Endothelial Adherens Junctions Research Articles

Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.

Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (2±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and optoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02783300.

The influences of ApoE isoforms on endothelial adherens junctions and actin cytoskeleton responding to mCRP.

Apolipoprotein E4 (ApoE4) plays an important role responding to monomeric C-reactive protein (mCRP) via binding to CD31 leading to cerebrovascular damage and Alzheimer's disease (AD). Using phosphor-proteomic profiling, we found altered cytoskeleton proteins in the microvasculature of AD brains, including increased levels of hyperphosphorylated tau (pTau) and the actin-related protein, LIMA1. To address the hypothesis that cytoskeletal changes serve as early pathological signatures linked with CD31 in brain endothelia in ApoE4 carriers, ApoE4 knock-in mice intraperitoneal injected with mCRP revealed that mCRP increased the expressions of phosphorylated CD31 (pCD31) and LIMA1, and facilitate the binding of pCD31 to LIMA1. mCRP combined with recombinant APOE4 protein decreased interaction of CD31 and VE-Cadherin at adherens junctions (AJs), along with altered the expression of various actin cytoskeleton proteins, causing microvasculature damage. Notably, the APOE2 protein attenuated these changes. Overall, our study demonstrates that ApoE4 responds to mCRP to disrupt the endothelial AJs which link with the actin cytoskeleton and this pathway could play a key role in the barrier dysfunction leading to AD risk.

Identification of hypoxic macrophages in glioblastoma: Unveiling therapeutic insights from tumour microenvironment analysis.

Tumor-associatedmacrophages (TAMs) exhibit remarkable heterogeneity in glioblastoma. Spatially resolved single-cell transcriptomic studies identified a monocyte-derived TAM subset localized in the peri-necrotic niche, driven by hypoxic cues to acquire ahypoxia response signature. These hypoxia-TAMs destabilize endothelial adherens junctions through adrenomedullin paracrine signaling, promoting the formation of hyperpermeable neovasculature that impedes drug delivery. Blocking adrenomedullin produced by hypoxia-TAMs restores vascular integrity, increases drug deliveryinto tumors, and provides combinatorial therapeutic benefits. Here we discuss the heterogeneity of TAMs regarding functional states and locations in glioblastomas, and propose future directions for studying the temporospatial dynamics of multifaceted TAM. HIGHLIGHTS: Single-cell omics reveal a functionally and spatially distinct hypoxia-TAM subset in glioblastoma. Adrenomedullin secreted by hypoxia-TAM destabilizes tumor vasculature and its blockade enhances vessel integrity and drug delivery.

Magnetically Integrated Tumor-Vascular Interface System to Mimic Pro-angiogenic Endothelial Dysregulations for On-Chip Drug Testing.

The tumor-vascular interface is a critical component of the tumor microenvironment that regulates all of the dynamic interactions between a growing tumor and the endothelial lining of the surrounding vasculature. In this paper, we report the design and development of a custom-engineered tumor-vascular interface system for investigating the early stage tumor-mediated pro-angiogenic dysfunctional behavior of the endothelium. Using representative endothelial cells and triple negative breast cancer cell lines, we established a biomimetic interface between a three-dimensional tumor tissue across a mature, functional endothelial barrier using a magnetically hybrid-integrated tumor-vascular interface system, wherein vasculature-like features containing a monolayer of endothelial cell culture on porous microfluidic channel surfaces were magnetically attached to tumor spheroids generated on a composite polymer-hydrogel microwell plate and embedded in a collagen matrix. Tumor-mediated endothelial microdynamics were characterized by their hallmark behavior such as loss of endothelial adherens junctions, increased cell density, proliferation, and changes in cell spreading and corroborated with endothelial YAP/TAZ nuclear translocation. We further confirm the feasibility of drug-mediated reversal of this pro-angiogenic endothelial organization through two different signaling mechanisms, namely, inhibition of the vascular endothelial growth factor pathway and the Notch signaling pathway, thereby demonstrating the utility of the tumor-vascular interface platform for rapid, early stage prediction of antiangiogenic drug efficacy. Overall, our work emphasizes the importance of our strategic engineering approach for identifying some unique, physiologically relevant aspects of the tumor-vascular interface, which are otherwise difficult to implement using standard in vitro approaches.

