Endothelial Cell Permeability Research Articles

Novel Therapies for ANCA-associated Vasculitis: Apilimod Ameliorated Endothelial Cells Injury through TLR4/NF-κB Pathway and NLRP3 Inflammasome.

Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a rapidly progressive form of glomerulonephritis for which effective therapeutic drugs are currently lacking, and its underlying mechanism remains unclear. This study aimed to investigate new treatment options for AAV through a combination of bioinformatics analysis and cell molecular experiments. The research utilized integrated bioinformatics analysis to identify genes with differential expression, conduct enrichment analysis, and pinpoint hub genes associated with AAV. Potential therapeutic compounds for AAV were identified using Connectivity Map and molecular docking techniques. In vitro experiments were then carried out to examine the impact and mechanism of apilimod on endothelial cell injury induced by MPO-ANCA-positive IgG. The findings revealed a set of 374 common genes from differentially expressed genes and key modules of WGCNA, which were notably enriched in immune and inflammatory response processes. A proteinprotein interaction network was established, leading to the identification of 10 hub genes, including TYROBP, PTPRC, ITGAM, KIF20A, CD86, CCL20, GAD1, LILRB2, CD8A, and COL5A2. Analysis from Connectivity Map and molecular docking suggested that apilimod could serve as a potential therapeutic cytokine inhibitor for ANCA-GN based on the hub genes. In vitro experiments demonstrated that apilimod could mitigate tight junction disruption, endothelial cell permeability, LDH release, and endothelial activation induced by MPO-ANCA-positive IgG. Additionally, apilimod treatment led to a significant reduction in the expression of proteins involved in the TLR4/NF-κB and NLRP3 inflammasome-mediated pyroptosis pathways. This study sheds light on the potential pathogenesis of AAV and highlights the protective role of apilimod in mitigating MPO-ANCA-IgG-induced vascular endothelial cell injury by modulating the TLR4/ NF-kB and NLRP3 inflammasome-mediated pyroptosis pathway. These findings suggest that apilimod may hold promise as a treatment for AAV and warrant further investigation.

FGF1 attenuates sepsis-induced coagulation dysfunction and hepatic injury via IL6/STAT3 pathway inhibition

Background & aimsSepsis, a globally prevalent and highly lethal condition, remains a critical medical challenge. This investigation aims to assess the relevance of FGF1 as a potential therapeutic target for sepsis. MethodsSepsis was induced in C57BL/6 mice through LPS administration to establish an in vivo animal model. Various in vitro assays were conducted using human umbilical vein endothelial cells to elucidate the role of FGF1 in the disruption of the coagulation system and liver injury associated with sepsis, as well as to explore its underlying molecular mechanisms. ResultsIn in vivo experiments, FGF1 ameliorated coagulation system disruption in septic mice by reducing the levels of pro-inflammatory and coagulation-related factors in the bloodstream. FGF1 also enhanced liver function in septic mice, mitigating liver inflammation and cell apoptosis, fostering liver vascular regeneration, increasing liver blood perfusion, and improving mouse survival. In vitro experiments demonstrated that FGF1 could inhibit LPS-induced inflammatory responses and apoptosis in endothelial cells, fortify endothelial cell barrier function, decrease endothelial cell permeability, promote endothelial cell proliferation, and restore endothelial cell tube-forming ability. Both in vivo and in vitro experiments substantiated that FGF1 improved sepsis by inhibiting the IL-6/STAT3 signaling pathway. ConclusionIn summary, our study indicates that FGF1 mitigates excessive inflammatory responses in sepsis by suppressing the IL-6/STAT3 signaling pathway, thereby improving systemic blood circulation and ameliorating liver damage in septic organisms. Consequently, this research identifies FGF1 as a potential clinical target for the treatment of human sepsis.

