Endothelial Monolayer Integrity Research Articles

Abstract 16: BcrKinase is a Novel Akt Kinase That Modulates Scavenger Receptor BI- and PDZK1-dependent Actions of HDL in Endothelium

High density lipoprotein cholesterol (HDL) has direct atheroprotective actions on endothelium. These are mediated by scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1, and they entail the activation of Akt kinase, which phosphorylates and thereby stimulates endothelial nitric oxide synthase (eNOS). In the present work we sought to determine how PDZK1 couples HDL/SR-BI to Akt and eNOS to modulate endothelial function. Using tandem affinity purification (TAP) following the infection of the human endothelial cell line EAhy926 with adenovirus expressing TAP-tagged PDZK1, we identified Breakpoint Cluster Region (Bcr) kinase as a PDZK1 interacting protein in endothelium. Whereas Bcr is well-known as a component of the Bcr-Abl fusion protein that results from translocation of the Philadelphia chromosome in chronic myelogenous leukemia, little is known of its function in endothelial cells. Bcr contains several distinctive domains including a C-terminal PDZ binding motif and a serine/threonine protein kinase domain. In primary human aortic endothelial cells (HAEC), we determined that endogenous Bcr interacts with PDZK1 in an HDL-dependent manner, and that Bcr is required for HDL-induced activation of eNOS and HDL stimulation of endothelial cell migration, which underlies the ability of the lipoprotein to promote endothelial monolayer integrity. Studies of mutant forms of Bcr with disruption of PDZK1 binding or kinase activity introduced into endothelial cells further revealed that Bcr-PDZK1 interaction and its kinase function are required for HDL activation of Akt and eNOS. Using a novel kinase assay that we recently developed that employs time-resolved Forster resonance energy transfer, we found that via SR-BI and PDZK1, HDL stimulates Bcr kinase activity in endothelial cells more than 20-fold. In addition, using Akt-based peptides in studies of the two known kinases for Akt, mTOR and PDK1, we determined that HDL activates Bcr kinase to directly phosphorylate Akt-Ser473 in an mTOR independent manner, and that Akt-Thr308 is a direct substrate of PDK1. These collective findings have identified Bcr to be a novel kinase for Akt, and they have revealed that Bcr is critically involved in HDL modulation of endothelial cell phenotype.

Secretome derived from breast tumor cell lines alters the morphology of human umbilical vein endothelial cells.

Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.

Endothelial p110γPI3K Mediates Endothelial Regeneration and Vascular Repair After Inflammatory Vascular Injury.

The integrity of endothelial monolayer is a sine qua non for vascular homeostasis and maintenance of tissue-fluid balance. However, little is known about the signaling pathways regulating regeneration of the endothelial barrier after inflammatory vascular injury. Using genetic and pharmacological approaches, we demonstrated that endothelial regeneration selectively requires activation of p110γPI3K signaling, which thereby mediates the expression of the endothelial reparative transcription factor Forkhead box M1 (FoxM1). We observed that FoxM1 induction in the pulmonary vasculature was inhibited in mice treated with a p110γ-selective inhibitor and in Pik3cg(-/-) mice after lipopolysaccharide challenge. Pik3cg(-/-) mice exhibited persistent lung inflammation induced by sepsis and sustained increase in vascular permeability. Restoration of expression of either p110γ or FoxM1 in pulmonary endothelial cells of Pik3cg(-/-) mice restored endothelial regeneration and normalized the defective vascular repair program. We also observed diminished expression of p110γ in pulmonary vascular endothelial cells of patients with acute respiratory distress syndrome, suggesting that impaired p110γ-FoxM1 vascular repair signaling pathway is a critical factor in persistent leaky lung microvessels and edema formation in the disease. We identify p110γ as the critical mediator of endothelial regeneration and vascular repair after sepsis-induced inflammatory injury. Thus, activation of p110γ-FoxM1 endothelial regeneration may represent a novel strategy for the treatment of inflammatory vascular diseases.

Relative contribution of physiological hydrostatic pressure and fluid shear stress to endothelial monolayer integrity

Purpose Vascular endothelial cells (ECs) are continuously subjected to mechanical forces such as fluid shear stress, stretching and hydrostatic pressure. The effect of hydrostatic pressure on EC responses has not been fully understood compared to that of the other two stimuli. The purpose of this study is to assess mechanical responses of ECs to these mechanical stimuli.

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

BackgroundPlasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.Methodology/Principal FindingsWe utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.Conclusions/SignificanceOur findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

GPR55 promotes migration and adhesion of colon cancer cells indicating a role in metastasis.

