Maintenance Of Endothelial Barrier Function Research Articles

Role of endothelial cell-selective adhesion molecule in the regulation of pulmonary vascular resistance

Endothelial cell-selective adhesion molecule (ESAM) is a member of the endothelial cell junction adhesion molecules family, which plays an important role in the maintenance of endothelial barrier function in the pulmonary circulation. The role of ESAM in affecting pulmonary vascular resistance remains unknown. To examine the role of ESAM in pulmonary vascular resistance, whole lungs were isolated from 16-18 weeks old ESAM knock out (KO) mice and wild type (WT) littermates, ventilated and perfused. We found that pulmonary vascular resistance is significantly increased in the lung of ESAM KO mice compared to WT mice. Bradykinin-induced, endothelium-dependent reduction in the pulmonary vascular resistance was impaired in ESAM KO mice, whereas the direct NO donor, sodium nitroprusside-induced endothelium independent responses remained similar in WT and ESAM KO mice. U46619 mediated increases in pulmonary vascular resistance was significantly higher in ESAM KO mice. Inhibition of the NO synthesis with the L-NAME (0.2 mM for 10 minutes) augmented the U46619-mediated increase in pulmonary vascular resistance in WT mice, but it did not affect the U46619 response in the ESAM KO mice. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor, was increased in the lungs of ESAM KO mice, whereas expression of collagen isoforms (type I and type III) and fibronectin were similar in WT and ESAM KO mice. Collectively, we propose that ESAM plays an important role in the maintenance of pulmonary vascular resistance, likely via preserving endothelial nitric oxide production in pulmonary arteries. AHA predoctoral award ID 917057. 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.

Protective effect of hydroxytyrosol and tyrosol metabolites in LPS-induced vascular barrier derangement in vitro.

The maintenance of endothelial barrier function is essential for vasal homeostasis and prevention of cardiovascular diseases. Among the toxic stimuli involved in the initiation of atherosclerotic lesions, Gram negative lipopolysaccharide (LPS) has been reported to be able to trigger endothelial dysfunction, through the alteration of barrier permeability and inflammatory response. Hydroxytyrosol (HT) and tyrosol (Tyr), the major phenolic compounds of extra virgin olive oil (EVOO), as wells as their circulating sulphated and glucuronidated metabolites have been shown to exert anti-inflammatory effects at endothelial level. In this study we investigated the protective effects of HT and Tyr metabolites on LPS-induced alteration of permeability in Human Umbilical Vein Endothelial Cells (HUVEC) monolayers and examined underlying signaling pathways, focusing on tight junction (TJ) proteins, mitogen-activated protein kinase (MAPK) and NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. It was shown that LPS-increased permeability in HUVEC cells was due to the alteration of TJ protein level, following the activation of MAPK and NLRP3. HT and Tyr sulphated and glucuronidated metabolites were able to limit the effects exerted by LPS, acting as signaling molecules with an efficacy comparable to that of their precursors HT and Tyr. The obtained results add a further piece to the understanding of HT and Tyr metabolites mechanisms of action in vascular protection.

KRIT1: A Traffic Warden at the Busy Crossroads Between Redox Signaling and the Pathogenesis of Cerebral Cavernous Malformation Disease.

Significance: KRIT1 (Krev interaction trapped 1) is a scaffolding protein that plays a critical role in vascular morphogenesis and homeostasis. Its loss-of-function has been unequivocally associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease of genetic origin characterized by defective endothelial cell-cell adhesion and ensuing structural alterations and hyperpermeability in brain capillaries. KRIT1 contributes to the maintenance of endothelial barrier function by stabilizing the integrity of adherens junctions and inhibiting the formation of actin stress fibers. Recent Advances: Among the multiple regulatory mechanisms proposed so far, significant evidence accumulated over the past decade has clearly shown that the role of KRIT1 in the stability of endothelial barriers, including the blood-brain barrier, is largely based on its involvement in the complex machinery governing cellular redox homeostasis and responses to oxidative stress and inflammation. KRIT1 loss-of-function has, indeed, been demonstrated to cause an impairment of major redox-sensitive mechanisms involved in spatiotemporal regulation of cell adhesion and signaling, which ultimately leads to decreased cell-cell junction stability and enhanced sensitivity to oxidative stress and inflammation. Critical Issues: This review explores the redox mechanisms that influence endothelial cell adhesion and barrier function, focusing on the role of KRIT1 in such mechanisms. We propose that this supports a novel model wherein redox signaling forms the common link between the various pathogenetic mechanisms and therapeutic approaches hitherto associated with CCM disease. Future Directions: A comprehensive characterization of the role of KRIT1 in redox control of endothelial barrier physiology and defense against oxy-inflammatory insults will provide valuable insights into the development of precision medicine strategies. Antioxid. Redox Signal. 38, 496-528.