GMPR2-MDA5 interaction bridged the cell junction and anti-GCRV immune

As a consequence of increased vascular endothelial permeability and vascular injury caused by GCRV (grass carp reovirus) infection, grass carp (Ctenopharyngodon idellus) could suffer from hemorrhagic disease and even death. MDA5 is a key RNA sensor that stimulates type I interferon as well as regulates vascular permeability. The guano monophosphate ductase 2 (GMPR2) plays a key role in regulating the permeability of vascular endothelial cells. This study examined the interaction between grass carp MDA5 and GMPR2 and their antiviral effects. GMPR2 interacted directly with MDA5 and promoted its protein level, whilst MDA5 conversely inhibited the GMPR2 protein expression. The expression of GCRV VP7 protein was inhibited by the increased levels of MDA5 and GMPR2. In addition, the cell junction molecule Claudin7b which is closely related to the change in vascular permeability, was inhibited by both MDA5 and GMPR2. Finally, in GMPR2 knockout zebrafish, the mRNA level of MDA5 was significantly down-regulated, which validated the positive functional relationship of GMPR2 on MDA5. Consistent changes of the Claudin 7b and VP7 protein levels were observed in the GMPR2 and MDA5 overexpression groups. In summary, MDA5-GMPR2 interaction affecting GCRV replication might through cell junction molecule Claudin7b. The results from this study provided systematic support for revealing the genetic basis of cell permeability change as well as the hemorrhagic diseases in grass carp.

Platelet releasates mitigate the endotheliopathy of trauma.

Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming. Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to trauma platelet releasates (TPR) and plasma (TP) was assessed via resistance measurement by Electric Cell-substrate Impedance Sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics. TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared to untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared to TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms. TPRs provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma. Prognostic/Epidemiological, Level III.

Interplay of hypoxia and host cells in the translocation, invasion and dissemination of Taenia solium in neurocysticercosis

Neurocysticercosis, caused by the invasion of the central nervous system (CNS) by Taenia solium larvae, poses a significant global health burden. Despite its prevalence and severe neurological consequences, understanding of the molecular and signalling pathways facilitating parasite dissemination and CNS invasion is limited. The lack of comprehensive knowledge of host-parasite interactions and associated proteins involved in T. solium infection hinders the development of targeted interventions to mitigate its ability to cross the epithelia barrier. This is complicated by reduced oxygen availability in the intestine, a phenomenon called hypoxia. Hypoxia can result in epithelial barrier disruption and cell damage, thereby promoting the translocation and dissemination of T. solium. This study aims to establish the role of hypoxia in T. solium invasion and disseminated infections. The effect of hypoxia on the migration, viability and morphological characteristics of T. solium would be determined using transwell invasion assays, flow cytometry and microscopy. T. solium oncosphere development and dissemination under hypoxic and normoxic conditions will be monitored using animal models. Also, host-parasite transcriptome and proteome profiling will be performed to determine pathways triggered under hypoxic conditions. It is expected that hypoxia would promote the invasion and dissemination of T. solium by enhancing epithelial and endothelial cell permeability. , hypoxia will induce the expression of binding and adhesion proteins and other virulence markers such as enolase, serpin, and glutathione transferases that are involved in host invasion. Understanding the role of hypoxia in the translocation mechanism of T. solium can be leveraged to provide insights into host tissues dissemination and the development of appropriate interventions.

Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody.

Subclinical vascular impairment can be exacerbated in individuals who experience sustained inflammation after COVID-19 infection. Our study explores the prevalence and impact of autoantibodies on vascular dysfunction in healthy COVID-19 survivors, an area that remains inadequately investigated. Focusing on autoantibodies against the atypical chemokine receptor 1 (ACKR1), COVID-19 survivors demonstrated significantly elevated anti-ACKR1 autoantibodies, correlating with systemic cytokines, circulating damaged endothelial cells, and endothelial dysfunction. An independent cohort linked these autoantibodies to increased vascular disease outcomes during a median 6.7-yr follow-up. We analyzed a single-cell transcriptome atlas of endothelial cells from diverse mouse tissues, identifying enriched Ackr1 expressions in venous regions of the brain and soleus muscle vasculatures, which holds intriguing implications for tissue-specific venous thromboembolism manifestations reported in COVID-19. Functionally, purified immunoglobulin G (IgG) extracted from patient plasma did not trigger cell apoptosis or increase barrier permeability in human vein endothelial cells. Instead, plasma IgG enhanced antibody-dependent cellular cytotoxicity mediated by patient PBMCs, a phenomenon alleviated by blocking peptide or liposome ACKR1 recombinant protein. The blocking peptide uncovered that purified IgG from COVID-19 survivors possessed potential epitopes in the N-terminal extracellular domain of ACKR1, which effectively averted antibody-dependent cellular cytotoxicity. Our findings offer insights into therapeutic development to mitigate autoantibody reactivity in blood vessels in chronic inflammation.