Tumour cell migration and adhesion constitute essential features of metastasis. G-protein coupled receptor 55 (GPR55), a lysophospholipid receptor, has been shown to play an important role in carcinogenesis. Here, we investigated the involvement of GPR55 in migration and metastasis of colon cancer cells. Adhesion and migration assays using the highly metastatic colon cancer cell line HCT116 and an in vivo assay of liver metastasis were performed. The GPR55 antagonist CID16020046, cannabidiol, a putative GPR55 antagonist and GPR55 siRNA were used to block GPR55 activity in HCT116 colon cancer cells. HCT116 cells showed a significant decrease in adhesion to endothelial cells and in migration after blockade with CID16020046 or cannabidiol. The inhibitory effects of CID16020046 or cannabidiol were averted by GPR55 siRNA knock down in cancer cells. The integrity of endothelial cell monolayers was increased after pretreatment of HCT116 cells with the antagonists or after GPR55 siRNA knockdown while pretreatment with lysophosphatidylinositol (LPI), the endogenous ligand of GPR55, decreased integrity of the monolayers. LPI also induced migration in GPR55 overexpressing HCT116 cells that was blocked by GPR55 antagonists. In a mouse model of metastasis, the arrest of HCT116 cancer cells in the liver was reduced after treatment with CID16020046 or cannabidiol. Increased levels of LPI (18:0) were found in colon cancer patients when compared with healthy individuals. GPR55 is involved in the migratory behaviour of colon carcinoma cells and may serve as a pharmacological target for the prevention of metastasis. © 2015 The British Pharmacological Society.

Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration Across Brain and Lung Microvascular Endothelium.

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin P (albumin) was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased P (albumin); more tumor cells adhered to and transmigrated through the endothelial monolayer with higher P (albumin); P (albumin) and tumor transmigration were increased by vascular endothelial growth factor, a representative of cytokines, and lipopolysaccharides, a typical systemic inflammatory factor, but reduced by adenosine 3',5'-cyclic monophosphate. These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation.

Expression of HMGB1 in septic serum induces vascular endothelial hyperpermeability.

The aim of the present study was to investigate the effects of high mobility group protein B1 (HMGB1), which is expressed in the serum of patients with sepsis, on vascular endothelial permeability. Sera from patients with sepsis were used to treat endothelial cells (ECs), and the effect on endothelial permeability was evaluated using immunofluorescence. The morphologies of endothelial cytoskeletal actin and vascular endothelial (VE)‑cadherin were assessed using laser scanning confocal microscopy. The protein expression levels of HMGB1, B‑cell lymphoma 2 (BCL‑2) and BCL‑2‑associated X protein (BAX) were detected using western blotting. EC apoptosis was measured using flow cytometry. The results demonstrated that HMGB1 was significantly expressed in the serum 24 h following the onset of sepsis, and the expression levels peaked at 48 h, which were sustained until 96 h post‑onset. Compared with the control group, treatment of the ECs with 20% septic serum in vitro significantly increased endothelial monolayer permeability (P<0.01), markedly induced transcellular filamentous (F)‑actin rearrangement with stress fiber formation, and resulted in the localization of VE‑cadherin fragmentations at the cell borders with increased gaps between ECs. Furthermore, flow cytometry showed that the apoptotic rate of ECs was significantly increased following treatment with septic serum. In addition, the expression levels of BAX were significantly increased, whereas the expression levels of BCL‑2 were significantly decreased. Pretreatment with an HMGBI inhibitor (ethyl pyruvate; 5 µM) 24 h prior to treatment with the septic serum attenuated the effects of septic serum treatment. Together, these findings suggested that treatment of ECs with sera from patients with sepsis may induce the loss of vascular endothelial monolayer integrity, elicit the formation of endothelial F‑actin stress fibers and initiate VE‑cadherin redistribution, which may be attributed to high levels of HMGB1 in the serum. This mechanism also appears to involve changes in the activation of BAX and BCL‑2, resulting in EC apoptosis.

Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity.

