Endothelial Junction Integrity Research Articles

Rnd3 Ameliorates Diabetic Cardiac Microvascular Injury via Facilitating Trim40-mediated Rock1 Ubiquitination.

Diabetic microvascular dysfunction is evidenced by disrupted endothelial cell junctions and increased microvascular permeability. However, effective strategies against these injuries remain scarce. In this study, the type 2 diabetes mouse model was established by high-fat diet combined with streptozotocin injection in Rnd3 endothelial- specific transgenic and knockout mice. Echocardiography was employed to evaluate cardiac function. Microvascular corrosion casts, lanthanum nitrate perfusion, trans- endothelial electrical resistance, FITC-dextran permeability assay and laser speckle contrast imaging were performed to evaluate the integrity of endothelial cell junctions and microvascular function. RNA sequencing, mass spectrometry, co-immunoprecipitation, immunofluorescence and molecular docking were used to explore the downstream regulators of Rnd3. Evidence from gain/loss-of-function studies denoted a protective role for Rnd3 against microvascular dysfunction in diabetic heart. Endothelial-specific deletion of Rnd3 significantly exacerbated coronary microvascular barrier dysfunction under diabetic conditions, while Rnd3 overexpression effectively prevented these effects. Furthermore, Rnd3 overexpression also attenuated cardiac dysfunction in diabetic mice, as indicated by increased LVEF, LVFS, and E/A ratio. Rnd3 overexpression inhibited CMECs apoptosis and increased CMECs migration in response to HG-PA challenge. Rnd3 overexpression inhibited Rock1 activity and MLC phosphorylation in CMECs treated with HG-PA stimulus. Mechanically, Rnd3 recruited and interacted with the E3 ubiquitin ligase Trim40 which further facilitated the degradation of Rock1, thus inhibiting endothelial barrier hyperpermeability in HG-PA-stimulated CMECs. However, the cardioprotective effects of Rnd3 were largely abrogated by Trim40 deficiency in diabetic conditions. Collectively, Rnd3 alleviates microvascular hyperpermeability, maintains endothelial barrier integrity, and mitigates cardiac dysfunction by regulating the Rock1/MLC signaling pathway in the state of DCM.

Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation.

Rickettsia spp. cause life-threatening diseases in humans. The fundamental pathophysiological changes in fatal rickettsial diseases are disrupted endothelial barrier and increased microvascular permeability. However, it remains largely unclear how rickettsiae induce microvascular endothelial injury. In the present study, we demonstrated that Rickettsia conorii infection disrupts the continuous immunofluorescence expression of the interendothelial tight junction protein, zonula occludens-1 (ZO-1), in infected monolayers of microvascular endothelial cells (MVECs), accompanied by significantly diminished total expression levels of ZO-1. Interestingly, R. conorii activated inflammasome in MVECs, as evidenced by cleaved caspase-1 and IL-1β in the cell lysates in association with significantly elevated expression levels of nucleotide binding and oligomerization domain, leucine-rich repeat, and pyrin containing protein 3 (NLRP3). Furthermore, selective inhibition of NLRP3 by MCC950 significantly suppressed the activation and cleavage of caspase-1 induced by R. conorii in endothelial cells, which further prevented the disruption of interendothelial junctions and reduction of ZO-1 expression. Of note, pharmaceutical inhibition of NLRP3 mitigated the disrupted endothelial integrity caused by R. conorii, measured by fluorescein isothiocyanate-dextran passage in a Transwell assay, independent of bacterial growth and cellular cytotoxicity. Taken together, our results suggest that R. conorii affected microvascular endothelial junction integrity likely via diminishing and interrupting the junctional protein ZO-1 in association with activating NLRP3 inflammasome. These data not only highlight the potential of ZO-1 as a biomarker for Rickettsia-induced microvascular injury but also provide insight into targeting NLRP3 inflammasome/ZO-1 signaling as a potentially adjunctive therapeutic approach for severe rickettsioses.