Notch transcriptional target tmtc1 maintains vascular homeostasis

Proper lung function requires the maintenance of a tight endothelial barrier while simultaneously permitting the exchange of macromolecules and fluids to underlying tissue. Disruption of this barrier results in an increased vascular permeability in the lungs, leading to acute lung injury. In this study, we set out to determine whether transcriptional targets of Notch signaling function to preserve vascular integrity. We tested the in vivo requirement for Notch transcriptional signaling in maintaining the pulmonary endothelial barrier by using two complementary endothelial-specific Notch loss-of-function murine transgenic models. Notch signaling was blocked using endothelial-specific activation of an inhibitor of Notch transcriptional activation, Dominant Negative Mastermindlike (DNMAML; CDH5CreERT2), or endothelial-specific loss of Notch1 (Notch1f/f; CDH5CreERT2). Both Notch mutants increased vascular permeability with pan-Notch inhibition by DNMAML showing a more severe phenotype in the lungs and in purified endothelial cells. RNA sequencing of primary lung endothelial cells (ECs) identified novel Notch targets, one of which was transmembrane O-mannosyltransferase targeting cadherins 1 (tmtc1). We show that tmtc1 interacts with vascular endothelial cadherin (VE-cadherin) and regulates VE-cadherin egress from the endoplasmic reticulum through direct interaction. Our findings demonstrate that Notch signaling maintains endothelial adherens junctions and vascular homeostasis by a transcriptional mechanism that drives expression of critical factors important for processing and transport of VE-cadherin.

Regulation of vascular endothelial integrity by mesenchymal stem cell extracellular vesicles after hemorrhagic shock and trauma

BackgroundPatients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses the systemic effects of HS/T on multiorgan EOT.MethodsIn vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H2O2) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer’s (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers.ResultsMSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice.ConclusionsIn conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.

Abstract 3112: Identifying NogoB Receptor As A Resilience Factor In Preventing Diabetes-associated Vasculature Complications Through The Regulation Of Histone Acetylation

The COVID-19 pandemic has underlined the link between cytokine storms and acute lung injury (ALI). Importantly, diabetic patients demonstrate a higher susceptibility to severe lung injury and increased mortality, indicating that hyperglycemia could compromise the host's resilience necessary to counteract cytokine-induced lung injury. Single-nucleus RNA-seq data from COVID-19 patients reveal a significant decline in endothelial cells expressing the Nogo-B receptor (NgBR). Our recent research confirms that NgBR plays a pivotal role in maintaining the structural integrity of blood vessels by regulating KAT7-mediated histone acetylation.To investigate NgBR's resilience role, we examined its expression in response to hyperglycemic stress. We found a reduction in NgBR expression levels in endothelial cells (ECs) isolated from db/db mice, mirroring observations in ECs from Streptozocin (STZ)-injected mice. Moreover, when human microvascular endothelial cells (HMECs) were exposed to high glucose levels, NgBR expression increased initially but subsequently declined with extended exposure. This inhibitory effect of high glucose on NgBR expression was both time- and dose-dependent and was mediated through the DNA methylation mechanism.We also examined the effects of NgBR loss on the integrity of pulmonary vasculature and the pathogenesis of lung injury. We induced genetic depletion of NgBR in ECs through tamoxifen administration and found severe lung hemorrhage in NgBR inducible endothelial cell-specific knockout (iecKO) mice. Impaired EC junctions were identified by reduced VE-cadherin immunostaining. Following exposure to a lipopolysaccharide (LPS), ALI model, we observed increased neutrophil accumulation in the alveolar or interstitial space, alveolar wall thickening, and edema in the lungs of either NgBR iecKO or diabetic mice. RNA-seq analysis revealed that NgBR knockdown resulted in decreased transcription of CDC42, a key gene regulating endothelial adherens junction through KAT7-mediated histone acetylation. Our findings indicate that NgBR is a resilience factor for maintaining vascular integrity, and epigenetic medicine can rescue NgBR loss in endothelial cells and the severity of diabetes-associated acute lung injury.

Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization

Monocyte-derived tumor-associated macrophages (Mo-TAMs) intensively infiltrate diffuse gliomas with remarkable heterogeneity. Using single-cell transcriptomics, we chart a spatially resolved transcriptional landscape of Mo-TAMs across 51 patients with isocitrate dehydrogenase (IDH)-wild-type glioblastomas or IDH-mutant gliomas. We characterize a Mo-TAM subset that is localized to the peri-necrotic niche and skewed by hypoxic niche cues to acquire a hypoxia response signature. Hypoxia-TAM destabilizes endothelial adherens junctions by activating adrenomedullin paracrine signaling, thereby stimulating a hyperpermeable neovasculature that hampers drug delivery in glioblastoma xenografts. Accordingly, genetic ablation or pharmacological blockade of adrenomedullin produced by Hypoxia-TAM restores vascular integrity, improves intratumoral concentration of the anti-tumor agent dabrafenib, and achieves combinatorial therapeutic benefits. Increased proportion of Hypoxia-TAM or adrenomedullin expression is predictive of tumor vessel hyperpermeability and a worse prognosis of glioblastoma. Our findings highlight Mo-TAM diversity and spatial niche-steered Mo-TAM reprogramming in diffuse gliomas and indicate potential therapeutics targeting Hypoxia-TAM to normalize tumor vasculature.

Identification of core genes of craniopharyngioma angiogenesis based on single-cell nuclear transcriptome sequencing.

This study aimed to explore the core genes of craniopharyngioma angiogenesis for targeted vascular therapy based on single-cell nuclear transcriptome sequencing. For single-cell nuclear transcriptome sequencing, we collected six samples from the tumor center and adjacent hypothalamic tumor tissues from three patients with craniopharyngioma, as well as four normal brain tissues based on Gene Expression Omnibus. We screened genes with differential up-regulation between vascular endothelial cells of craniopharyngioma and those of normal brain tissues, performed GO and KEGG analysis, constructed the protein-protein interaction network, and selected key genes verified using immunofluorescence. After data cleaning and quality control, 623 craniopharyngioma endothelial cells and 439 healthy brain endothelial cells were obtained. Compared with normal brain endothelial cells, craniopharyngioma endothelial cells were screened for 394 differentially up-expressed genes (DEGs). GO and KEGG results showed that DEGs probably modulated endothelial cells, adherens junction, focal adhesion, migration, actin cytoskeleton, and invasion via the PI3K-AKT, Rap1, Ras, Wnt, and Hippo pathways. The core genes screened were CTNNB1, PTK2, ITGB1, STAT3, FYN, HIF1A, VCL, SMAD3, PECAM1, FOS, and CDH5. This study obtained possible anti-angiogenic genes in craniopharyngioma. Our results shed novel insights into molecular mechanisms and craniopharyngioma treatment.

Cortactin is in a complex with VE-cadherin and is required for endothelial adherens junction stability through Rap1/Rac1 activation

Vascular permeability is mediated by Cortactin (Cttn) and regulated by several molecules including cyclic-adenosine-monophosphate, small Rho family GTPases and the actin cytoskeleton. However, it is unclear whether Cttn directly interacts with any of the junctional components or if Cttn intervenes with signaling pathways affecting the intercellular contacts and the cytoskeleton. To address these questions, we employed immortalized microvascular myocardial endothelial cells derived from wild-type and Cttn-knock-out mice. We found that lack of Cttn compromised barrier integrity due to fragmented membrane distribution of different junctional proteins. Moreover, immunoprecipitations revealed that Cttn is within the VE-cadherin-based adherens junction complex. In addition, lack of Cttn slowed-down barrier recovery after Ca2+ repletion. The role of Cttn for cAMP-mediated endothelial barrier regulation was analyzed using Forskolin/Rolipram. In contrast to Cttn-KO, WT cells reacted with increased transendothelial electrical resistance. Absence of Cttn disturbed Rap1 and Rac1 activation in Cttn-depleted cells. Surprisingly, despite the absence of Cttn, direct activation of Rac1/Cdc42/RhoA by CN04 increased barrier resistance and induced well-defined cortical actin and intracellular actin bundles. In summary, our data show that Cttn is required for basal barrier integrity by allowing proper membrane distribution of junctional proteins and for cAMP–mediated activation of the Rap1/Rac1 signaling pathway.

Trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and invivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions

Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (ChfrΔEC) mice. We also observe abrogation of LPS-induced degradation of VE-cadherin in ChfrΔEC mice, suggesting the pathophysiological relevance of CHFR in regulating the endothelial junctional barrier in inflammation. Lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality induced by LPS were all suppressed in ChfrΔEC mice. We find that the transcription factor FoxO1 is a key upstream regulator of CHFR expression. These findings demonstrate the requisite role of the endothelial cell-expressed E3-ligase CHFR in regulating the expression of VE-cadherin, and thereby endothelial junctional barrier integrity.

Engineering tumoral vascular leakiness with gold nanoparticles

Delivering cancer therapeutics to tumors necessitates their escape from the surrounding blood vessels. Tumor vasculatures are not always sufficiently leaky. Herein, we engineer therapeutically competent leakage of therapeutics from tumor vasculature with gold nanoparticles capable of inducing endothelial leakiness (NanoEL). These NanoEL gold nanoparticles activated the loss of endothelial adherens junctions without any perceivable toxicity to the endothelial cells. Microscopically, through real time live animal intravital imaging, we show that NanoEL particles induced leakiness in the tumor vessels walls and improved infiltration into the interstitial space within the tumor. In both primary tumor and secondary micrometastases animal models, we show that pretreatment of tumor vasculature with NanoEL particles before therapeutics administration could completely regress the cancer. Engineering tumoral vasculature leakiness represents a new paradigm in our approach towards increasing tumoral accessibility of anti-cancer therapeutics instead of further increasing their anti-cancer lethality.

CAMP: A master regulator of cadherin-mediated binding in endothelium, epithelium and myocardium.

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3',5'-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised.

Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src deficiency, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

SHOCK INDUCES ENDOTHELIAL PERMEABILITY AFTER TRAUMA THROUGH INCREASED ACTIVATION OF RHOA GTPASE.

Introduction: Severely injured patients develop a dysregulated inflammatory state characterized by vascular endothelial permeability, which contributes to multiple organ failure. To date, however, the mediators of and mechanisms for this permeability are not well established. Endothelial permeability in other inflammatory states such as sepsis is driven primarily by overactivation of the RhoA GTPase. We hypothesized that tissue injury and shock drive endothelial permeability after trauma by increased RhoA activation leading to break down of endothelial tight and adherens junctions. Methods: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, whereas continuous resistance was measured using electrical cell-substrate impedance sensing (ECIS) Z-Theta technology, 10% ex vivo plasma from severely injured trauma patients was added, and resistance measurements continued for 2 hours. Areas under the curve (AUCs) were calculated from resistance curves. For GTPase activity analysis, HUVECs were grown to confluence and incubated with 10% trauma plasma for 5 minutes before harvesting of cell lysates. Rho and Rac activity were determined using a G-LISA assay. Significance was determined using Mann-Whitney tests or Kruskal-Wallis test, and Spearman ρ was calculated for correlations. Results: Plasma from severely injured patients induces endothelial permeability with plasma from patients with both severe injury and shock contributing most to this increased permeability. Surprisingly, Injury Severity Score (ISS) does not correlate with in vitro trauma-induced permeability (-0.05, P > 0.05), whereas base excess (BE) does correlate with permeability (-0.47, P = 0.0001). The combined impact of shock and injury resulted in a significantly smaller AUC in the injury + shock group (ISS > 15, BE < -9) compared with the injury only (ISS > 15, BE > -9; P = 0.04) or minimally injured (ISS < 15, BE > -9; P = 0.005) groups. In addition, incubation with injury + shock plasma resulted in higher RhoA activation ( P = 0.002) and a trend toward decreased Rac1 activation ( P = 0.07) compared with minimally injured control. Conclusions: Over the past decade, improved early survival in patients with severe trauma and hemorrhagic shock has led to a renewed focus on the endotheliopathy of trauma. This study presents the largest study to date measuring endothelial permeability in vitro using plasma collected from patients after traumatic injury. Here, we demonstrate that plasma from patients who develop shock after severe traumatic injury induces endothelial permeability and increased RhoA activation in vitro . Our ECIS model of trauma-induced permeability using ex vivo plasma has potential as a high throughput screening tool to phenotype endothelial dysfunction, study mediators of trauma-induced permeability, and screen potential interventions.

Endothelial CYP2J2 overexpression restores the BRB via METTL3-mediated ANXA1 upregulation.

Blood-retinal barrier (BRB) breakdown is responsible for multiple ocular diseases, such as diabetic retinopathy, age-related macular degeneration, and retinal vascular occlusive diseases. Increased vascular permeability contributes to vasogenic edema and tissue damage, with consequent adverse effects on vision. Herein, we found that endothelial CYP2J2 overexpression maintained BRB integrity after ischemia-reperfusion injury and consequently protected against retinal ganglion cell loss. Oxidative stress repressed endothelial ANXA1 expression in vivo and in vitro. CYP2J2 upregulated methyltransferase-like 3 (METTL3) expression and hence promoted ANXA1 translation via ANXA1 m6 A modification in endothelium under oxidative stress. CYP2J2 maintained the distribution of endothelial tight junctions and adherens junctions in an ANXA1-dependent manner. Endothelial ANXA1 plays an indispensable role in vascular homeostasis and stabilization during development. Endothelial ANXA1 deletion disrupted retinal vascular perfusion as well as BRB integrity. CYP2J2 metabolites restored BRB integrity in the presence of ANXA1. Our findings identified the CYP2J2-METTL3-ANXA1 pathway as a potential therapeutic target for relieving BRB impairments.

Role of moesin and its phosphorylation in VE-cadherin expression and distribution in endothelial adherens junctions

Background and aimVascular endothelial cadherin (VE-cadherin) is an important element of adherens junctions (AJs) between endothelial cells. Its expression and proper distribution are critical for AJ formation and vascular integrity. Our previous studies have demonstrated that moesin phosphorylation mediated the hyper-permeability in endothelial monolayer and microvessels. However, the role of moesin and its phosphorylation in VE-cadherin expression and distribution is not clear. Methods and resultsIn vivo, expression of VE-cadherin was significantly reduced in retina and other various tissues in moesin knock out mice (Msn-/Y). In vitro, by regulating moesin expression with siRNA and adenovirus transfection, we verified that moesin has an effect on VE-cadherin expression in HUVECs, while transcription factor KLF4 may participate in this process. In addition, treatment of advanced glycation end products (AGEs) induced abnormal distribution of VE-cadherin in retinal microvessels from C57BL/6 wild type mice, and in vitro studies indicated that moesin Thr558 phosphorylation had a critical role in AGE-induced VE-cadherin internalization from cytomembrane to cytoplasm. Further investigation demonstrated that the inhibition of F-actin polymerization with cytochalasin D could abolish AGE- and Thr558 phosphor-moesin-mediated VE-cadherin internalization. ConclusionThis study suggests that moesin regulates VE-cadherin expression through KLF4 and the state of moesin phosphorylation at Thr558 affects the integrity of VE-cadherin-based AJs. Thr558 phosphor-moesin mediates AGE-induced VE-cadherin internalization through cytoskeleton reassembling.

Human Neutrophil Defensins Disrupt Liver Interendothelial Junctions and Aggravate Sepsis

Human neutrophil peptides 1-3 (HNP1-3), also known as human α-defensins, are the most abundant neutrophil granule proteins. The genes that encode HNP1-3, DEFA1/DEFA3, exhibit extensive copy number variations, which correlate well with their protein levels. Human and mouse studies have shown that increased copy numbers of DEFA1/DEFA3 worsen sepsis outcomes. Additionally, high concentrations of HNP1-3 in body fluids have been reported in patients with sepsis. However, direct evidence for the pathogenic role of HNP1-3 proteins during sepsis progression is lacking. In current study, sepsis was induced by means of cecal puncture and ligation. Various doses of HNP-1 (low dose with 0.5 mg/kg body weight and high dose with 10 mg/kg body weight) or phosphate buffer saline were intraperitoneally administered to mice at six hours after sepsis onset. Survival rate was monitored, and vascular permeability, endothelial cell pyroptosis, and immunofluorescence of endothelial adherens junction protein vascular endothelial-cadherin were evaluated. The administration of a high dose of HNP-1 after sepsis onset led to increased mortality, more severe liver injury, and increased vascular permeability in the liver and mesentery. The injection of high dose of HNP-1 did not directly induce liver endothelial cell death but destroyed interendothelial junctions in the liver. Moreover, genetic deficiency of nucleotide-binding oligomerization domain-like receptor protein-3 or caspase-1 abrogated the high mortality and disrupted liver interendothelial junctions caused by high dose of HNP-1 during sepsis. This study directly demonstrates that neutrophil defensins play a key role in regulating endothelial stability during sepsis development.

Claudin5 protects the peripheral endothelial barrier in an organ and vessel-type-specific manner.

Dysfunctional and leaky blood vessels resulting from disruption of the endothelial cell (EC) barrier accompanies numerous diseases. The EC barrier is established through endothelial cell tight and adherens junctions. However, the expression pattern and precise contribution of different junctional proteins to the EC barrier is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability and commonalities of EC barrier patterning. Across tissues, Claudin5 exhibited diminishing expression along the arteriovenous axis, correlating with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage properties and response to the leakage agonist histamine. Loss of Claudin5 enhanced histamine-induced leakage in an organotypic and vessel type-specific manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss left junction ultrastructure unaffected but altered its composition, with concomitant loss of zonula occludens-1 and upregulation of VE-Cadherin expression. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds, providing insights to modify EC barrier stability in a targeted, organ-specific manner.