Complications arising from dengue virus infection include potentially fatal vascular leak, and severe disease has been linked with excessive immune cell activation. An understanding of the triggers of this activation is critical for the development of appropriately targeted disease control strategies. We show here that the secreted form of the dengue virus nonstructural protein 1 (NS1) is a pathogen-associated molecular pattern (PAMP). Highly purified NS1 devoid of bacterial endotoxin activity directly activated mouse macrophages and human peripheral blood mononuclear cells (PBMCs) via Toll-like receptor 4 (TLR4), leading to the induction and release of proinflammatory cytokines and chemokines. In an in vitro model of vascular leak, treatment with NS1 alone resulted in the disruption of endothelial cell monolayer integrity. Both NS1-mediated activation of PBMCs and NS1-induced vascular leak in vitro were inhibited by a TLR4 antagonist and by anti-TLR4 antibody treatment. The importance of TLR4 activation in vivo was confirmed by the reduction in capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. These results pinpoint NS1 as a viral toxin counterpart of the bacterial endotoxin lipopolysaccharide (LPS). Similar to the role of LPS in septic shock, NS1 might contribute to vascular leak in dengue patients, which highlights TLR4 antagonists as a possible therapeutic option.

Mechanosensitive PPAP2B Regulates Endothelial Responses to Atherorelevant Hemodynamic Forces.

PhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis. To establish the critical role of PPAP2B in endothelial responses to hemodynamics. Reduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells. Atherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Role of N-WASP in Endothelial Monolayer Formation and Integrity

Endothelial cells (ECs) form a monolayer that serves as a barrier between the blood and the underlying tissue. ECs tightly regulate their cell-cell junctions, controlling the passage of soluble materials and immune cells across the monolayer barrier. We studied the role of N-WASP, a key regulator of Arp2/3 complex and actin assembly, in EC monolayers. We report that N-WASP regulates endothelial monolayer integrity by affecting the organization of cell junctions. Depletion of N-WASP resulted in an increase in transendothelial electrical resistance, a measure of monolayer integrity. N-WASP depletion increased the width of cell-cell junctions and altered the organization of F-actin and VE-cadherin at junctions. N-WASP was not present at cell-cell junctions in monolayers under resting conditions, but it was recruited following treatment with sphingosine-1-phosphate. Taken together, our results reveal a novel role for N-WASP in remodeling EC junctions, which is critical for monolayer integrity and function.

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways.

Exposure of pulmonary artery endothelial cells (PAECs) to hyperoxia results in a compromise in endothelial monolayer integrity, an increase in caspase-3 activity, and nuclear translocation of apoptosis-inducing factor (AIF), a marker of caspase-independent apoptosis. In an endeavor to identify proteins involved in hyperoxic endothelial injury, we found that the protein expression of heat-shock protein 70 (Hsp70) was increased in hyperoxic PAECs. The hyperoxia-induced Hsp70 protein expression is from hspA1B gene. Neither inhibition nor overexpression of Hsp70 affected the first phase barrier disruption of endothelial monolayer. Nevertheless, inhibition of Hsp70 by using the Hsp70 inhibitor KNK437 or knock down Hsp70 using siRNA exaggerated and overexpression of Hsp70 prevented the second phase disruption of lung endothelial integrity. Moreover, inhibition of Hsp70 exacerbated and overexpression of Hsp70 prevented hyperoxia-induced apoptosis, caspase-3 activation, and increase in nuclear AIF protein level in PAECs. Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic PAECs. Inhibition of Hsp70/AIF association by KNK437 correlated with increased nuclear AIF level and apoptosis in KNK437-treated PAECs. Finally, the ROS scavenger NAC prevented the hyperoxia-induced increase in Hsp70 expression and reduced the interaction of Hsp70 with AIF in hyperoxic PAECs. Together, these data indicate that increased expression of Hsp70 plays a protective role against hyperoxia-induced lung endothelial barrier disruption through caspase-dependent and AIF-dependent apoptotic pathways. Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis. The hyperoxia-induced increase in Hsp70 expression and Hsp70/AIF interaction is contributed to ROS formation.

Abstract 351: Mechano-sensitive Ppap2b Regulates Endothelial Responses to Athero-relevant Hemodynamic Forces

Rationale: PhosPhatidic-Acid-Phosphatase-type-2B (PPAP2B), an integral membrane protein that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies (GWAS). However, it is unclear whether GWAS-identified CAD genes including PPAP2B participate in mechanotransduction mechanisms by which vascular endothelia respond to local athero-relevant blood flows that contribute to the regional nature of atherosclerosis. Approach and Results: Reduced endothelial PPAP2B was detected in vivo in swine aortic arch exposed to chronic disturbed flow and in mouse carotid artery subjected to surgically-induced acute disturbed flow. In culture, elevated PPAP2B was measured in human aortic endothelial cells (HAEC) under athero-protective flow mimicking hemodynamics of human distal carotid artery when compared with athero-susceptible waveform representing flow in carotid sinus. Disturbed flow-induced miR-92a was identified as a direct posttranscriptional inhibitor of mechano-sensitive PPAP2B. PPAP2B suppression abrogated athero-protection of unidirectional flow, shown by elevation of inflammatory genes. Inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. Moreover, PPAP2B inhibition resulted in myosin-light-chain phosphorylation and intercellular gaps, which were abolished by inhibition of lysophosphatidic acid receptors 1 and 2. Expression-quantitative-trait-locus-mapping demonstrated PPAP2B CAD risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in HAEC. Conclusions: Athero-relevant flows dynamically modulate endothelial PPAP2B expression through miR-92a regulation. Mechano-sensitive PPAP2B plays an indispensable role in mediating cell alignment, promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under athero-protective flow.

