor analysis showed that toll-like receptor (TLR) 4 but not TLR1/2 or receptor for advanced glycation end inhibition significantly attenuated deleterious effects of H3 and H4 histones. Inhibitor of Rho-kinase was without effect, while inhibition of Src kinase caused partial preservation of cell-cell junctions, H3/H4-induced permeability and inflammation. Deleterious effects of H3/H4 were blocked by heparin. Activation of Epac-Rap1 signaling restored EC barrier properties after histone challenge. Intravenous injection of histones in mice caused elevation of inflammatory markers and increased vascular leak. Post-treatment with pharmacological Epac/Rap1 activator suppressed injurious effects of histones in vitro and in vivo. These results identify H3 and H4 as key histone subunits exhibiting deleterious effects on pulmonary vascular endothelium via TLR4-dependent mechanism. In conclusion, elevation of circulating histones may represent a serious risk of exacerbated acute lung injury (ALI) and multiple organ injury during severe trauma and infection.

Germline soma communication mediated by gap junction proteins regulates epithelial morphogenesis.

Gap junction (GJ) proteins, the primary constituents of GJ channels, are conserved determinants of patterning. Canonically, a GJ channel, made up of two hemi-channels contributed by the neighboring cells, facilitates transport of metabolites/ions. Here we demonstrate the involvement of GJ proteins during cuboidal to squamous epithelial transition displayed by the anterior follicle cells (AFCs) from Drosophila ovaries. Somatically derived AFCs stretch and flatten when the adjacent germline cells start increasing in size. GJ proteins, Innexin2 (Inx2) and Innexin4 (Inx4), functioning in the AFCs and germline respectively, promote the shape transformation by modulating calcium levels in the AFCs. Our observations suggest that alterations in calcium flux potentiate STAT activity to influence actomyosin-based cytoskeleton, possibly resulting in disassembly of adherens junctions. Our data have uncovered sequential molecular events underlying the cuboidal to squamous shape transition and offer unique insight into how GJ proteins expressed in the neighboring cells contribute to morphogenetic processes.

Mitofusin-2 stabilizes adherens junctions and suppresses endothelial inflammation via modulation of \u03b2-catenin signaling

Endothelial barrier integrity is ensured by the stability of the adherens junction (AJ) complexes comprised of vascular endothelial (VE)-cadherin as well as accessory proteins such as β-catenin and p120-catenin. Disruption of the endothelial barrier due to disassembly of AJs results in tissue edema and the influx of inflammatory cells. Using three-dimensional structured illumination microscopy, we observe that the mitochondrial protein Mitofusin-2 (Mfn2) co-localizes at the plasma membrane with VE-cadherin and β-catenin in endothelial cells during homeostasis. Upon inflammatory stimulation, Mfn2 is sulfenylated, the Mfn2/β-catenin complex disassociates from the AJs and Mfn2 accumulates in the nucleus where Mfn2 negatively regulates the transcriptional activity of β-catenin. Endothelial-specific deletion of Mfn2 results in inflammatory activation, indicating an anti-inflammatory role of Mfn2 in vivo. Our results suggest that Mfn2 acts in a non-canonical manner to suppress the inflammatory response by stabilizing cell–cell adherens junctions and by binding to the transcriptional activator β-catenin.

Mechanosensitive Piezo1 channel activation promotes ventilator-induced lung injury via disruption of endothelial junctions in ARDS rats

ObjectiveThis study aimed to investigate the role of endothelial Piezo1 in mediating ventilator-induced lung injury secondary to acute respiratory distress syndrome (ARDS). MethodsRats and lung endothelial cells (ECs) were transfected with Piezo1 shRNA (shPiezo1) and Piezo1 siRNA, respectively, to knock down Piezo1. Intratracheal instillation or incubation with lipopolysaccharide (LPS) was used to establish an ARDS model, and high tidal volume (HVT) ventilation or 20% cyclic stretch (CS) was administered to simulate a two-hit injury. Lung injury, alterations in lung endothelial barrier, disruption of adherens junctions (AJs), and Ca2+ influx were assessed. ResultsLung vascular hyperpermeability was further increased in ARDS rats following HVT ventilation, which was abrogated in shPiezo1-treated rats. 20% CS led to severer rupture of AJs following LPS stimulation as indicated by immunofluorescence staining. The internalization and degradation of VE-cadherin were blocked by knockdown of Piezo1. Additionally, 20% CS induced Piezo1 activation, manifesting as elevated intracellular Ca2+ concentration in LPS-treated ECs, and subsequently increased calcium-dependent calpain activity. Pharmacological inhibition of calpain or Piezo1 knockdown prevented the loss of VE-cadherin, p120-catenin, and β-catenin in ARDS rats undergoing HVT ventilation and LPS-treated ECs exposed to 20% CS. ConclusionExcessive mechanical stretch during ARDS induces the activation of Piezo1 channel and its downstream target, calpain, via Ca2+ influx. This results in the disassembly of endothelial AJs and further facilitates lung endothelial barrier breakdown and vascular hyperpermeability.