PERK specific inhibitor AMG-44 protects against Trimethylamine N-oxide (TMAO)-induced Endoplasmic Reticular Stress and Endothelial Dysfunction

The gut microbiota is known to release metabolites detrimental to the endothelial cell lining of the vascular wall leading to endothelial dysfunction. We have earlier reported that Trimethylamine N-oxide (TMAO), an intestinal microbe derived metabolite, caused endothelial dysfunction. In the present study we hypothesized that protein kinase R-like endoplasmic reticulum kinase (PERK)/eIF2α/ATF4 signaling is involved in TMAO-induced endoplasmic reticular (ER) stress and that treatment with PERK inhibitor AMG-44 protects against TMAO-induced ER stress. The endothelial cells were treated with a range of increasing doses of TMAO (0-300 μM) to induce ER stress. PERK inhibitor AMG-44 (20 – 200nM) was used to treat the TMAO treated and control cells. TAMO showed a dose-dependent upregulation of ER stress marker proteins involved in the PERK/eIF2α/ATF4 ER-stress signaling pathway. Western blot analysis showed significant increase in p-PERK/PERK, p-eIF2α/eIF2α, and IRE1α, AFT-4 protein expression which was further confirmed by immunofluorescent analysis in endothelial cells. Treatment with AMG-44 significantly attenuated the PERK/eIF2α/ATF4 axis also protected against increased endothelial cell permeability induced by TMAO (n=6/group; p<0.05). Our results demonstrate that TMAO-induced endothelial dysfunction via the ER stress-mediated PERK- eIF2 α -ATF4 signaling pathway and that treatment with PERK specific inhibitor AMG-44 could be a novel therapeutic strategy in protection against TMAO-induced ER stress and endothelial dysfunction. NIH R01HL148711 to Dr. Sai Sudha Koka. This is the full abstract presented at the American Physiology Summit 2024 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.

Remodeled Extracellular Matrix Guides Endothelial Cell Phenotype and Transcriptome to Fibrotic Niche

Impairment of endothelial barrier integrity causes vascular injury leading to acute lung injury (ALI), which, if not resolved, can compromise a patient’s lung functions and mortality. While it is known that both endothelial cells (EC) and their underlying extracellular matrix (ECM) are required for preserving lung homeostasis and functions, much of the efforts to understand mechanisms regulating these functions have focused mainly on EC. A fundamental unresolved question is whether lung-remodeled ECM formed due to leaky endothelium impacts EC functions, and if so, how? Herein, using lung ECM derived from a mouse lacking focal adhesion kinase (FAK), specifically in the endothelium (remodeled ECM), we assessed ECM components and their role in regulating EC functions and their relevance in vivo. Previously, we have shown that conditional deletion of FAK in endothelium increases EC permeability and induces vascular injury at homeostasis. Mass spectrometric analysis showed 805 proteins in control ECM (FAKfl/fl mice lung derived ECM) consisting of glycoproteins, matrisome-associated proteins, and secreted factors. However, the remodeled ECM had 722 proteins and was highly enriched with fibrillin 1, fibrinogen, and collagen I. Moreover, the remodeled ECM was two-fold stiffer than the control ECM. Remodeled ECM with its inherent composition recapitulates the 3D microenvironment, encouraging EC morphological alteration, increased cell stiffness, and enhanced focal adhesion (FA) formation compared to control ECM. In addition, RNA sequencing identified increased expression of genes linked with fibrosis, including integrin b8, TGFb2, GDF15, SMAD7, COL1A1, IL33, ID3, BMPR1A, and ADAMTS4 in EC seeded on remodeled ECM. Interestingly, fibrotic signaling observed in EC plated on remodeled ECM mimicked in vivo in EC-FAK−/− mice as they showed increased hydroxyproline content and collagen deposition in the lungs, indicating loss of EC-FAK as the crucial initiator of fibrotic signaling. In summary, studies for the first time show disrupted EC signaling as the instructor of fibrotic niche in the lungs for targeting pulmonary fibrosis. NIH. This is the full abstract presented at the American Physiology Summit 2024 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.

