Endothelial Barrier Dysfunction Research Articles

Unfolded protein response in endothelial injury

ABSTRACT Endothelial barrier dysfunction is associated with sepsis and lung injury, both direct and indirect. We discuss the involvement of unfolded protein response in the protective effects of heat shock protein 90 inhibitors and growth hormone releasing hormone antagonists in the vascular barrier, to reveal new possibilities in acute respiratory distress syndrome treatment.

Association between vascular endothelial growth factor-mediated blood-brain barrier dysfunction and stress-induced depression.

Several lines of evidence suggest that stress induces the neurovascular dysfunction associated with increased blood-brain barrier (BBB) permeability, which could be an important pathology linking stress and psychiatric disorders, including major depressive disorder (MDD). However, the detailed mechanism resulting in BBB dysfunction associated in the pathophysiology of MDD still remains unclear. Herein, we demonstrate the role of vascular endothelial growth factor (VEGF), a key mediator of vascular angiogenesis and BBB permeability, in stress-induced BBB dysfunction and depressive-like behavior development. We implemented an animal model of depression, chronic restraint stress (RS) in BALB/c mice, and found that the BBB permeability was significantly increased in chronically stressed mice. Immunohistochemical and electron microscopic observations revealed that increased BBB permeability was associated with both paracellular and transcellular barrier alterations in the brain endothelial cells. Pharmacological inhibition of VEGF receptor 2 (VEGFR2) using a specific monoclonal antibody (DC101) prevented chronic RS-induced BBB permeability and anhedonic behavior. Considered together, these results indicate that VEGF/VEGFR2 plays a crucial role in the pathogenesis of depression by increasing the BBB permeability, and suggest that VEGFR2 inhibition could be a potential therapeutic strategy for the MDD subtype associated with BBB dysfunction.

METTL3-Mediated N6-Methyladenosine Modification of Trim59 mRNA Protects Against Sepsis-Induced Acute Respiratory Distress Syndrome.

N6-methyladenosine (m6A) RNA modification is a fundamental determinant of mRNA metabolism in eukaryotic cells and is involved in numerous physiological and pathological processes. However, the specific role of m6A modification in sepsis-induced acute respiratory distress syndrome(ARDS) remains unknown. Here, we show that the levels of m6A RNA were significantly decreased in septic lungs and that METTL3 was the main regulator involved in the absence of m6A RNA modification. Pulmonary endothelial barrier damage is a critical process in the pathogenesis of acute lung injury during sepsis. METTL3 regulated endothelial barrier dysfunction and inflammatory responses in sepsis-induced ARDS in vivo and in vitro. Furthermore, we identified tripartite motif-containing (Trim)59 as a key m6A effector and Trim59 deficiency exacerbated lung injury. Mechanistically, METTL3 inhibited endothelial injury in sepsis-induced ARDS through Trim59-associated NF-κB inactivation. Our findings revealed novel insights into epitranscriptional mechanisms in sepsis-induced ARDS via m6A modifications, which has important application value in the diagnosis, prognosis, and molecular-targeted therapy of sepsis-associated lung injury.

Flavivirus NS1 Triggers Tissue-Specific Disassembly of Intercellular Junctions Leading to Barrier Dysfunction and Vascular Leak in a GSK-3β-Dependent Manner.

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins β-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of β-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of β-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3β (GSK-3β). Supporting this model, we found that a chemical inhibitor of GSK-3β reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3β as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.

Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.