Control of inflammatory lung injury and repair by metabolic signaling in endothelial cells.

Sepsis-induced inflammatory lung injury includes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). There are currently no effective treatments for ALI/ARDS, but clinical outcomes could be improved by inhibiting lung injury and/or promoting post-sepsis vascular repair. In this review, we describe studies of endothelial cell metabolic pathways in sepsis-induced ALI/ARDS and vascular repair and identify areas of research that deserve attention in future studies. We also describe studies of metabolic interventions that aim to inhibit ALI/ARDS and/or promote post-sepsis vascular repair, including those that target endothelial cell metabolites, endothelial cell metabolic signaling pathways, and endothelial cell metabolism. Endothelial cells are integral to both the injury and repair phases of ALI/ARDS. During the injury phase of ALI/ARDS, lung endothelial cell survival decreases, and lung endothelial cell-to-endothelial cell (EC-EC) junctions are weakened. During the repair phase after sepsis-induced lung injury, lung endothelial cell proliferation and lung EC-EC junction reannealing occur. These crucial aspects of ALI/ARDS and post-sepsis vascular repair, that is, endothelial cell viability, growth, and junction integrity, are controlled by a myriad of metabolites and metabolic signaling pathways in endothelial cells. Metabolic signaling pathways in endothelial cells represent a novel class of putative targets for the prevention and treatment of sepsis-induced inflammatory lung injury. Therapies that target metabolic signaling in endothelial cells are currently being explored as potential treatments for sepsis-induced inflammatory lung injury.

Off-targets of BRAF inhibitors disrupt endothelial signaling and vascular barrier function.

Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. The blood vessel lining is in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to approved BRAFi for melanoma in the vascular endothelium. We showed that clinically approved BRAFi induced a paradoxical activation of endothelial MAPK signaling. Moreover, phosphoproteomics revealed distinct sets of off-targets per inhibitor. Endothelial barrier function and junction integrity were impaired upon treatment with vemurafenib and the next-generation dimerization inhibitor PLX8394, but not with dabrafenib or encorafenib. Together, these findings provide insights into the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality. Better understanding of off-target effects could help to identify molecular mechanisms behind AEs and guide the continued development of therapies for BRAF-mutant melanoma.

Acetyl-NPKY of integrin-β1 binds KINDLIN2 to control endothelial cell proliferation and junctional integrity

Integrin-dependent crosstalk between cell-matrix adhesions and cell-cell junctions is critical for controlling endothelial permeability and proliferation in cancer and inflammatory diseases but remains poorly understood. Here, we investigated how acetylation of the distal NPKY-motif of Integrin-β1 influences endothelial cell physiology and barrier function. Expression of an acetylation-mimetic β1-K794Q-GFP mutant led to the accumulation of immature cell-matrix adhesions accompanied by a transcriptomic reprograming of endothelial cells, involving genes associated with cell adhesion, proliferation, polarity, and barrier function. β1-K794Q-GFP induced constitutive MAPK signaling, junctional impairment, proliferation, and reduced contact inhibition at confluence. Structural analysis of Integrin-β1 interaction with KINDLIN2, biochemical pulldown assay, and binding energy determination by using molecular dynamics simulation showed that acetylation of K794 and the K794Q-mutant increased KINDLIN2 binding affinity to the Integrin-β1. Thus, enhanced recruitment of KINDLIN2 to Lysine-acetylated Integrin-β1 and resulting modulation of barrier function, offers new therapeutic possibilities for controlling vascular permeability and diseaseconditions.