CircRNA-PTPN4 mediated regulation of FOXO3 and ZO-1 expression: implications for blood–brain barrier integrity and cognitive function in uremic encephalopathy

Uremic encephalopathy (UE) poses a significant challenge in neurology, leading to the need to investigate the involvement of non-coding RNA (ncRNA) in its development. This study employed ncRNA-seq and RNA-seq approaches to identify fundamental ncRNAs, specifically circRNA and miRNA, in the pathogenesis of UE using a mouse model. In vitro and in vivo experiments were conducted to explore the circRNA-PTPN4/miR-301a-3p/FOXO3 axis and its effects on blood–brain barrier (BBB) function and cognitive abilities. The research revealed that circRNA-PTPN4 binds to and inhibits miR-301a-3p, leading to an increase in FOXO3 expression. This upregulation results in alterations in the transcriptional regulation of ZO-1, affecting the permeability of human brain microvascular endothelial cells (HBMECs). The axis also influences the growth, proliferation, and migration of HBMECs. Mice with UE exhibited cognitive deficits, which were reversed by overexpression of circRNA-PTPN4, whereas silencing FOXO3 exacerbated these deficits. Furthermore, the uremic mice showed neuronal loss, inflammation, and dysfunction in the BBB, with the expression of circRNA-PTPN4 demonstrating therapeutic effects. In conclusion, circRNA-PTPN4 plays a role in promoting FOXO3 expression by sequestering miR-301a-3p, ultimately leading to the upregulation of ZO-1 expression and restoration of BBB function in mice with UE. This process contributes to the restoration of cognitive abilities.Graphical 1. The circRNA-PTPN4/miR-301a-3p/FOXO3 axis is identified as a key regulator of blood–brain barrier integrity and cognitive function in uremic encephalopathy.2. circRNA-PTPN4 sequestration of miR-301a-3p enhances FOXO3 expression, leading to upregulation of ZO-1 and improved endothelial permeability.3. Overexpression of circRNA-PTPN4 in uremic mice restores cognitive abilities and reduces neuronal loss and inflammatory infiltration.

The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Nuciferine reduces vascular leakage and improves cardiac function in acute myocardial infarction by regulating the PI3K/AKT pathway

The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI). Nuciferine has a potentially beneficial effect in the treatment of cardiovascular disease, albeit its role in microvascular structure and function during AMI remains unclear. This study aimed to investigate the protective effect and the related mechanisms of nuciferine in microvascular injury during AMI. Cardiac functions and pathological examination were conducted in vivo to investigate the effect of nuciferine on AMI. The effect of nuciferine on permeability and adherens junctions in endothelial cells was evaluated in vitro, and the phosphorylation level of the PI3K/AKT pathway (in the presence or absence of PI3K inhibitors) was also analyzed. In vivo results indicated that nuciferine inhibited ischemia-induced cardiomyocyte damage and vascular leakage and improved cardiac function. In addition, the in vitro results revealed that nuciferine could effectively inhibit oxygen–glucose deprivation (OGD) stimulated breakdown of the structure and function of human coronary microvascular endothelial cells (HCMECs). Moreover, nuciferine could significantly increase the phosphorylation level of the PI3K/AKT pathway. Finally, the inhibitor wortmannin could reverse the protective effect of nuciferine on HCMECs. Nuciferine inhibited AMI-induced microvascular injury by regulating the PI3K/AKT pathway and protecting the endothelial barrier function in mice.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.

UNVEILING THE PROTECTIVE MECHANISMS OF PUERARIN AGAINST ACUTE LUNG INJURY: A COMPREHENSIVE EXPLORATION OF THE ROLES AND MECHANISMS OF MST1/ERS SIGNALING.