DDAH1 Promotes Lung Endothelial Barrier Repair by Decreasing Leukocyte Transendothelial Migration and Oxidative Stress in Explosion-Induced Lung Injury

Explosion-induced injury is the most commonly encountered wound in modern warfare and incidents. The vascular inflammatory response and subsequent oxidative stress are considered the key causes of morbidity and mortality among those in blast lung injury. It has been reported dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays important roles in regulating vascular endothelial injury repair and angiogenesis, but its role in explosion-induced injury remains to be explained. To explore the mechanism of vascular injury in blast lung, 40 C57BL/6 wild type mice and 40 DDAH1 knockout mice were randomly equally divided into control group and blast group, respectively. Body weight, lung weight, and dry weight of the lungs were recorded. Diffuse vascular leakage was detected by Evans blue test. The serum inflammatory factors, nitric oxide (NO) contents, and ADMA level were determined through ELISA. Hematoxylin-eosin staining and ROS detection were performed for histopathological changes. Western blot was used to detect the proteins related to oxidative stress, cell adhesion molecules and leukocyte transendothelial migration, vascular injury, endothelial barrier dysfunction, and the DDAH1/ADMA/eNOS signaling pathway. We found that DDAH1 deficiency aggravated explosion-induced body weight reduction, lung weight promotion, diffuse vascular leakage histopathological changes, and the increased levels of inflammatory-related factors. Additionally, DDAH1 deficiency also increased ROS generation, MDA, and IRE-1α expression. Regarding vascular endothelial barrier dysfunction, DDAH1 deficiency increased the expression of ICAM-1, Itgal, Rac2, VEGF, MMP9, vimentin, and N-cadherin, while lowering the expression of occludin, CD31, and dystrophin. DDAH1 deficiency also exacerbated explosion-induced increase of ADMA and decrease of eNOS activity and NO contents. Our results indicated that explosion could induce severe lung injury and pulmonary vascular insufficiency, whereas DDAH1 could promote lung endothelial barrier repair and reduce inflammation and oxidative stress by inhibiting ADMA signaling which in turn increased eNOS activity.

Sufentanil ameliorates oxygen-glucose deprivation/reoxygenation-induced endothelial barrier dysfunction in HCMECs via the PI3K/Akt signaling pathway.

Ischemic heart disease, a chronic myocardial damage disease caused by coronary artery ischemia, is the leading cause of death worldwide. The aim of the present study was to explore the efficacy of sufentanil in myocardial ischemia/reperfusion (I/R) injury. Oxygen and glucose deprivation/reoxygenation (OGD/R) was utilized to induce human cardiac microvascular endothelial cells (HCMECs) to simulate myocardial I/R injury in vitro. The Cell Counting Kit-8 assay was used to detect the effects of sufentanil on HCMECs and OGD/R-induced HCMECs. The TUNEL, lactate dehydrogenase (LDH) activity, immunofluorescence and in vitro permeability assays, were used to assess apoptosis, LDH activity, VE-cadherin protein expression levels and endothelial barrier function in OGD/R-induced HCMECs, respectively. Moreover, western blotting was performed to assess the protein expression levels of apoptosis, endothelial barrier function and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)-related proteins. The results demonstrated that sufentanil had no significant influence on the viability of HCMECs but increased the viability of OGD/R-induced HCMECs in a dose-dependent manner. Furthermore, sufentanil inhibited cell apoptosis and permeability of OGD/R-induced HCMECs but enhanced the protein expression levels of tight junction proteins, including ZO-1, Occludin, VE-cadherin and Claudin-5. Sufentanil was also demonstrated to activate the PI3K/Akt signaling pathway. In addition, the use of LY294002, an inhibitor of the PI3K/Akt signaling pathway, partially abolished the protective effects of sufentanil on apoptosis, permeability and tight junction protein expression levels. These results indicated that sufentanil ameliorated OGD/R-induced endothelial barrier dysfunction in HCMECs, potentially via the PI3K/Akt signaling pathway. The present study therefore suggested that sufentanil may serve as a novel therapeutic option for the improvement of myocardial I/R injury.