E-cadherin to P-cadherin switching in lobular breast cancer with tubular elements

Loss of E-cadherin expression due to mutation of the CDH1 gene is a characteristic feature of invasive lobular breast cancer (ILBC). Beta-catenin, which binds to the cytoplasmic domain of E-cadherin, is simultaneously downregulated, reflecting disassembly of adherens junctions (AJs) and loss of cell adhesion. E-cadherin to P-cadherin expression switching can rescue AJs and cell adhesion. However, P-cadherin has not been implicated in ILBC, so far. We aimed to characterize 13 ILBCs with exceptional histomorphology, which we termed ILBCs with tubular elements. The CDH1 mutational status was determined by next generation sequencing and whole-genome copy number (CN) profiling. Expression of cadherins was assessed by immunohistochemistry. ILBCs with tubular elements were ER-positive (13/13) and HER2-negative (13/13) and harbored deleterious CDH1 mutations (11/13) accompanied by loss of heterozygosity due to deletion of chromosome 16q22.1 (9/11). E-cadherin expression was lost or reduced in noncohesive tumor cells and in admixed tubular elements (13/13). Beta-catenin expression was lost in noncohesive tumor cells, but was retained in tubular elements (11/13), indicating focal rescue of AJ formation. N-cadherin and R-cadherin were always negative (0/13). Strikingly, P-cadherin was commonly positive (12/13) and immunoreactivity was accentuated in tubular elements. Adjacent lobular carcinoma in situ (LCIS) was always P-cadherin-negative (0/7). In a reference cohort of LCIS specimens, P-cadherin was constantly not expressed (0/25). In a reference cohort of invasive mammary carcinomas, P-cadherin-positive cases (36/268, 13%) were associated with triple-negative nonlobular breast cancer (P < 0.001). Compared with ILBCs from the reference cohort, P-cadherin expression was more common in ILBCs with tubular elements (12/13 versus 7/84, P < 0.001). In summary, E-cadherin to P-cadherin switching occurs in a subset of ILBCs. P-cadherin is the molecular determinant of a mixed-appearing histomorphology in ILBCs with tubular elements.

YAP activity protects against endotoxemic acutelung injury by activating multiple mechanisms.

The roles of the Hippo‑Yes‑associated protein (YAP) pathway in lung injury and repair remain elusive. The present study examined the effects of systemic inhibition or stimulation of YAP activity on lung injury, repair and inflammation in a mouse model of lipopolysaccharide(LPS)‑induced lung injury. Mice were treated with or without YAP inhibitor, verteporfin, or with or without YAP stimulator, XMU‑MP‑1, and intraperitoneally injected with LPS (7.5mg/kg). Lung injury and repair were evaluated by histological analysis and by testing for markers of lung injury. Lung inflammation was assessed by measuring tissue levels of inflammatory mediators. Lung injury was associated with a decreased, whereas lung repair was associated with an increased YAP activity evidenced by nuclear translocation. Lung injury was associated with a high level of lung inflammation and epithelial adherens junction disassembly, but not with cell proliferation or epithelial cell regeneration. The injury phase was defined as 0‑48h post‑LPS injection, and the 48‑168h time period was considered the repair phase. Inhibition of YAP activity at the injury phase, using verteporfin, exacerbated, whereas its stimulation, using XMU‑MP‑1, alleviated lung injury, lung inflammation and epithelial adherens junction disassembly. Inhibition or stimulation of YAP activity at the injury phase had no effects on cell proliferation or epithelial regeneration. By contrast, lung repair was associated with inflammation resolution, increased cell proliferation, epithelial regeneration and reassembly of epithelial adherens junctions. Inhibition of YAP activity at the repair phase delayed inflammation resolution, impeded lung recovery, inhibited cell proliferation and epithelial regeneration, and inhibited epithelial adherens junction reassembly. Stimulation of YAP activity at the repair phase reversed all these processes. The results of the current study demonstrated that the Hippo‑YAP activity serves a protective role against endotoxemic lung injury. The Hippo‑YAP activity alleviated lung inflammation and injury at the injury phase and promoted inflammation resolution and lung repair at the repair phase.

IGF2BP1 is a targetable SRC/MAPK-dependent driver of invasive growth in ovarian cancer

ABSTRACT Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HGSOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here, we show that the IGF2 mRNA-binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HGSOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in an RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signalling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation, only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rationale for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HGSOC.

A Novel Pharmacological Approach to Enhance the Integrity and Accelerate Restitution of the Intestinal Epithelial Barrier.