Abstract 8: RNA-binding Protein Quaking Maintains Endothelial Barrier Function Through β-catenin and VE-cadherin

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is determined largely by adherens junctions. Remarkably little is known about their regulation at the post[[Unable to Display Character: &amp;#8208;]]transcriptional level. We find that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent adult endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently we aimed to investigate the role of QKI in EC vascular integrity. Silencing of QKI markedly impaired (0.65 fold ±0.13; p&lt;0.05) the capacity to form a high resistance endothelial monolayer, as measured using Electric cell-substrate impedance sensing. To confirm a role for QKI in maintaining EC barrier function in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p&lt;0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to wild type littermates. Interestingly, the mRNA of both β-catenin and VE-cadherin, the prime adhesion proteins in EC adherens junctions, contain conserved QKI-binding sites. Moreover the targeted reduction of QKI resulted in a reduction of β-catenin and VE-cadherin protein expression. Importantly, we identified a direct role for QKI in regulating mRNA biology of β-catenin and VE-cadherin, as RNA immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the mRNA and induces transcript translation, respectively. This effects was perturbed upon reduced QKI expression. In conclusion, we show that QKI functions as a critical regulator of β-catenin and VE-cadherin in endothelial cells, and the modulation of QKI expression affects endothelial monolayer integrity, in vitro and in vivo. These studies provide novel insight into the importance of post-transcriptional regulation of components of the endothelial adherens junction, and may have wide ranging implications for the preservation of vascular integrity in disease.

Interaction between mesenchymal stem cells and endothelial cells restores endothelial permeability via paracrine hepatocyte growth factor in vitro.

IntroductionMesenchymal stem cells (MSCs) have potent stabilising effects on vascular endothelium injury, inhibiting endothelial permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secrete more factors by MSC-endothelial cell (MSC-EC) interactions. We hypothesised that MSC-EC interactions restore endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF.MethodsWe investigated the endothelial permeability induced by LPS under two co-culture conditions. Human pulmonary microvascular endothelial cells (HPMECs) were added into the upper chambers of cell-culture inserts, while two different co-culture conditions were used in the lower side of the transwells, as follows: (1) MSC-EC interaction group: MSCs and HPMECs contact co-culture; (2) MSC group: MSCs only. The endothelial paracellular and transcellular permeabilities in the upper side of transwells were detected. Then the concentration of HGF was measured in the culture medium by using an enzyme-linked immunosorbent assay kit, followed by neutralisation of HGF with anti-HGF antibody in the co-culture medium. In addition, adherens junction and cytoskeleton protein expressions were measured by Western blot and immunofluorescence. HPMEC proliferation was analysed by bromodeoxyuridine incorporation assay.ResultsThe paracellular permeability significantly increased after LPS stimulation in a dose-dependent and time-dependent manner. Meanwhile, MSC-EC interaction more significantly decreased endothelial paracellular and transcellular permeability induced by LPS. Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group. However, neutralising HGF with anti-HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability. Compared with the MSC group, MSC-EC interaction increased vascular endothelial (VE)-cadherin and occludin protein expression, reduced caveolin-1 protein expression in HPMECs, and restored remodelling of F-actin and junctional localisation of VE-cadherin. Furthermore, the proliferation ratio in the MSC-EC interaction group was higher than that of the MSC group. However, the effects of MSCs were significantly blocked by anti-HGF antibody.ConclusionsThese data suggested that MSC-EC interaction decreased endothelial permeability induced by LPS, which was attributed mainly to HGF secreted by MSCs. The main mechanisms by which HGF restored the integrity of endothelial monolayers were remodelling of endothelial intercellular junctions, decreasing caveolin-1 protein expression, and inducing proliferation in HPMECs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0025-1) contains supplementary material, which is available to authorized users.

Hyperglycemia abolishes the protective effect of ischemic preconditioning in glomerular endothelial cells invitro.