Endothelial Cell Protein Targeting by Myeloperoxidase-Derived 2-Chlorofatty Aldehyde.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

S1PR2/RhoA/ROCK1 pathway promotes inflammatory bowel disease by inducing intestinal vascular endothelial barrier damage and M1 macrophage polarization

Vascular and immune dysfunctions are thought to be related to the pathogenesis of inflammatory bowel disease (IBD), but behind this, the exact mechanism of mucosal vascular endothelial barrier dysfunction and macrophage phenotypic transition is not fully understood. Here, we explored the mechanistic role of sphingosine 1-phosphate receptor 2 (S1PR2) and its downstream G protein RhoA/Rho kinase 1 (ROCK1) signaling pathway in the intestinal endothelial barrier damage and M1 macrophage polarization in IBD. We found that the expression of S1PR2 in intestinal mucosal vascular endothelial cells and macrophages of IBD patients and DSS-induced colitis mice as well as vascular endothelial cells and macrophages treated with LPS in vitro was significantly increased. Knocking down or pharmacologically inhibiting S1PR2 significantly downregulated the expression of RhoA and ROCK1 in vascular endothelial cells and macrophages. Furthermore, inhibition of S1PR2 and ROCK1 reversed the impaired vascular barrier function and M1 macrophage polarization in vivo and in vitro, while reducing ER stress in vascular endothelial cells and glycolysis in macrophages. In addition, inhibition of ER stress or glycolysis reversed LPS-induced impairment of vascular endothelial cell barrier function and M1 macrophage polarization. Collectively, our results indicate that the S1PR2/RhoA/ROCK1 signaling pathway may participate in the pathogenesis of IBD by regulating vascular endothelial barrier function and M1 macrophage polarization.

Sulfated β-glucan from Agaricus subrufescens inhibits flavivirus infection and nonstructural protein 1-mediated pathogenesis

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1–6,1-3)-β-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 μg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 μg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 μg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

A Protein Tyrosine Phosphatase 4A3 (PTP4A3) inhibitor prevents and repairs pulmonary endothelial barrier dysfunction and ameliorates acute lung injury provoked by the SARS‐CoV‐2 Spike protein subunit 1 in k18‐hACE2 transgenic mice

PTP4A3 regulates various inflammatory and cytoskeletal signaling pathways (e.g., STAT3, RhoA, ERK, AKT) that are involved in endothelial barrier dysfunction and acute lung injury (ALI). We have investigated the actions of JMS‐053, a small PTP4A3 inhibitor, in both in vitro and in vivo models of SARS‐CoV‐2 Spike protein subunit 1 (S1SP)‐induced endothelial inflammation and acute lung injury. S1SP (10nM) caused a time‐dependent endothelial barrier dysfunction in human lung microvascular endothelial cell monolayers that was ameliorated by JMS‐053 (12.5µM) administered either as pre‐treatment (4 hours prior to S1SP) or post‐treatment (5 hours after S1SP). In K18‐hACE2 transgenic mice, which express the human ACE2 receptor for SARS‐CoV‐2 S1SP, we instilled S1SP intratracheally, post‐treated with JMS‐053 (10mg/kg i.p. at 1, 24 and 48 hours after S1SP) or vehicle, and investigated molecular, functional and histological outcomes 72 hours later. JMS‐053 mice displayed lower alveolar cellularity, proteinosis and “cytokine storm” (i.e., IL‐6, IL‐1β, IL‐17), and lower levels of phosphorylated (activated) STAT3 and NF‐kB, compared to mice instilled with S1SP and treated with vehicle. Moreover, JMS‐053 greatly reduced quantifiable histological evidence of injury, compared to vehicle‐treated animals. This data suggests that inhibition of PTP4A3 could represent a critical target for novel therapeutic approaches towards endothelial dysfunction, inflammation and acute lung injury.

Adiponectin Ameliorates Hyperglycemia-Induced Retinal Endothelial Dysfunction, Highlighting Pathways, Regulators, and Networks.