Disruption of the gut barrier is an essential mechanism of inflammatory bowel diseases (IBDs) contributing to the development of mucosal inflammation. A hallmark of barrier disruption is the disassembly of epithelial adherens junctions (AJs) driven by decreased expression of a major AJ protein, E-cadherin. A group of isoxazole compounds, such as E-cadherin-upregulator (ECU) and ML327, were previously shown to stimulate E-cadherin expression in poorly differentiated human cancer cells. This study was designed to examine whether these isoxazole compounds can enhance and protect model intestinal epithelial barriers in vitro. The study was conducted using T84, SK-CO15, and HT-29 human colonic epithelial cell monolayers. Disruption of the epithelial barrier was induced by pro-inflammatory cytokines, tumor necrosis factor-α, and interferon-γ. Barrier integrity and epithelial junction assembly was examined using different permeability assays, immunofluorescence labeling, and confocal microscopy. Epithelial restitution was analyzed using a scratch wound healing assay. E-cadherin-upregulator and ML327 treatment of intestinal epithelial cell monolayers resulted in several barrier-protective effects, including reduced steady-state epithelial permeability, inhibition of cytokine-induced barrier disruption and junction disassembly, and acceleration of epithelial wound healing. Surprisingly, these effects were not due to upregulation of E-cadherin expression but were mediated by multiple mechanisms including inhibition of junction protein endocytosis, attenuation of cytokine-induced apoptosis, and activation of promigratory Src and AKT signaling. Our data highlight ECU and ML327 as promising compounds for developing new therapeutic strategies to protect the integrity and accelerate the restitution of the intestinal epithelial barrier in IBD and other inflammatory disorders.

EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria.

Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

Interleukin-8 Secreted by Glioblastoma Cells Induces Microvascular Hyperpermeability Through NO Signaling Involving S-Nitrosylation of VE-Cadherin and p120 in Endothelial Cells.

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

Time-Variant SRC Kinase Activation Determines Endothelial Permeability Response

In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) viaphosphorylation of VE cadherin, and thereby increasejunctional permeability. Here, using a chemical biology approach to temporally control SRC activation,we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.

Novel Junctional and Nuclear Roles of the Mitochondrial Protein Mitofusin 2 During Inflammation

BackgroundSevere inflammation can cause widespread damage to the endothelium and result in fatal illnesses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Vascular endothelial barrier dysfunction is the major vascular complication in ALI. One of the key mechanisms of barrier disruption is the disassembly of adherens junction (AJ) complexes consisting of VE‐cadherin as well as accessory proteins such as β‐catenin and p120‐catenin. We observed that Mfn2, which has previously been primarily studied as a mitochondrial protein, is also located at the endothelial plasma membrane and co‐localizes with VE‐cadherin as well as translocates into nucleus under inflammatory condition, suggesting novel roles in AJs.Methods and ResultsBy using three‐dimensional structured illumination microscopy (3D‐SIM) which provides high spatial resolution (□130 nm), we studied Mfn2 localization in human lung microvascular ECs (HLMVECs). Mfn2 predominantly co‐localizes with the mitochondrial membrane protein Tom20 but it also co‐localizes at the plasma membrane with VE‐cadherin in resting ECs. However, upon pro‐inflammatory stimulation with TNF‐α, Mfn2 is dissociated from AJs and no longer co‐localizes with VE‐cadherin. Mechanistically, Mfn2 directly interacts with VE‐cadherin and β‐catenin in resting ECs as assessed by immunoprecipitation assay and a proximity ligation assay (PLA). To determine whether Mfn2 affects endothelial barrier integrity and function, Mfn2 was depleted in HLMVECs with a doxycycline inducible lentiviral shMfn2 RNA. Mfn2 silencing disrupted AJ junctions as visualized by VE‐cadherin and β‐catenin immunostaining. Mfn2 depletion significantly increased transendothelial permeability of ECs as assessed by a FITC‐conjugated albumin tracer assay and transendothelial resistance (TER). We also identified novel binding partners of Mfn2 using the high affinity tandem mass spectrometry (LC‐MS/MS). The analysis of gene ontology indicates that Mfn2 interacts with cell‐cell interaction proteins including cadherin binding and nuclear factor binding proteins with &gt;1.5‐fold increase. Finally, the complex of Mfn2 and β‐catenin translocates into the nucleus upon TNF‐α stimulation.ConclusionThese data suggest that Mfn2 may directly regulate AJ stability in ECs by interacting with the AJ complex proteins VE‐cadherin and β‐catenin. Mfn2 may act as a co‐regulator with β‐catenin, a key transcription factor in inflammatory condition. These studies enhance our understanding of how the “mitochondrial” protein Mfn2 regulates inflammatory activation via novel “non‐mitochondrial” functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spatiotemporal Control of Glycolysis Modulates ATP Generation and Enhances Restoration of Endothelial Barrier Function Following Inflammatory Injury