Abstract 9695: RASA3 as a Novel Candidate Gene in Sickle Cell Disease-associated Pulmonary Hypertension and Pulmonary Arterial Hypertension

Introduction: Sickle cell disease (SCD)-associated PH is associated with increased mortality when tricuspid regurgitant jet velocity (TRV) is ≥2.5m/s and mPAP is ≥25mmHg on right heart catheterization. RASA3, a ubiquitously expressed GTPase-activating protein, is integral to angiogenesis and maintenance of endothelial barrier function. Hypothesis: Loss of RASA3 leads to the development of PH in patients with SCD-associated PH and PAH. Methods: Cis-eQTLs were queried for RASA3 using whole genome genotype arrays and gene expression profiles from PBMCs of subjects with SCD from 3 cohorts. Genome-wide SNPs near or in the RASA3 gene that may associate with lung RASA3 expression were identified using data from the Genotype-Tissue Expression project, reduced to 9 tagging SNPs for RASA3 and associated with TRV≥2.5m/s and RHC hemodynamics. Associations between the top RASA3 SNP and RHC hemodynamics were tested using whole genome genotype data from subjects in the PAH Biobank. PAECs collected from explanted lungs from patients with IPAH or control lungs were cultured with RASA3 expression evaluated on Western blot. Results: PBMC-derived RASA3 expression was significantly lower in patients with SCD-associated PH using TRV≥2.5m/s and RHC hemodynamics, with significantly higher mortality (Figure 1). The risk allele (T) at rs9525228, an eQTL for RASA3, correlated with PH risk, higher TRV and higher PVR among PBMCs associated with decreased RASA3 expression in patients with SCD-associated PH. The T allele at rs9525228 was significantly associated with increased mPAP (p=0.014), PVR (p=0.041) and mean right atrial pressure (p=0.04) in patients from the PAH Biobank. RASA3 expression was significantly lower in PAECs from patients with IPAH compared to control. Conclusions: RASA3 is a novel candidate gene in SCD-associated PH and PAH, with RASA3 expression appearing to be protective. Further studies are ongoing to delineate the role of RASA3 in PH.

Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function.

Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

Generation and characterization of human soluble VE‐cadherin (sVE‐cadherin)

Endothelial barrier function is maintained by tight and adherens junctions. Adherens junction protein Vascular Endothelial (VE)‐cadherin plays a critical role for maintenance of endothelial barrier function by providing the mechanical strength between adjacent endothelial cells. Previously, it was shown that inflammation‐induced loss of endothelial barrier function is associated with the formation of soluble VE‐cadherin fragments (sVE‐cadherin) that comprise the extracellular domains (EC) 1‐5. Moreover, it was proposed that sVE‐cadherin could be involved in the loss of endothelial barrier function. Here, we generated a recombinant sVE‐cadherin EC1‐5 construct to test whether the presence of sVE‐cadherin alone and independent of pro‐inflammatory stimuli would affect the integrity of endothelial barrier function.DNA templates of extracellular domains EC1‐5 of human VE‐cadherin were amplified via PCR and ligated into different plasmids. Then, Chinese‐Hamster Ovary cells (CHO) were transfected with these vectors which resulted in CHO cells secreting functional active sVE‐cadherin. This sVE‐cadherin in CHO supernatants was purified and used for biochemical characterization. Thereafter, functional analysis was performed on human microvascular endothelial cells (HDMECs).First, sequencing of the purified products from CHO cells that had been transfected with the plasmids containing EC1‐5 confirmed the sequence of the extracelIular domains EC1‐5 of VE‐cadherin. In the cell culture supernatants of transfected CHO cells the presence of sVE‐cadherin was further verified using Western Blots where a 90 kDa protein in the cell culture supernatant was detected that proved to be sVE‐cadherin as revealed by an antibody directed against the extracellular domain of VE‐cadherin. In addition, by using an ELISA‐based system that specifically recognizes extracellular domains of VE‐cadherin, levels of 100–200 ng/ml of sVE‐cadherin were found in the cell culture supernatants of transfected CHO cells which in summary confirmed the successful generation of human sVE‐cadherin.To further test the hypothesis that sVE‐cadherin compromises endothelial barrier function HDMECs were incubated with different concentrations of sVE‐cadherin followed by measurements of transendothelial electrical resistance (TER) and immunostaining. In TER measurements sVE‐cadherin led to a significant loss of TER compared to controls after 2h hours of incubation beginning at concentrations of 75 ng/ml. In immunostaining sVE‐cadherin application on endothelial monolayers resulted in intercellular gap formation, loss of VE‐cadherin at the cell borders and led to the formation of stress fibers in the actin cytoskeleton. Preliminary experiments using immunoprecipitation assays showed that sVE‐cadherin binds to vascular endothelial protein tyrosine phosphatase (VE‐PTP) pointing towards a potential mechanism by which sVE‐cadherin induces loss of VE‐cadherin.In summary, we successfully generated human sVE‐cadherin. Our data in endothelial cells demonstrate that sVE‐cadherin disrupts endothelial barrier function in a dose‐dependent manner independent of the presence of pro‐inflammatory stimuli.Support or Funding InformationDFG SCHL1962/4‐2