Objectives: Puerarin, the principal active constituent extracted from Pueraria, is believed to confer protection against sepsis-induced lung injury. The study aimed to elucidate the role and mechanism of Mst1/ERS in puerarin-mediated protection against acute lung injury (ALI). Methods: Monolayer vascular endothelial cell permeability was assessed by gauging the paracellular flow of FITC-dextran 40,000 (FD40). ELISA was employed for the quantification of inflammatory cytokines. Identification of target proteins was conducted through western blotting. Histological alterations and apoptosis were scrutinized using hematoxylin-eosin staining and TUNEL staining, respectively. The ultrastructure of the endoplasmic reticulum was observed via transmission electron microscopy. Results: Puerarin significantly protected mice from LPS-induced ALI, reducing lung interstitial width, neutrophil and lymphocyte infiltration, pulmonary interstitial and alveolar edema, and lung apoptosis. Puerarin treatment also markedly attenuated levels of TNF-α and IL-1β in both alveolar lavage fluid and serum. Furthermore, puerarin significantly attenuated LPS-induced increases in Mst1, GRP78, CHOP, and Caspase12 protein expression and blunted LPS-induced decrease in ZO-1 protein expression in lung tissues. Puerarin obviously reduced endoplasmic reticulum expansion and vesiculation. Similarly, puerarin significantly mitigated the LPS-induced reduction in HUVEC cell viability and ZO-1 expression. Puerarin also attenuated LPS-induced increase in apoptosis, TNF-α and IL-1β, FD40 flux, and Mst1, GRP78, CHOP, and Caspase12 expression in HUVEC cells. Nevertheless, the inhibitory impact of puerarin on vascular endothelial cell injury, lung injury, and endoplasmic reticulum stress (ERS) was diminished by Mst1 overexpression. Conclusion: These findings demonstrated that the Mst1/ERS signaling pathway played a pivotal role in the development of LPS-induced vascular endothelial cell dysfunction and ALI. Puerarin exhibited the ability to attenuate LPS-induced vascular endothelial cell dysfunction and ALI by inhibiting the Mst1/ERS signaling pathway.

Geraniol attenuates oxygen-glucose deprivation/reoxygenation-induced ROS-dependent apoptosis and permeability of human brain microvascular endothelial cells by activating the Nrf-2/HO-1 pathway.

Blood-brain barrier breakdown and ROS overproduction are important events during the progression of ischemic stroke aggravating brain damage. Geraniol, a natural monoterpenoid, possesses anti-apoptotic, cytoprotective, anti-oxidant, and anti-inflammatory activities. Our study aimed to investigate the effect and underlying mechanisms of geraniol in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human brain microvascular endothelial cells (HBMECs). Apoptosis, caspase-3 activity, and cytotoxicity of HBMECs were evaluated using TUNEL, caspase-3 activity, and CCK-8 assays, respectively. The permeability of HBMECs was examined using FITC-dextran assay. Reactive oxygen species (ROS) production was measured using the fluorescent probe DCFH-DA. The protein levels of zonula occludens-1 (ZO-1), occludin, claudin-5, β-catenin, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were determined by western blotting. Geraniol showed no cytotoxicity in HBMECs. Geraniol and ROS scavenger N-acetylcysteine (NAC) both attenuated OGD/R-induced apoptosis and increase of caspase-3 activity and the permeability to FITC-dextran in HBMECs. Geraniol relieved OGD/R-induced ROS accumulation and decrease of expression of ZO-1, occludin, claudin-5, and β-catenin in HBMECs. Furthermore, we found that geraniol activated Nrf2/HO-1 pathway to inhibit ROS in HBMECs. In conclusion, geraniol attenuated OGD/R-induced ROS-dependent apoptosis and permeability in HBMECs through activating the Nrf2/HO-1 pathway.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Inter- and Intra-donor Variability in Bone Marrow-Derived Mesenchymal Stromal Cells: Implications for Clinical Applications

Mesenchymal stromal cells (MSCs) are attractive as a therapeutic modality in multiple disease conditions characterized by inflammation and vascular compromise. Logistically they are advantageous since they can be isolated them from adult tissue sources, such as bone marrow (BM). The phase 2a START clinical trial determined BM-MSCs to be safe in patients with moderate to severe acute respiratory distress syndrome (ARDS). Herein, we examine a subset of the clinical doses of MSCs generated for the phase 2a START trial from three unique donors (1-3), where one of the donors’ donated BM on two separate occasions (Donor 3 and 3W). The main objective of this study was to correlate properties of the cells from the four lots with plasma biomarkers from treated patients and relevant to ARDS outcomes. To do this we evaluated MSC donor lots for (1) post-thaw viability, (2) growth kinetics, (3) metabolism, (4) surface marker expression, (5) protein expression, (6) immunomodulatory ability, and (7) their functional effects on regulating endothelial cell permeability. MSC-specific marker expression and protection of thrombin-challenged endothelial barrier permeability was similar among all four donor lots. Inter and intra-donor variability was observed in all the other in vitro assays. Furthermore, patient plasma ANG-2 and Protein C levels at 6 hours post-transfusion were correlated to cell viability in an inter- and intra-donor dependent manner. These findings highlight the potential of donor dependent (inter-) and collection dependent (intra-) effects in patient biomarker expression.

Linagliptin's impact on lymphatic barrier and lymphangiogenesis in oral cancer with high glucose.

Uncertainties remain regarding the effect of elevated glucose levels on lymphatic metastasis of cancer cells. Our study elucidated the mechanisms linking high glucose to lymphangiogenesis and lymphatic barrier-related factors and investigated the protective role of linagliptin against lymphatic barrier dysfunction. A CAL-27-LEC co-culture system was established. Sodium fluorescein permeability assay observed lymphatic endothelial cell permeability. Western blotting and RT-qPCR detected protein and mRNA expression under different conditions, respectively. CCK-8, scratch wound healing, and transwell assays revealed cell migration and proliferation. Tube formation experiment tested capacity for endothelial tube formation. Immunohistochemical staining analyzed tissue sections from 43 oral cancer individuals with/without diabetes. In high-glucose co-culture system, we observed increased lymphatic barrier permeability and decreased expression of ZO-1 and occludin, two tight-junction proteins; conversely, the expression of PAR2, a high permeability-related protein, was increased. Following linagliptin treatment, the expression levels of VEGF-C, VEGFR-3, and PAR2 decreased, while those of ZO-1 and occludin increased. Considerably higher levels of LYVE-1 expression in individuals with diabetes than in those without diabetes. By ameliorating the high glucose-induced disruption of the lymphatic endothelial barrier, linagliptin may reduce lymphangiogenesis and exhibit an inhibitory effect on lymphatic metastasis in oral cancer patients with diabetes.

Sevoflurane alleviates inflammation, apoptosis and permeability damage of human umbilical vein endothelial cells induced by lipopolysaccharide by inhibiting endoplasmic reticulum stress via upregulating RORα

Endothelial dysfunction often accompanies sepsis. Sevoflurane (Sev) is a widely used inhaled anesthetic that has a protective effect on sepsis-associated damage. We aimed to elucidate the role of Sev in endothelial dysfunction by using a model of LPS induced HUVECs. Sev increased the viability and decreased the apoptosis of HUVECs exposed to LPS. Inflammation and endothelial cell adhesion were improved after Sev addition. Besides, Sev alleviated LPS-induced endothelial cell permeability damage in HUVECs. RORα served as a potential protein that bound to Sev. Importantly, Sev upregulated RORα expression and inhibited endoplasmic reticulum (ER) stress in LPS-treated HUVECs. RORα silencing reversed the impacts of Sev on ER stress. Moreover, RORα deficiency or tunicamycin (ER stress inducer) treatment restored the effects of Sev on the viability, apoptosis, inflammation and endothelial permeability damage of HUVECs exposed to LPS. Taken together, Sev ameliorated LPS-induced endothelial cell damage by targeting RORα to inhibit ER stress.

PEDF inhibits VEGF-induced vascular leakage through binding to VEGFR2 in acute myocardial infarction

Pigment epithelium-derived factor (PEDF) could bind to vascular endothelial growth factor receptor 2 (VEGFR2) and inhibit its activation induced by VEGF. But how PEDF affects VEGFR2 pathway is still poorly understood. In this study, we elucidated the precise mechanism underlying the interaction between PEDF and VEGFR2, and subsequently corroborated our findings using a rat AMI model. PEDF prevented endocytosis of VE-cadherin induced by hypoxia, thereby protecting the endothelium integrity. A three-dimensional model of the VEGFR2-PEDF complex was constructed by protein-protein docking method. The results showed that the VEGFR2-PEDF complex was stable during the simulation. Hydrogen bonds, binding energy and binding modes were analyzed during molecular dynamics simulations, which indicated that hydrogen bonds and hydrophobic interactions were important for the recognition of VEGFR2 with PEDF. In addition, the results from exudation of fibrinogen suggested that PEDF inhibits vascular leakage in acute myocardial infarction and confirmed the critical role of key amino acids in the regulation of endothelial cell permeability. This observation is also supported by echocardiography studies showing that the 34mer peptide sustained cardiac function during acute myocardial infarction. Besides, PEDF and 34mer could inhibit the aggregation of myofiber in the heart and promoted the formation of a dense cell layer in cardiomyocytes, which suggested that PEDF and 34mer peptide protect against AMI-induced cardiac dysfunction. These results suggest that PEDF inhibits the phosphorylation of downstream proteins, thereby preventing vascular leakage, which provides a new therapeutic direction for the treatment of acute myocardial infarction. Communicated by Ramaswamy H. Sarma