Dulaglutide restores endothelial progenitor cell levels in diabetic mice and mitigates high glucose-induced endothelial injury through SIRT1-mediated mitochondrial fission

Type 2 diabetes mellitus (T2DM) significantly impairs the functionality and number of endothelial progenitor cells (EPCs) and resident endothelial cells, critical for vascular repair and regeneration, exacerbating the risk of vascular complications. GLP-1 receptor agonists, like dulaglutide, have emerged as promising therapeutic agents due to their multifaceted effects, including the enhancement of EPC activity and protection of endothelial cells. This study investigates dulaglutide's effects on peripheral blood levels of CD34+ and CD133+ cells in a mouse model of lower limb ischemia and its protective mechanisms against high-glucose-induced damage in endothelial cells. Results demonstrated that dulaglutide significantly improves blood flow, reduces tissue damage and inflammation in ischemic limbs, and enhances glycemic control. Furthermore, dulaglutide alleviated high-glucose-induced endothelial cell damage, evident from improved tube formation, reduced reactive oxygen species accumulation, and restored endothelial junction integrity. Mechanistically, dulaglutide mitigated mitochondrial fission in endothelial cells under high-glucose conditions, partly through maintaining SIRT1 expression, which is crucial for mitochondrial dynamics. This study reveals the potential of dulaglutide as a therapeutic option for vascular complications in T2DM patients, highlighting its role in improving endothelial function and mitochondrial integrity.

Umbilical cord mesenchymal stem cells from gestational diabetes show impaired ability to up-regulate paracellular permeability from sub-endothelial niche.

In vitro studies have shown that Wharton's jelly mesenchymal stem cells (WJ-MSCs) can cross umbilical and uterine endothelial barriers and up-regulate endothelial junctional integrity from sub-endothelial niches. This pericytic behaviour may be lost in pregnancies complicated by gestational diabetes (GDM), where increased vascular permeability and junctional disruption are reported. The aim of the present study was to investigate whether WJ-MSCs isolated from GDM pregnancies displayed any changes in morphology, proliferation, VEGF-A secretion, and their ability to influence paracellular junctional composition and permeability. WJ-MSCs were isolated from human umbilical cords from normal pregnancies (nWJ-MSCs, n=13) and those complicated by GDM (gWJ-MSCs), either diet-controlled (d-GDM, n=13) or metformin-treated (m-GDM, n=9). We recorded that 4-fold more WJ-MSCs migrated from m-GDM, and 2.5-fold from d-GDM cord samples compared with the normal pregnancy. gWJ-MSCs showed a less predominance of spindle-shaped morphology and secreted 3.8-fold more VEGF-A compared with nWJ-MSCs. The number of cells expressing CD105 (Endoglin) was higher in gWJ-MSCs compared with nWJ-MSCs (17%) at P-2. The tracer leakage after 24 h across the HUVEC + gWJ-MSCs bilayer was 22.13% and 11.2% higher in the m-GDM and d-GDM, respectively, HUVEC + nWJ-MSCs. Transfection studies with siRNAs that target Endoglin were performed in n-WJ-MSCs; transfected cells were co-cultured with HUVEC followed by permeability studies and VE-cadherin analyses. Loss of Endoglin also led to increased VEGF-A secretion, increased permeability and affected endothelial stabilization. These results reinforce the pericytic role of nWJ-MSCs to promote vascular repair and the deficient ability of gWJ-MSCs to maintain endothelial barrier integrity.

SURG-22. MECHANISMS OF BBB PERMEABILITY CHANGES TRIGGERED BY LASER THERAPY

Abstract The blood-brain barrier (BBB) severely limits access of systemically delivered drugs to the brain and brain tumors. Laser interstitial thermal therapy (LITT) has previously been shown to increase BBB permeability in both patients and in mice. The exact mechanisms of LITT-induced BBB permeability changes and their temporal features have yet to be elucidated. To answer this question, we used an established mouse model of LITT to evaluate changes in endothelial cell tight junction integrity and transcytosis over time in normal brain and demonstrate changes in transcytosis in human GBM tumor samples. Mean vesicular density (MVD) was calculated using electron microscopy to evaluate transcytosis. Immunohistochemistry (IHC) was used to assess tight junction integrity (Claudin 5 area/CD31 area) and Mfsd2a levels (Mfsd2a area/CD31 area) along endothelial cells in the laser penumbra. Tight junction integrity decreased 2.5 fold 3 days after LITT, with continued decrease 14 days post-LITT. Mean vesicular density (MVD) in endothelial cells within the laser penumbra increased 3-4 fold compared to sham treated mice, with a peak increase 14 days after LITT before returning to baseline levels 21 days after treatment. These changes in transcytosis correlated with a significant decrease in the intensity of Mfsd2a IHC in the laser penumbra at day 14. Finally, we found similar low levels of transcytosis in non-enhancing and enhancing human GBM tumor samples, confirming that transcytosis is potentially targetable to manipulate BBB permeability. CONCLUSION: Disruptions in tight junction integrity continue up to 14 days after LITT, while LITT-induced increases in transcytosis occur in a delayed fashion, peaking 14 days after laser treatment. Increased transcytosis is inversely correlated with decreased Mfsd2a expression, suggesting a role for Mfsd2a in regulation of LITT-induced BBB changes. Importantly, baseline levels of transcytosis in human GBM appear to be low, such that transcytosis could be targeted to increase BBB permeability.

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.

Cortistatin deficiency reveals a dysfunctional brain endothelium with impaired gene pathways, exacerbated immune activation, and disrupted barrier integrity

BackgroundBrain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood–brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment.MethodsWild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS.ResultsThe endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage.ConclusionsThe neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB.

Telomerase reverse transcriptase restores pancreatic microcirculation profiles and attenuates endothelial dysfunction by inhibiting mitochondrial superoxide production: A potential target for acute pancreatitis therapy

BackgroundAcute pancreatitis (AP) is a potentially lethal disease related to prominent microcirculation dysfunction. Pancreatic microvascular endothelial dysfunction enhances oxidative stress with tissue damage. Increased superoxide production disrupts endothelial junction integrity and increases endothelial permeability. Endothelial mitochondrial ROS (mtROS) represent a major intracellular source of superoxide anions. The non-canonical function of telomerase reverse transcriptase (TERT) involves the maintenance of cellular redox homeostasis in somatic tissues. MethodsWe investigated whether TERT restores microcirculation dysfunction and attenuates the endothelium injury by inhibiting superoxide production during AP progression. We established TERT transgenic and TERT knock-down mice and used cerulein (CER) and lipopolysaccharide (LPS) injections to induce AP models. In addition, we exposed HUVECs to LPS following TERT overexpression or silencing to explore the role of TERT in endothelial dysfunction. We also performed flow cytometry and confocal microscopy assays by using HUVECs. And a mtROS inhibitor, MitoTempo, was used to scavenge mitochondria superoxide and alkyl. ResultsTERT transgenic mice were found to have restored pancreatic microcirculation profiles and microvascular endothelial morphology compared with wild-type mice under cerulein injection. In contrast, TERT silencing displayed the opposite effect in response to cerulein. Subsequently, we showed that TERT overexpression attenuates mtROS production and mitochondrial dysfunction during LPS-stimulated endothelial dysfunction. Furthermore, we found that TERT overexpression maintains the balance between mitochondrial contents and ATP level during endothelial dysfunction. In addition, the protective trend of MitoTempo is impeded after TERT silencing. ConclusionTERT restores pancreatic microcirculation dysfunction and attenuates microvascular endothelium lesions by inhibiting the increase of superoxide production and mitochondrial dysfunction.

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.

Hyperinsulinemia alters Notch1 and ZO-1 expression in human brain microvascular endothelial cells

Alzheimer’s disease (AD) is an irreversible disease of aging that progressively impairs cognitive function. Although the pathogenesis of AD remains elusive, dysfunction of the blood-brain barrier (BBB) appears to be an important contributing factor. Of the cells that form the BBB, brain microvascular endothelial cells play an important role in maintaining the integrity of the barrier. Disruption of endothelial tight junctions may contribute to the BBB dysfunction observed in AD. Complete understanding of how this may occur remains unclear, but the intersection between AD and type 2 diabetes mellitus (T2DM) suggests possibilities. Individuals with T2DM are at greater risk of developing AD, and the hyperinsulinemia that characterizes untreated T2DM may contribute to endothelial damage and BBB dysfunction by disrupting tight junctions. The cellular mechanisms by which this dynamic occurs continue to be explored, but one signaling pathway that may be involved is the Notch signaling pathway. The objective of this study was to investigate the effects of hyperinsulinemia on Notch1 and zonula occludens-1 (ZO-1, a key tight junctional protein) expression in human brain microvascular endothelial cells. We hypothesized hyperinsulinemia disrupts tight junctions in brain endothelial cells by decreasing ZO-1 protein expression in a Notch1-dependent manner. To explore this hypothesis, human cerebral microvascular endothelial cells (hCMEC/d3 cell line) were exposed to hyperinsulinemic conditions (100 nM) for 0.5, 1, 2, and 4 hours. Following treatment, total cellular protein was isolated, quantified, and separated by SDS-PAGE. Notch1 and ZO-1 protein expression were analyzed by western blot, and expression levels normalized to GAPDH. Our results indicate hyperinsulinemia increased protein expression of Notch1 by almost 5-fold and decreased ZO-1 expression by 3-fold in a time-dependent manner compared to untreated control cells. These results demonstrate hyperinsulinemia alters both Notch1 and ZO-1 protein expression along a parallel timeline, suggesting ZO-1 expression may be dependent on Notch1 signaling; however, further studies are needed to determine whether Notch1 signaling is required for decreased ZO-1 expression during hyperinsulinemic conditions. Disruption of cerebral microvascular endothelial tight junctional integrity due to hyperinsulinemia in T2DM may negatively impact BBB functionality, lending insight into the development and progression of AD. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The alpha7 integrin subunit in astrocytes promotes endothelial blood-brain barrier integrity.

The blood-brain barrier (BBB) is a vascular endothelial cell boundary that partitions the circulation from the central nervous system to promote normal brain health. We have a limited understanding of how the BBB is formed during development and maintained in adulthood. We used quantitative transcriptional profiling to investigate whether specific adhesion molecules are involved in BBB functions, with an emphasis on understanding how astrocytes interact with endothelial cells. Our results reveal a striking enrichment of multiple genes encoding laminin subunits as well as the laminin receptor gene Itga7, which encodes the alpha7 integrin subunit, in astrocytes. Genetic ablation of Itga7 in mice led to aberrant BBB permeability and progressive neurological pathologies. Itga7-/- mice also showed a reduction in laminin protein expression in parenchymal basement membranes. Blood vessels in the Itga7-/- brain showed separation from surrounding astrocytes and had reduced expression of the tight junction proteins claudin 5 and ZO-1. We propose that the alpha7 integrin subunit in astrocytes via adhesion to laminins promotes endothelial cell junction integrity, all of which is required to properly form and maintain a functional BBB.

Contribution of astrocytic MMP9 toward progression of VCID pathology

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. High levels of plasma homocysteine or hyperhomocysteinemia has been characterized as a risk factor for VCID however, the mechanism underlying the connection between hyperhomocysteinemia and development of VCID pathology remains elusive. I hypothesize that hyperhomocysteinemia initiates a pro‐inflammatory cascade that increases the activity of MMP9 causing both perivascular astrocytes to dissociate from their vessels and initiating the degradation of endothelial cell tight junction proteins, leading to blood brain barrier dysfunction and the progression toward VCID pathology.MethodFor in vivo studies, C57BL6 WT and MMP9KO mice were placed on a control diet or a diet deficient in folate, vitamins B6 and B12 and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12, and 16 weeks. For in vitro, experiments, primary astrocytes from WT and MMP9KO mice were treated with a 50μM homocysteine stimulus or an inflammatory stimulus (TNF‐a, IL‐1b and C1q) for 48 hours and the conditioned media was collected. This homocysteine or inflammatory astrocyte conditioned media was then used to treat WT endothelial cells. Immunohistochemistry and gene expression analysis were used to determine neuroinflammatory changes while histology was used to identify changes in astrocytic end‐feet proteins, tight junction proteins, vascular density and microhaemorrhages. Both gel and in situ zymography were used to assess proteinase activity of MMP9 and related gelatinases. Western blots were used to investigate substrates of MMP9 including claudin, occludin and β‐dystroglycan. Behaviour was assessed using rotarod, novel object recognition and spontaneous alternation testing. Transendothelial electrical resistance and sodium fluorescein assays were used to measure the integrity of endothelial cell tight junctions in vitro.ResultStudies are underway to examine changes in cognition, microhaemorrhages, neuroinflammation, astrocyte end‐foot integrity, tight junction protein integrity and activity of MMP9.ConclusionCollectively, our findings suggest that astrocytic MMP9 may play an integral role in the mechanism associating homocysteine induced neuroinflammation with vascular pathogenesis leading to VCID, highlighting this pathway as an important subject for future study.

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.

Feedback between mechanosensitive signaling and active forces governs endothelial junction integrity

The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the regulation of endothelial junctions as dependent on the feedback between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Simulations reveal that active cell tension can stabilize cadherin bonds, but excessive RhoA signaling can drive bond dissociation and junction failure. While actin polymerization aids gap closure, high levels of Rac1 can induce junction weakening. Combining the modeling framework with experiments, our model predicts the influence of pharmacological treatments on the junction state and identifies that a critical balance between RhoA and Rac1 expression is required to maintain junction stability. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.

TMIC-46. DISRUPTING TRICELLULAR TIGHT JUNCTIONS TO ENHANCE BLOOD-BRAIN-BARRIER PERMEABILITY IN GLIOMA MODELS

Abstract BACKGROUND The blood-brain-barrier (BBB) is predominantly regulated by brain endothelial cells held together by junctional proteins. The BBB poses a significant hinderance to CNS drug entry of effective chemotherapy agents. Previous BBB studies have shown that disruption of tricellular and/or bicellular tight junctional proteins transiently increased CNS drug permeability. OBJECTIVE To evaluate the effect of drug proteins angubindin-1, C-CPE, and C-CPEmt on tight junction proteins angulin-1, claudin-3, and claudin 5, respectively, aimed at transiently enhancing BBB permeability and decreasing glioma growth. METHODS We evaluated junctional disruption with drug proteins, angubindin-1 (600µg/mL), C-CPE (200µg/mL), and C-CPEmt (200µg/mL) on rat brain endothelium. Endothelial junctional integrity studies were assessed by immunoblotting and cell-cell electrical impedance assays. Treatment effects on rat malignant glioma (S635) was measured via migration and rat glioma models. RESULTS Overall, we observed a time-dependent effect of drug proteins on junctional expression and function. Immunoblotting demonstrated a significant decrease (p< 0.05) in angulin-1 expression 5-hours after angubindin-1 treatment, while claudin-3 and claudin-5 expression barely decreased between 2 and 24-hours after C-CPE and C-CPEmt treatment, respectively. Cell-cell integrity was disrupted by 73%, 52%, and 69% compared with control, 3-hours after angubindin-1, C-CPE, and C-CPEmt treatment, respectively. Studies assessing qualitative junctional expression changes after drug proteins are ongoing. Gliomas cells expressed high angulin-1, and interestingly, migration was 50% decreased with angubindin-1 treatment, yet no migration changes were evident with C-CPE or C-CPEmt treatment. Rat glioma model studies evaluating liposomal doxorubicin combined with angubindin-1 are currently being explored to display effects on CNS drug concentrations, cytotoxicity, junctional expression, and survival. DISCUSSION Transient disruption of BBB tricellular junctions was seen with angubindin-1 treatment. Additionally, cell migration was hindered in angulin-1 overexpressing gliomas. These findings are promising and demonstrate the need for more combinational therapies aimed at increasing BBB permeability while also impairing glioma progression.

Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1

Directly targeting caveolar caveolin-1 is a potential mechanism to regulate endothelial permeability, especially during oxidative stress, but little evidence on the topic limits therapeutics discoveries. In this study, we investigated the pharmacological effect of an antioxidant LM49 (5,2′-dibromo-2,4′,5′-trihydroxydiphenylmethanoe) and its five diphenylmethanone derivatives on endothelial permeability and establish two distinct mechanisms of action. Multiplex molecular assays with theoretical modeling indicate that diphenylmethanone molecules, including LM49, directly bind the caveolin-1 steric pocket of ASN53/ARG54, ILE49/ASP50, ILE18, LEU59, ASN60, GLU48 and ARG19 residues. They also indicated dynamic binding-affinity for diphenylmethanone derivatives. First, this molecular interaction at caveolin-1 pocket inhibits its phosphorylation at TYR14 residue in H2O2-injured endothelial cell. A positive correlation was established between diphenylmethanone derivative binding-affinity and caveolin-1 phosphorylation inhibition. Inhibition of caveolin-1 phosphorylation, however, was independent of the LM49-mediated variation of protein tyrosine kinase activity, suggesting a direct blockage of adenosine triphosphate substrate diffusion into cavelion-1 structure. Second, LM49 increases the expression of cellular adhesive and tight junction proteins, VE-cadherin and occludin, in H2O2-injured cell, in a dose dependent manner. A leakage assay of fluorescein isothiocyanate-labeled dextran 40 across cell monolayer suggested improvement in endothelial barrier integrity with diphenylmethanone treatments. Our results demonstrate a direct targeting effect of caveolin-1 on endothelial permeability, and should guide the diphenylmethanone therapy against oxidative stress-induced junction dysfunction, especially at caveolar membrane invagination.

LACTATE IMPAIRS VASCULAR PERMEABILITY BY INHIBITING HSPA12B EXPRESSION VIA GPR81-DEPENDENT SIGNALING IN SEPSIS.

ABSTRACTIntroduction: Sepsis impaired vascular integrity results in multiple organ failure. Circulating lactate level is positively correlated with sepsis-induced mortality. We investigated whether lactate plays a role in causing endothelial barrier dysfunction in sepsis. Methods: Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP). Lactic acid was injected i.p. (pH 6.8, 0.5 g/kg body weight) 6 h after CLP or sham surgery. To elucidate the role of heat shock protein A12B (HSPA12B), wild-type, HSPA12B-transgenic, and endothelial HSPA12B-deficient mice were subjected to CLP or sham surgery. To suppress lactate signaling, 3OBA (120 μM) was injected i.p. 3 h before surgery. Vascular permeability was evaluated with the Evans blue dye penetration assay. Results: We found that administration of lactate elevated CLP-induced vascular permeability. Vascular endothelial cadherin (VE-cadherin), claudin 5, and zonula occluden 1 (ZO-1) play a crucial role in the maintenance of endothelial cell junction and vascular integrity. Lactate administration significantly decreased VE-cadherin, claudin 5, and ZO-1 expression in the heart of septic mice. Our in vitro data showed that lactate (10 mM) treatment disrupted VE-cadherin, claudin 5, and ZO-1 in endothelial cells. Mechanistically, we observed that lactate promoted VE-cadherin endocytosis by reducing the expression of HSPA12B. Overexpression of HSPA12B prevented lactate-induced VE-cadherin disorganization. G protein–coupled receptor 81 (GPR81) is a specific receptor for lactate. Inhibition of GPR81 with its antagonist 3OBA attenuated vascular permeability and reversed HSPA12B expression in septic mice. Conclusions: The present study demonstrated a novel role of lactate in promoting vascular permeability by decreasing VE-cadherin junctions and tight junctions in endothelial cells. The deleterious effects of lactate in vascular hyperpermeability are mediated via HSPA12B- and GPR81-dependent signaling.