BackgroundThe pathophysiology of diabetic retinopathy (DR) is multifaced. A low level of circulating adiponectin (APN) in type 2 diabetes is associated with microvasculature complications, and its role in the evolution of DR is complex.AimThis study is designed to explore the potential impact of APN in the pathogenesis of DR, linking the changes in cellular and biological processes with the pathways, networks, and regulators involved in its actions.MethodsHuman microvascular retinal endothelial cells (HMRECs) were exposed to 30mM glucose (HG) and treated with globular adiponectin (30μg/mL) for 24 hours. The cells were evaluated for reactive oxidative stress (ROS) and apoptosis. RT-PCR profile arrays were utilized to evaluate the profile of genes involved in endothelial functions, angiogenesis, extracellular matrix, and adhesion molecules for hyperglycemic HMRECs treated with adiponectin. In addition, the barrier function, leukocyte migration, and angiogenesis were evaluated. The differential expressed genes (DEGs) were outlined, and bioinformatic analysis was applied.ResultsAdiponectin suppresses ROS production and apoptosis in HMRECs under HG conditions. Adiponectin improved migration and barrier functions in hyperglycemic cells. The bioinformatic analysis highlighted that the signaling pathways of integrin, HMGB1, and p38 AMPK, are mainly involved in the actions of APN on HMRECs. APN significantly affects molecular functions, including the adhesion of cells, chemotaxis, migration of WBCs, and angiogenesis. STAT3, NFKB, IKBKB, and mir-8 are the top upstream regulators, which affect the expressions of the genes of the data set, while TNF and TGFB1 are the top regulators.ConclusionAdiponectin significantly counteracts hyperglycemia at various cellular and molecular levels, reducing its impact on the pathophysiological progression towards DR in vitro using HMRECs. Adiponectin ameliorates inflammatory response, oxidative stress, and endothelial barrier dysfunction using a causal network of NFBk complex, TNF, and HMGB1 and integrin pathways.

Amelioration of acute lung injury by peptides targeting scavenger receptor CD36

CD36 belongs to scavenger receptor class B and is a fatty acid translocase. Our previous studies have suggested that the receptor may be involved in regulation of responses to endotoxin and inflammation of the lungs in mice. In current studies, we have designed a group of helical peptides that interact with and modulate the activities of CD36 and tested their activities against endothelial barrier dysfunction and acute lung injuries in mice. Peptides that inhibit CD36 activities including L37pA, ELR‐B and ELR‐BP reversed or mitigated the barrier disruptive activities induced by LPS, HKSA, truncated phospholipids and histone H3 as measured by TER (trans‐endothelial electric resistance). In consistence with this, the peptides also enhanced the barrier protective activities of OxPAPC. The peptides were delivered intravenously to mice treated with LPS or HKSA to induce acute lung injuries. It has been observed that among peptides that antagonize the activities of CD36, L37pA, ELR‐B and ELR‐BP have demonstrated inhibitory effects on acute lung injuries induced by LPS characterized by reduced extravagance of inflammatory cells from circulation to bronchoalveolar lavage (BAL) and a decrease in protein concentration in BAL fluid from the peptide‐treated mice. The observation indicates that modulation of CD36 activities by peptide inhibitors is a novel strategy to reduce acute lung injuries induced by pro‐inflammatory agents.

Endothelial PP2AC prevents endotoxemia‐induced acute lung injury by suppressing STAT3 activity

Acute lung injury (ALI) is a critical hypoxemic respiratory failure characterized by alveolar‐capillary damage, unchecked inflammation, vascular leak, and pulmonary edema secondary to endothelial barrier dysfunction. Identifying the candidate molecules targeting mechanisms for excessive production of proinflammatory cytokines and endothelial dysfunction are warranted for therapeutic intervention. Recent studies show that the signal transducers and activators of transcription 3 (STAT3) phosphorylation in critical for inflammatory cytokines production and systemic inflammation. Protein phosphatase 2A (PP2A) is a hetero‐trimeric, ubiquitously expressed Ser/Thr phosphatase. The phosphatase activity of PP2A is regulated by its catalytic C subunit (PP2AC). PP2A plays an essential role in modulating tissue inflammation by maintaining the phosphorylation/de‐phosphorylation balance of multiple proteins. So far, however, no studies have deciphered PP2AC isoform‐a function in regulating pulmonary vascular endothelial permeability and inflammatory signaling. We hypothesize that PP2AC expression in vascular endothelial cell (EC) protects the lung against sepsis‐induced lung injury. Wild‐type (WT) and endothelial specific PP2AC‐deficient mice (PP2ACCKO) were challenged with lipopolysaccharide. We found that lipopolysaccharide‐induced inflammatory response, characterized by up‐regulated level of IL‐1b, IL‐6, MPO activity and pulmonary vascular hyper‐permeability in lung tissue of PP2ACCKO mice was significantly elevated as compared to WT littermate control. Mechanistically, we showed that PP2AC deletion in mouse lung‐EC and in human lung‐EC markedly augmented Ser727‐phosphorylation‐mediated STAT3 activation and MMP9 level in response to lipopolysaccharide. Intriguingly, administration of a STAT3 inhibitor suppressed lipopolysaccharide‐induced production of MMP9 and proinflammatory cytokines IL‐1b, IL‐6, MPO activity and pulmonary vascular hyper‐permeability and thus rescued the augmented inflammatory phenotype in PP2ACCKO mice lung. In conclusion, these data showed the protective role of endothelial‐PP2AC in limiting endothelial barrier dysfunction and inflammation, and thereby it attenuates sepsis‐induced lung injury by preventing hyper‐activated STAT3 signaling.

In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID‐19

Study objectiveEndothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells.MethodsCitrate plasma was sampled as part of the Pa‐COVID‐19 cohort study (ethics approval EA2/066/20) from patients with severe COVID‐19 (high flow O2 or mechanically ventilated; WHO severity score: 5‐7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell‐substrate impedance sensing (ECIS), immunofluorescence for endothelial VE‐cadherin and F‐actin, western blot analyses of PAR‐1 cleavage, and real‐time Ca2+ imaging. Plasma from healthy donors served as control.ResultsCOVID‐19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti‐coagulant with thromboprotective function were decreased. COVID‐19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca2+ influx in response to COVID‐19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC‐067047).ConclusionHere, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID‑19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID‐19.

Dysfunctional Mitochondria and Hemolysis are Linked by Impaired Maturation of Reticulocytes: Possible Role in Pulmonary Hypertension

IntroductionWe previously demonstrated that significantly elevated levels of circulating free heme (CFH) is associated with preclinical (Sugen/Hypoxia in rats) pulmonary hypertension (PH) and clinical pulmonary arterial hypertension (PAH). However, the pathogenic mechanisms of free heme release in PAH patients and animal models in the absence of hemolytic disease are yet to be elucidated.HypothesisThe mitochondria play a crucial role in reticulocyte maturation (precursors to red blood cells). Our hypothesis tests if mitochondrial dysfunction (MD) impaired reticulocyte maturation increases its fragility and causes rupture, elevating CFH levels that promote PAH.MethodsRecently reported genetic PH rat models carrying the NFU1G206C mutation spontaneously develop PH with severe MD. Erythropoiesis activation, reticulocyte counts, and CFH levels were monitored in this model. ChemiDoc imaging system was used to visualize FITC‐dextran extravasation in fresh transversely cut pulmonary slices 24 hours after FITC‐dextran injection (60 mg/kg. i.v.). Changes in right ventricle (RV) hemodynamics were used to determine the level of disease severity. Pulmonary artery smooth muscle cells (PASMCs) isolated from wild‐type (WT) and NFU1G206C rats were used to evaluate effects of free heme in vitro. Matrigel assay was used to evaluate the tube formation potential of endothelial cells (EC’s).ResultsThe NFU1G206C rats showed significantly higher erythropoietin levels and immature reticulocyte counts compared to WTs. Correspondingly, significantly elevated plasma free heme and extravasation of FITC‐dextran into lung parenchyma were observed, indicating pulmonary endothelial barrier dysfunction, previously demonstrated as an essential contributor to PH pathobiology. Notably, treatment of human pulmonary artery EC’s with 50µM heme completely abolished EC tube formation, indicating that CFH could trigger a cellular environment leading to PH. Additionally, exposing isolated PASMC from NFU1G206C to 50µM heme increased the proliferation rate by ~1.5 and ~2.9 folds compared to untreated NFU1G206C or WT PASMC. Chronic lipoic acid (LA) supplementation to restore mitochondrial function (1.7mM orally), normalized total/immature reticulocyte counts in NFU1G206C rats (WT/NFU1G206C/NFU1G206C+LA for total and immature reticulocyte, p<0.002 and p<0.001) and fully reversed the PAH phenotype. Alternatively, the RV systolic pressure of NFU1G206C rats was significantly (p<0.0001) attenuated by blocking the heme transporter (HCP1) with sulfasalazine (SL, 20mg/ml/kg, i.p.).ConclusionWe show that mitochondrial dysfunction impaired reticulocyte maturation promotes hemolysis resulting in increased CFH. Elevated CFH can trigger pathogenic pathways leading to PH/PAH via EC barrier dysfunction and increased PASMC proliferation. Treatment approaches that improve mitochondrial metabolism (LA) or inhibit free heme entrance to the cell (SL) abrogate the PH phenotype, highlighting the critical role of free heme‐induced signaling in disease pathobiology.

Beneficial Effects of LOX‐1 Inhibition on Brain Endothelial Proinflammatory Mediators and Barrier Proteins Following OxLDL Plus Acute Ischemic Injury

Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular injury is elevated levels of oxidized low‐density lipoprotein (OxLDL). Via the lectin‐like OxLDL receptor 1 (LOX‐1), OxLDL contributes to mechanisms associated with vascular dysfunction and inflammation. The impact of OxLDL/LOX‐1 on cerebrovascular endothelial dysfunction and inflammation in the setting of AIS remains to be elucidated. The aim of this study is to investigate the effect of OxLDL via LOX‐1 on endothelial proinflammatory mediator and integral barrier protein levels during in vitro ischemic‐like conditions. We hypothesized that acute ischemic injury would increase endothelial barrier dysfunction as well as inflammation and that OxLDL would exacerbate these responses in a LOX‐1 dependent manner. Primary male human brain microvascular endothelial cells (HBMEC) were preconditioned with human OxLDL (50μg/dL; 12h) or vehicle using a serum dose reported in AIS patients. Next cells were treated with BI‐0115 (selective LOX‐1 inhibitor; 10μM) or vehicle (<0.1% DMSO) for 0.5h prior to being exposed to normoxia (21% O2) or hypoxia plus glucose deprivation (HGD; 1% O2) for 6h in the continued presence of OxLDL or vehicle. Levels of HBMEC barrier protein (ZO‐1), adhesion molecule (VCAM‐1), and inflammatory proteins (iNOS, IL‐1β) were examined using FIJI mediated immunocytochemical analysis. In terms of barrier protein levels, we observed that ZO‐1 levels were decreased following HGD. Contrary to our hypothesis, OxLDL plus HGD resulted in increased levels of ZO‐1; and moreover, we observed a greater increase in ZO‐1 levels when treated with BI‐0115 alone or BI‐0115 plus OxLDL. During normoxic conditions OxLDL increased levels of ZO‐1 and this response was LOX‐1 independent. Concomitantly, HGD increased levels of the adhesion molecule, VCAM‐1. The presence of HGD plus OxLDL or OxLDL plus BI‐0115 had no effect on VCAM‐1 expression. During normoxic conditions, OxLDL increased VCAM‐1 levels in a LOX‐1 dependent manner. We next observed that HGD increased the levels of proinflammatory mediators, iNOS and IL‐1β. OxLDL under normoxic conditions significantly increased the levels of iNOS in a LOX‐1 dependent manner but had no effect on the levels of IL‐1β. HGD plus OxLDL had no effect on iNOS levels; however, treatment with BI‐0115 alone or BI‐0115 plus OxLDL significantly attenuated HGD‐mediated increases in iNOS levels. Moreover during HGD, OxLDL increased levels of IL‐1β in a LOX‐1 dependent manner suggesting that OxLDL acting through LOX‐1 leads to an increase in a central mediator of vascular inflammation and permeability. In conclusion, the beneficial effect of LOX‐1 inhibition on cerebrovascular endothelial permeability and inflammation suggests that this receptor may be a novel, viable therapeutic target in the treatment of AIS.

Reduction of Reactive Oxygen Species by a Growth Hormone‐Releasing Hormone Antagonist in Endothelial Cells

INTRODUCTIONGrowth hormone‐releasing hormone (GHRH) is a hypothalamic hormone regulating the secretion of Growth Hormone (GH) from the anterior pituitary gland. The expression of GHRH and its receptors (full length pituitary‐type and splice variants) in tissues such as breast, prostate and lung, suggested that the activities of that hypothalamic hormone is not limited to the GH ‐ insulin‐like growth factor‐I axis. Reactive Oxygen Species (ROS) are involved in inflammation, disrupt inter‐endothelial junctions, promote cytoskeletal remodeling and induce pro‐inflammatory cytokines (e.g. interleukin [IL]‐1β, IL‐6). Those events have been associated to endothelial hyper‐permeability, the hallmark of Acute Respiratory Distress Syndrome (ARDS). The purpose of the present study is to investigate the effects of GHRH and GHRH antagonists (GHRHAnt) in ROS generation of three different endothelial cell lines.METHODSBovine pulmonary artery endothelial cells (BPAEC), human lung microvascular endothelial cells (HMVEC‐L), human cerebral microvascular endothelial cells (hCMEC/D3) and mouse embryonic fibroblasts (NIH/3T3) were maintained at 37°C in a humidified atmosphere of 5% CO2‐ 95% air per suppliers’ instructions. Cells seeded in 96‐well plates (1×104 cells/well, n=4 wells per group) were treated with GHRH (1µM), or GHRHAnt (1µM), or GHRHAnt (1µM) prior to H2O2 (0.1mM) exposure. To measure ROS levels, the cells were incubated with 25µM of DCFDA for 45 min, and fluorescence intensity was measured by a fluorescence microplate reader using excitation and emission wavelength of 485 nm and 535 nm respectively. Protein expression was measured utilizing Western Blot analysis. The densitometry of immunoblots was accomplished by using Image J software (NIH), and Student's t test was used to determine the statistically significant differences among the groups. A value of p < 0.05 was considered significant.RESULTSThe expression of GHRH receptors in BPAEC, hCMEC/D3 and HMVEC‐L was confirmed by Western Blot. NIH/3T3 cells were used as negative controls, since they do not express receptors responding to GHRH. All cells were exposed to vehicle (0.1% DMSO), GHRH (1µM), or GHRHAnt (1µM) for 8 hours. Our results demonstrate that GHRH significantly increases ROS generation in all three endothelial cell lines, and GHRHAnt reduces ROS production. We also measured the effects of GHRHAnt against H2O2‐induced oxidative stress. Cells were treated with vehicle (0.1% DMSO), or GHRHAnt (1µM) for 8 hours, prior to vehicle (PBS) or H2O2 (0.1mM) exposure (8 hours). H2O2 potentiated ROS generation, and that effect was counteracted by GHRHAnt. Those peptides failed to suppress the H2O2‐induced ROS production in NIH/3T3 cells, since they do not express GHRH receptors.CONCLUSIONSOur results reveal that GHRHAnt suppress ROS production in the endothelium, suggesting that those compounds may be of therapeutic value in diseases related to endothelial barrier dysfunction and oxidative stress (e.g. Acute Respiratory Distress Syndrome and sepsis).

Serum from rats subjected to traumatic brain injury and hemorrhagic shock induces profound endothelial barrier dysfunction

IntroductionTraumatic brain injury (TBI) has been associated with development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanism linking TBI with development of ARDS, we characterized effects of serum factors released during TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS.MethodsThe adult male rat polytrauma model consisted of controlled cortical impact (CCI)–induced TBI followed by 30 minutes of hemorrhage shock (HS) induced by blood withdrawal. The HS phase was followed by a 1‐hour “prehospital” Hextend fluid resuscitation phase and then a 1‐hour “hospital phase” when shed blood was reinfused. 48 hours after the TBI, blood was drawn from right ventricle and serum samples were obtained. Serum was collected from the TBI or sham rats, and its effects on barrier properties of pulmonary endothelial cell culture were assessed by measurements of transendothelial electrical resistance (TER) using the electrical cell impedance sensor and visualization of fluorescent tracer penetration through EC monolayer using XPerT assay.ResultsWe found pronounced barrier‐disruptive effects of serum samples from animals exposed to controlled cortical impact (CCI) and HS, but not from sham operated rats. Rapid and sustained decrease in EC monolayer transendothelial resistance was observed within minutes after serum addition and remained below basal TER levels after incubation with CCI‐HS sera. Thrombin has been implicated in the early‐phase barrier disruptive activity of CCI‐HS serum, as both thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of serum‐induced TER decline, but did not affect TER decline at later time points. These findings suggest that thrombin elevation may play a role in early‐phase barrier disruptive activity of CCI‐HS serum. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The late phase barrier disruptive effect of CCI‐HS was also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated with serum from CCI‐HS rats for 3 hours have demonstrated significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody.ConclusionThese results suggest that exposure to CCI‐HS leads to production of humoral factors causing early and late‐phase barrier disruptive effects in lung vascular endothelium. While thrombin‐PAR1 signaling has been identified as mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remain to be determined.

Cell‐free Hemoglobin‐Oxidized LDL Axis Contributes to Microvascular Endothelial Barrier Dysfunction and Poor Outcomes During Sepsis

IntroductionSepsis, a dysregulated host response to infection with high morbidity and mortality, is characterized by a systemic inflammatory response and widespread vascular hyperpermeability leading to edema, organ dysfunction, and death with no specific treatments. Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis‐induced organ dysfunction driven by circulating inflammatory mediators, oxidants, and proteolytic enzymes. Cell‐free hemoglobin (CFH) is one such factor, released from damaged red blood cells during sepsis due to their increased deformability and fragility. Though CFH is not detectable in healthy subjects, plasma CFH is elevated in patients with sepsis and independently associated with mortality. CFH contributes to endothelial dysfunction, but the signaling mechanisms are incompletely understood. CFH can oxidize circulating low‐density lipoproteins (oxLDL), which could potentiate endothelial barrier disruption. We hypothesized that elevated levels of CFH during sepsis are associated with increased oxLDL and contribute to microvascular endothelial barrier dysfunction through the oxLDL receptor, lectin‐like oxidized LDL receptor 1 (LOX‐1).MethodsCirculating levels of CFH and oxLDL were measured in sepsis patients. In human lung microvascular endothelial cells (HLMVECs), transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell‐substrate Impedance Sensing (ECIS).Results (Figure)In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686; p=0.016, n=12) and were higher in patients who died in hospital (33.24 U/L [IQR 28.08, 48.28] vs. 27.19 U/L [IQR 16.24, 38.24]; p=0.045, n=25). HLMVEC barrier dysfunction induced by CFH (p<0.0001, n=6‐7) or oxLDL (p=0.002, n=4‐6) was attenuated by blocking the LOX‐1 receptor with small molecule inhibitor BI‐0115 (Boehringer Ingelheim, 20 µM; p=0.018 vs CFH, n=5; p=0.0002 vs oxLDL, n=6).ConclusionIncreased plasma CFH and oxLDL are associated with HLMVEC barrier dysfunction and poor clinical outcomes during sepsis; one mechanism by which CFH may cause vascular hyperpermeability during sepsis is through oxidation of LDL which can drive signaling through the LOX‐1 receptor.