SIRT2 stabilizes endothelial adherens junctions

Endothelial barrier functional is essential for maintenance of tissue‐fluid homeostasis in lung. Disruption of the endothelial barrier leads to pulmonary edema, acute lung injury, and respiratory failure. Adherens junction (AJ) protein complex, and associated cytoskeleton are the major components regulating barrier integrity. Post‐translational protein modifications of AJ constituents, such as phosphorylation, nitration, and nitrosylation, have been well known to regulate endothelial barrier function. More recently, lysine acetylation/deacetylation has emerged as an important and physiologically significant posttranslational protein modification involved in pathogenesis of human diseases. Sirtuins (SIRTs) are NAD+‐dependent deacetylases, and critical regulators of energy metabolism and oxidative stress response in different cell types. Here, we studied the function of SIRT2, the only cytosolic sirtuin family member, in regulating endothelial barrier function in the context of inflammatory lung disease. We have showed that SIRT2 is the most abundant sirtuin in human lung endothelial cells. Importantly, SIRT2 protein level is significantly increased in lung endothelial cells upon challenging mice with a sublethal dose of lipopolysaccharides, a gram‐negative bacteria endotoxin which causes systemic inflammatory responses resulting in acute lung injury. Increase in SIRT2 protein level was also observed in human lung microvascular endothelial cells (HLMVEC) upon challenge with permeability‐increasing agents such as human α‐thrombin and TNF‐α, further emphasizing the potentially important role of SIRT2 in regulating adaptation of endothelial cells to inflammatory responses. Furthermore, depletion of SIRT2 in HLMVECs monolayers led to destabilization of VE‐cadherin junctions suggesting that SIRT2 regulates integrity of endothelial barrier. SIRT2 depleted monolayers also demonstrated an increased gap area following TNF‐α treatment whereas cells overexpressing human SIRT2 attenuated TNF‐α‐induced disruption of VE‐cadherin junctions. Our data suggest that SIRT2 plays a critical role in the maintenance of endothelial barrier function under basal conditions and upon inflammatory response. In order to explore the underlying mechanism by which SIRT2 regulates endothelial barrier integrity, we performed mass‐spectrometry analysis of acetylated proteins in HLMVECs treated with control siRNA or depleted of SIRT2. Interestingly, we found that acetylation of BORG5, a Cdc42 effector protein 1, was markedly increased after SIRT2 knockdown as well as in cells treated with hTNF‐α. We observed that human SIRT2‐FLAG interacted with BORG5‐V5 overexpressed in HEK293T cells and the level of acetyl‐BORG5 was increased in cells depleted of SIRT2. Furthermore, knockdown BORG5 reversed gap formation in endothelial cell with SIRT2 knockdown suggesting that SIRT2 stabilizes AJs through deacetylation of BORG5. Overall, our studies describe the novel role of SIRT2 in the mechanisms regulating the endothall barrier integrity through deacetylation of BORG5 allowing the assembly of stable AJs.Support or Funding InformationXiaoyan Yang is supported by AHA Award #17SDG33410608This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucagon-like peptide-1 receptor activation alleviates lipopolysaccharide-induced acute lung injury in mice via maintenance of endothelial barrier function

Glucagon-like peptide-1 (GLP-1), which is well known for regulating glucose homeostasis, exhibits multiple actions in cardiovascular disorders and renal injury. However, little is known about the effect of GLP-1 receptor (GLP-1R) activation on acute lung injury (ALI). In this study, we investigated the effect of GLP-1R on ALI and the potential underlying mechanisms with the selective agonist liraglutide. Our results show that GLP-1 levels decreased in serum, though they increased in bronchoalveolar lavage fluid (BALF) and lung tissue in a mouse model of lipopolysaccharide (LPS)-induced ALI. Liraglutide prevented LPS-induced polymorphonuclear neutrophil (PMN) extravasation, lung injury, and alveolar-capillary barrier dysfunction. In cultured human pulmonary microvascular endothelial cells (HPMECs), liraglutide protected against LPS-induced endothelial barrier injury by restoring intercellular tight junctions and adherens junctions. Moreover, liraglutide prevented PMN–endothelial adhesion by inhibiting the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and thereafter suppressed PMN transendothelial migration. Furthermore, liraglutide suppressed LPS-induced activation of Rho/NF-κB signaling in HPMECs. In conclusion, our results show that GLP-1R activation protects mice from LPS-induced ALI by maintaining functional endothelial barrier and inhibiting PMN extravasation. These results also suggest that GLP-1R may be a potential therapeutic target for the treatment of ALI.

Epac1 is required for cAMP‐mediated endothelial barrier stabilization

Regulation of intercellular junctional complexes is critical for the control of endothelial barrier function and dependent on cAMP‐mediated Rac1 activation. Recently, the exchange protein activated by cAMP (Epac1) has been shown to serve as a tonic stabilizer of the endothelial barrier. Here, we further elucidate the role of Epac1 in cAMP and Rac1‐mediated endothelial barrier regulation.Transendothelial‐electrical‐resistance (TER) measurements in newly generated immortalized myocardial endothelial cells derived from Epac1‐knockout (KO) mice revealed significant reduction in the baseline TER, when compared to wild‐type (WT) cells, indicating that Epac1 is required for maintenance of endothelial barrier function. This effect was associated with fragmented VE‐cadherin staining and reduced localization of tight junctional proteins ZO‐1 and occludin along cell borders. In WT‐cells, application of forskolin (F) and rolipram (R) resulted in increased TER associated with linearization and augmentation of VE‐cadherin immunostaining at adherens junctions. In contrast, Epac1‐KO‐cells did not respond to F/R treatment indicating that Epac1 is crucial for cAMP‐mediated endothelial barrier stabilization. In comparison to WT‐cells, Epac1‐KO‐cells revealed significantly increased protein levels of Rac1, whereas Rac1 basal activity was not enhanced, indicating a defect in Rac1 activation. However, in Epac1‐KOcells, cAMP‐mediated Rac1 activation was not abolished and the basal cAMP‐concentration was significantly increased. Additionally, in Epac1‐deficient cells F/R‐mediated increase in cAMP levels was less compared to WT cells. Taken together, our data show that Epac1 is crucial for cAMP‐mediated barrier stabilization by mechanisms which at least in part are independent of Rac1 regulation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Cytoplasmic Deacetylase, SIRT2, in Stabilizing Lung Endothelial Barrier Function

Lung microvascular endothelial functional and structural integrity is essential for maintenance of tissue‐fluid homeostasis and normal function of the respiratory system. Disruption of the pulmonary endothelial barrier leads to pulmonary edema, acute lung injury, and respiratory failure. Adherens junction (AJ) proteins, actin cytoskeleton, and microtubules are major components required for maintenance of barrier integrity. Post‐translational protein modifications of AJ constituents, such as phosphorylation, nitration, and nitrosylation, have been well known to regulate endothelial barrier function. More recently, lysine acetylation/deacetylation has emerged as an important and physiologically significant posttranslational protein modification in physiological and pathological conditions. However, its role in endothelial barrier function remains largely unknown. Sirtuins (SIRTs) are NAD+‐dependent deacetylases, and critical regulators of energy metabolism and oxidative stress response in different cell types. We studied the function of SIRT2, the only cytosolic sirtuin family member, in regulating pulmonary endothelial barrier function during inflammatory response. We showed that SIRT2 is the most abundant sirtuin in human lung endothelial cells. Importantly, SIRT2 protein level is significantly increased in lung endothelial cells upon challenging mice with a sublethal dose of lipopolysaccharides, a gram‐negative bacteria endotoxin which causes systemic inflammatory responses resulting in acute lung injury. Increase in SIRT2 protein level was also observed in human lung microvascular endothelial cells (HLMVEC) upon challenge with permeability‐increasing agents such as human α‐thrombin and TNF‐α, further emphasizing the potentially important role of SIRT2 in regulating adaptation of endothelial cells to inflammatory responses. To determine the role of SIRT2 activity in the maintenance of lung endothelial barrier function, we knocked down SIRT2 in HLMVECs and assessed changes in endothelial permeability upon challenge with α‐thrombin. We found that SIRT2 knock down significantly augmented α‐thrombin‐induced permeability increases as compared to control siRNA‐treated cells, suggesting that SIRT2 activity may play a critical role in regulating endothelial barrier integrity. Furthermore, treatment of endothelial cells with AGK2, a SIRT2‐selective inhibitor, significantly decreased assembly of endothelial junctions due to increased accumulation of VE‐cadherin in the cytoplasm. Our data suggest that SIRT2 plays a critical role in the maintenance of endothelial barrier function under basal conditions and upon inflammatory response.Support or Funding InformationXiaoyan Yang : T32HL007829‐23, AHA 17SDG33410608.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sphingosine-1-Phosphate Receptor-1 Agonist Sew2871 Causes Severe Cardiac Side Effects and Does Not Improve Microvascular Barrier Breakdown in Sepsis.

Endothelial barrier dysfunction is a hallmark in the pathogenesis of sepsis. Sphingosine-1-phosphate (S1P) has been proposed to be critically involved in the maintenance of endothelial barrier function predominately by activating S1P receptor-1 (S1P1). Previous studies have shown that the specific S1P1 agonist SEW2871 improves endothelial barrier function under inflammatory conditions. However, the effectiveness of SEW2871 and potential side effects remained largely unexplored in a clinically relevant model of sepsis. Therefore, this study aimed to evaluate the effects of SEW2871 in the Colon ascendens stent peritonitis (CASP) model. Polymicrobial sepsis was induced in Sprague-Dawley rats using CASP model that enabled the monitoring of macro-hemodynamic parameters. Twelve hours after surgery, animals received either SEW2871 or sodium chloride. Mesenteric endothelial barrier function was evaluated 24 h after sepsis induction by intravital microscopy. Organ pathology was assessed in lungs. S1P levels, blood gas analyses, and blood values were measured at different time points. In parallel the effect of SEW2871 was evaluated in human dermal microvascular endothelial cells. In vitro SEW2871 partially stabilized TNF-α-induced endothelial barrier breakdown. However, in vivo SEW2871 caused severe cardiac side effects in septic animals leading to an increased lethality. Sepsis-induced endothelial barrier dysfunction was not attenuated by SEW2871 as revealed by increased FITC-albumin extra-vasation, requirement of intravasal fluid replacement, and pulmonary edema. Interestingly, Sham-operated animals did not present any side effects after SEW2871 treatment. Our study demonstrates that the application of SEW2871 causes severe cardiac side effects and cannot attenuate the inflammation-induced endothelial barrier breakdown in a clinically relevant sepsis model, suggesting that the time point of administration and the pro-inflammatory milieu play a pivotal role in the therapeutic benefit of SEW2871.

Vascular protection by high-density lipoprotein-associated sphingosine-1-phosphate.

Epidemiological studies and animal experiments have consistently demonstrated cardiovascular protection by high-density lipoprotein (HDL). Findings from a growing number of studies further indicate that sphingosine-1-phosphate (S1P) mediates many of the beneficial effects of HDL on the cardiovascular system, including vasodilatation, angiogenesis, maintenance of endothelial barrier function, and protection against atherosclerosis and ischemia/reperfusion injury. In this review, we summarize the most recent literature investigating the effects of HDL-S1P on cardiovascular health and highlight potential opportunities for clinical translation of these findings.

Cigarette Smoke Disrupted Lung Endothelial Barrier Integrity and Increased Susceptibility to Acute Lung Injury via Histone Deacetylase 6.

Epidemiologic evidence indicates that cigarette smoke (CS) is associated with the development of acute lung injury (ALI). We have previously shown that brief CS exposure exacerbates lipopolysaccharide (LPS)-induced ALI in vivo and endothelial barrier dysfunction in vitro. In this study, we found that CS also exacerbated Pseudomonas-induced ALI in mice. We demonstrated that lung microvascular endothelial cells (ECs) isolated from mice exposed to CS had a greater permeability or incomplete recovery after challenges by LPS and thrombin. Histone deacetylase (HDAC) 6 deacetylates proteins essential for maintenance of endothelial barrier function. We found that HDAC6 phosphorylation at serine-22 was increased in lung tissues of mice exposed to CS and in lung ECs exposed to cigarette smoke extract (CSE). Inhibition of HDAC6 attenuated CSE-induced increases in EC permeability and CS priming of ALI. Similar barrier protection was provided by the microtubule stabilizer taxol, which preserved α-tubulin acetylation. CSE decreased α-tubulin acetylation and caused microtubule depolymerization. In coordination with increased HDAC6 phosphorylation, CSE inhibited Akt and activated glycogen synthase kinase (GSK)-3β; these effects were ameliorated by the antioxidant N-acetyl cysteine. Our results suggest that CS increases lung EC permeability, thereby enhancing susceptibility to ALI, likely through oxidative stress-induced Akt inactivation and subsequent GSK-3β activation. Activated GSK-3β may activate HDAC6 via phosphorylation of serine-22, leading to α-tubulin deacetylation and microtubule disassembly. Inhibition of HDAC6 may be a novel therapeutic option for ALI in cigarette smokers.

Reciprocal Regulation of eNOS and Caveolin‐1 Function in Endothelial Cells

Endothelial nitric oxide (eNOS)‐mediated NO production plays a critical role in the regulation of vascular tone, adhesivity, and maintenance of endothelial barrier function. Caveolin‐1 (Cav‐1), which is found highly expressed in endothelia, is an important regulator of vascular tone via modulation of eNOS activity. eNOS‐derived NO may also affect Cav‐1 functions directly and indirectly through nitrosation and phosphorylation‐dependent modifications, although the exact molecular mechanisms and effects on eNOS expression and activity in endothelial cells are not clear. Here, reciprocal regulation by the two proteins was investigated. First, we determined by semi‐quantitative Western blotting that the ratio Cav‐1 to eNOS expression in primary culture endothelial cells was approximately 200:1. Secondly, upon stimulation of ECs with Ca2+ ionophore A23187 to activate Ca2+‐dependent signaling, Cav‐1 nitrosation, phosphorylation, and monomerization occurred within minutes which could be blocked by L‐NAME (eNOS inhibitor) and PP2 (Src kinase inhibitor). Furthermore, oligmerization of Cav‐1 was enhanced when eNOS expression was reduced by siRNA. These data suggest that eNOS, through a NO‐Src signaling pathway, induces a conformational change to the functional oligomeric form of Cav‐1. Thirdly, Cav‐1 knockdown by siRNA reduced eNOS expression level, although NO production and eNOS phosphorylation per molecule were elevated 2‐fold. Finally, in HEK cells overexpressing eNOS, transfection of Cav‐1 resulted in a concentration‐dependent increase followed by a decrease in eNOS expression and activity. Thus, eNOS‐derived NO production promotes Cav‐1 nitrosylation, phosphorylation and monomerization, while lower levels of Cav‐1 expression stabilize eNOS expression and higher levels inhibit eNOS activity via phosphorylation‐dependent binding as part of a negative feedback regulatory mechanism.Support or Funding InformationFinancial Support: P01 HL60678 and Department of Anesthesiology

Vascular Endothelia Mechanically Sense Barrier Quality and Maintain Functional Integrity through ROS‐Dependent Actin Remodeling

Objective: Mechanisms for maintenance of endothelial integrity are of enormous biomedical importance. Despite its tenuous structure and constitutive exposure to disruptive strains, the endothelium normally exhibits extremely robust barrier properties. The objective of this study was to elucidate fundamental mechanisms by which the endothelium senses and responds to integrity disruptions in order to maintain its barrier function. Results: We show that in response to ~5-50 micron-scale disruptions in the endothelium, induced by transmigrating leukocytes or a mechanical probe, endothelial cells generate unique ‘ventral lamellipodia' that propagate via integrins toward and across these ‘micro-wounds' to close them and promote re-annealing of the adherens junctions. Experiments combining probe-induced micro-wounding, pharmacologic modulation of contractility & substrate stretching manipulations demonstrate that endothelia 'sense' breaches in its barrier as force imbalance and specifically loss of isometric tension upon rupture of adhesions. Such loss if tension was acutely/locally translated into biomechanical signals for reparative actin remodeling. Indeed, ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of H2O2. Together with Apr2/3, these were functionally required for effective micro-wound healing and maintenance of endothelial barrier function. Conclusion We propose that barrier disruptions are detected as local release of isometric tension, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.

Single nucleotide variants in the protein C pathway and mortality in dialysis patients.

BackgroundThe protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients.MethodsSingle nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort.ResultsFactor V Leiden was associated with a 1.5-fold (95% CI 1.1–1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0–1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality.ConclusionOur study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients.

Abl Family Kinases Regulate Endothelial Barrier Function In Vitro and in Mice

The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg), as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR), Kit, colony stimulating factor 1 receptor (CSF1R), and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca2+ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use of Abl kinase inhibitors may have potential for the treatment of disorders involving pathological vascular leakage.

Abstract 296: The RNA-Binding Protein Quaking Maintains Endothelial Barrier Function Through Targeting Vascular Endothelial Cadherin mRNA

Endothelial cells (ECs) form a vital barrier between the blood and the artery wall, and play a major role in the onset of atherosclerosis. Endothelial monolayer integrity is determined largely by EC-cell interactions, where vascular endothelial cadherin (VE-cadherin) is the central adhesive component at these endothelial adherens junctions. While many of the receptors and signaling proteins involved in regulating these adherens junctions have been identified, surprisingly little is known regarding their regulation at the post[[Unable to Display Character: ‐]]transcriptional level. The RNA-binding protein Qauking (QKI), originally known for its function in the nervous system, has been demonstrated to be essential for blood vessel formation. We find that QKI is highly expressed in quiescent ECs, in vitro and in vivo. In contrast, human umbilical vein ECs displayed reduced levels of QKI in response to the inflammatory stimuli TNF-α as well as in cells lacking cell-cell contacts, suggesting that QKI may act to enhance barrier function. To test this, we specifically abrogated QKI expression in ECs and measured their capacity to form a high-resistance monolayer with Electrical Cell-substrate Impedance Sensing. Silencing of QKI did not affect EC adhesion or spreading, but markedly affected the capacity to form a high resistance endothelial monolayer. Consistent with these data, and the fact that VE-cadherin mRNA contains a putative QKI-response element, the targeted reduction in QKI was accompanied by a significant reduction in VE-cadherin expression at cell junctions. Importantly, we identified a direct role for QKI in regulating VE-cadherin mRNA biology, as RNA immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the VE-cadherin mRNA and regulate transcript stability, respectively. In conclusion, we show that the modulation of QKI expression levels affects endothelial monolayer integrity by functioning as a critical regulator of the VE-cadherin mRNA. These studies provide novel insight into a role for post-transcriptional regulation in the maintenance of endothelial barrier function, and may have wide ranging implications for the preservation of vascular integrity in disease.

Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

Cytosolic free calcium concentration ([Ca2+]i) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca2+]i overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca2+]i overload can be prevented by lithium treatment. [Ca2+]i and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-d-glucose (5mM; 2-DG) plus sodium cyanide (5mM; NaCN) caused a significant decrease in cellular ATP content (14±1nmol/mg protein vs. 18±1nmol/mg protein in the control, n=6 culture dishes, P<0.05), an increase in [Ca2+]i (278±24nM vs. 71±2nM in the control, n=60 cells, P<0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10mM 2-DG led to a similar decrease in ATP content (14±2nmol/mg vs. 18±1nmol/mg in the control, P<0.05) with a delay of 5min. The [Ca2+]i response of EC was biphasic with a peak after 1min (183±6nM vs. 71±1nM, n=60 cells, P<0.05) followed by a sustained increase in [Ca2+]i. A 24-h pre-treatment with 10mM of lithium chloride before the inhibition of ATP synthesis abolished both phases of the 2-DG-induced [Ca2+]i increase. This effect was not observed when lithium chloride was added simultaneously with 2-DG.We conclude that lithium chloride abolishes the injurious [Ca2+]i overload in EC and that this most likely occurs by preventing inositol 3-phosphate-sensitive Ca2+-release from the endoplasmic reticulum. Though further research is needed, these findings provide a novel option for therapeutic strategies to protect the endothelium against imminent barrier failure.