BackgroundAcute inflammatory injury is characterized by dysfunction of the vascular endothelium, resulting in disruption of endothelial adherens junctions, increased vascular permeability and leukocyte infiltration. While the mechanisms of assembly and disassembly of endothelial adherens junctions have been extensively studied, little is known about the bioenergetics of the barrier repair process. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial metabolism and function and could thus serve as a potential mediator of barrier repair.Methods and ResultsTo investigate how the endothelial metabolic profile changes during inflammation, we performed a Seahorse bioenergetic flux assay on Human Lung Microvascular Endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased by 36% following treatment with the inflammatory mediator TNFα, while oxidative phosphorylation was not affected. Pharmacological inhibition of PFKFB3 abolished TNFα mediated upregulation of glycolysis. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p&lt;0.0001). Whole cell ATP levels, measured by live cell microscopy using the fluorescent ATP biosensor Perceval HR, increased 31% following TNFα treatment (p&lt;0.0001), largely concentrated at the cell periphery. We hypothesized that this regional ATP generation is PFKFB3 dependent. To address this question, we engineered a novel construct in which PFKFB3 is targeted to the cell membrane in an inducible manner and thus regulating metabolism in a spatiotemporal manner. Importantly, PFKFB3 membrane targeting enhanced endothelial barrier restoration by increasing the extent and rate of recovery (p&lt;0.0001) following barrier disruption.ConclusionsThis data suggests that spatial and temporal regulation of PFKFB3 mediated glycolysis is important for restoration of the endothelial barrier after inflammatory injury. A better understanding of the endothelial barrier bioenergetics could allow for the development of novel therapeutic approaches to treat diseases in which endothelial barrier function is acutely compromised.Support or Funding InformationAHA 18PRE34070092 National Institute of Health P01HL060678This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Galectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly.

The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking β1-integrin antibodies point to β1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.

Abstract 16654: The Role of Glycolytic ATP Generation in the Restoration of Vascular Endothelial Barrier Function

Introduction: Acute Lung Injury (ALI) is characterized by endothelial barrier dysfunction of the lung vasculature, leading to disassembly of endothelial adherens junctions, increased vascular permeability and fluid accumulation in the lungs. Little is known about the bioenergetics of the barrier disruption and repair processes. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial glucose metabolism and could thus serve as a potential mediator of barrier repair. Methods and Results: To quantitatively measure metabolic changes in response to an inflammatory stimulus, we performed a Seahorse bioenergetic flux assay using human lung microvascular endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased acutely by 36% (from 9.1 to 12.4 mpH/min) following treatment with the inflammatory mediator TNFα. This upregulation was abolished in the presence of PFK15, a specific inhibitor of PFKFB3. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p&lt;0.0001). Live cell microscopy of endothelial cells expressing the ATP biosensor Perceval HR revealed that ATP is generated at cell junctions following TNFα treatment. Additionally, controlled translocation of PFKFB3 to the plasma membrane resulted in enhanced barrier restoration after injury, both in terms of the extent of barrier integrity as well as the rate of recovery (p&lt;0.0001). Finally, PFKFB3 was pharmacologically inhibited in vivo following lung injury with the bacterial toxin LPS in mice. This inhibition resulted in significantly impaired recovery of lung vascular barrier function as measured by an Evans blue albumin (EBA) permeability assay (p&lt;0.001). Endothelial cell-specific overexpression of PFKFB3 in mice by liposomal delivery resulted in an enhanced rate of recovery from LPS mediated injury. Lung vascular leakiness decreased from 29.0 to 18.3 ng EBA/g body weight at two days following the LPS injury (p&lt;0.001), thus achieving pre-injury levels of barrier function. Conclusions: Glycolytic ATP generation regulated by PFKFB3 is essential for restoration of the endothelial barrier after inflammatory injury. A better understanding of the endothelial barrier bioenergetics could allow for the development of novel therapeutic approaches which enhance barrier function in severe diseases such as acute lung injury.

IL-1β promotes transendothelial migration of PBMCs by upregulation of the FN/α5β1 signalling pathway in immortalised human brain microvascular endothelial cells

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain.The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the β1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether β1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1β (IL-1β)-induced inflammatory in vitro BBB model. We demonstrate that IL-1β increases cell-matrix adhesion and induces a redistribution of active β1 integrins to the basal surface. In particular, binding of α5β1 integrin to its ligand fibronectin is enhanced and α5β1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5β1 integrin reduces the IL-1β-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs).These data imply that IL-1β-induced inflammation not only destabilizes tight junctions but also increases α5β1 integrin-dependent cell-matrix adhesion to fibronectin.