In preclinical investigations, ischemic preconditioning (IPC) protects kidneys from ischemia/reperfusion injury. The direct effects of IPC on glomerular endothelial cells have not been studied in detail. Most investigations of IPC have focused on healthy cells and animals, and it remains unknown whether IPC is renoprotective in the setting of medical comorbidities such as diabetes. In this study, we determined the preventive potential of IPC in healthy glomerular endothelial cell monolayers, and compared these results to monolayers cultured under hyperglycemic conditions. We exposed glomerular endothelial monolayers to 1 h of IPC 24 h prior to oxygen–glucose deprivation (OGD), an in vitro model of ischemia/reperfusion injury. Glomerular endothelial monolayer integrity was assessed by measuring transendothelial electrical resistance, albumin flux, and cell survival. We found that IPC protected healthy but not hyperglycemic glomerular endothelial monolayers from ischemia/reperfusion injury. Furthermore, not only was the protective effect of IPC lost in the setting of hyperglycemia, but IPC was actually deleterious to the integrity of hyperglycemic glomerular endothelial cell monolayers.

Effect of proinflammatory activation on F-actin distribution in cultured human endothelial cells under conditions of experimental microgravity.

We compared the state of actin cytoskeleton, morphology, and expression of VE-cadherin in endothelial cells of human umbilical cord vein under conditions of TNF-α-mediated activation and microgravity modeling and found that 3D-clinorotation for 24 h impaired the integrity of endothelial monolayer, altered cell morphology, induced cytoskeleton reorganization, and reduced the expression of VE-cadherin. The combination of experimental microgravity and proinflammatory activation led to more pronounced clearing of the perinuclear space from microfilaments and accumulation of depolymerized actin, which confirms additive effect of the studied factors on actin cytoskeleton of endothelial cells.

The Impact of Insulin Treatment on the Expression of Vascular Endothelial Cadherin and Beta-Catenin in Human Fetoplacental Vessels

Vascular endothelial cadherin and β-catenin play a key role in establishment and maintenance of the endothelial monolayer integrity, regulation of vascular barrier function, and initiation of angiogenesis. The cadherin-catenin complex has been shown to be reduced in type 1 diabetic placenta, but the exact relationship between histopathologic findings and clinical data is not known. Immunohistochemistry of placental tissue from type 1, type 2, and gestational diabetes showed that diabetes per se might be compatible with normal levels of vascular endothelial (VE)-cadherin and β-catenin in fetoplacental vessels as long as the patient has not been treated with insulin. Immunoreactivity of VE-cadherin did correlate poorly with maternal glycemic control, as was investigated in this study, by birth weight, body mass index, and hemoglobin A1c (HbA1c). There was no correlation found between the immunoreactivity of β-catenin and birth weight, body mass index, or HbA1c. However our data did show a strong correlation between immunoreactivity and whether or not the patient had been treated with insulin. Patients diagnosed with gestational diabetes who had not been treated with insulin had similar levels of VE-cadherin and β-catenin to the control group, thus indicating that diabetes per se must not necessarily lead to a reduction. Our study suggests that therapeutic intervention using insulin in pregnancies complicated by diabetes might have potentially harmful effects on placental morphology. Future studies should further investigate these findings.

Moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia.

Anti-inflammatory functions of antibiotics may counteract deleterious hyperinflammation in pneumonia. Moxifloxacin reportedly exhibits immunomodulatory properties, but experimental evidence in pneumonia is lacking. Therefore, we investigated moxifloxacin in comparison with ampicillin regarding pneumonia-associated pulmonary and systemic inflammation and lung injury. Ex vivo infected human lung tissue and mice with pneumococcal pneumonia were examined regarding local inflammatory response and bacterial growth. In vivo, clinical course of the disease, leucocyte dynamics, pulmonary vascular permeability, lung pathology and systemic inflammation were investigated. In addition, transcellular electrical resistance of thrombin-stimulated endothelial cell monolayers was quantified. Moxifloxacin reduced cytokine production in TNF-α-stimulated, but not in pneumococci-infected, human lung tissue. In vivo, moxifloxacin treatment resulted in reduced bacterial load as compared with ampicillin, whereas inflammatory parameters and lung pathology were not different. Moxifloxacin-treated mice developed less pulmonary vascular permeability during pneumonia, but neither combination therapy with moxifloxacin and ampicillin in vivo nor examination of endothelial monolayer integrity in vitro supported direct barrier-stabilizing effects of moxifloxacin. The current experimental data do not support the hypothesis that moxifloxacin exhibits potent anti-inflammatory properties in pneumococcal pneumonia.

Microtubule dynamics control HGF-induced lung endothelial barrier enhancement.

Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics.