Reduction In Transendothelial Electrical Resistance Research Articles

Catalpol downregulates vascular endothelial‑cadherin expression and induces vascular hyperpermeability.

Catalpol, an iridiod glucoside isolated from Rehmannia glutinosa, has been reported to possess anti‑inflammatory properties. However, the molecular mechanisms underlying this effect have not been fully elucidated. This study aimed to investigate the effects of catalpol on vascular permeability. Using Transwell permeability assays and measurements of trans‑endothelial electrical resistance (TEER), it was demonstrated that 1 mM catalpol induces a significant increase in the permeability of the monolayers of human umbilical vein endothelial cells (HUVECs). Western blotting and immunofluorescence demonstrated that catalpol inhibits the expression of vascular endothelial (VE)‑cadherin, the key component of adherens junctions, but not occludin, the major constituent of tight junctions. In addition, catalpol inhibits the ETS transcription factor ERG, a positive regulator of VE‑cadherin. Knockdown of ERG expression compromised the catalpol‑induced reduction of TEER in HUVECs. The present study revealed a novel effect of catalpol on vascular permeability and gave insight into the multifaceted roles of catalpol in inflammation.

Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.

Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF-induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC-dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF-induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF-induced redistribution of ZO-1 and the recruitment of detergent-soluble occludin and claudin-5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time- and dose-dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF-induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant-negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF-mediated changes in paracellular permeability.

Doxycycline hyclate protects lipopolysaccharide-induced endothelial barrier dysfunction by inhibiting the activation of p38 mitogen-activated protein kinase.

Doxycycline hyclate (DOX-h) attenuates inflammatory conditions independent of its antimicrobial effect. This study aimed to observe the effects of DOX-h on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. The endothelial monolayer permeability of human umbilical vein endothelial cells (HUVECs) was monitored by transendothelial electrical resistance (TEER). The phosphorylation of mitogen-activated protein kinases (MAPKs) and the arrangement of F-actin were detected. The results showed that both pretreatment and simultaneous treatment with DOX-h markedly attenuated the LPS-induced reduction in TEER and the disorganization of F-actin on HUVECs in a dose- and time-dependent manner. LPS mediated the phosphorylation of all three MAPKs (p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK)), but DOX-h was only able to inhibit the LPS-induced phosphorylation of p38 and JNK. The data further suggested that DOX-h alleviated LPS-evoked TEER reduction and F-actin redistribution by inhibiting the phosphorylation of p38 and its downstream target, heat shock protein (HSP)27. Thus, DOX-h attenuates LPS-induced endothelial barrier dysfunction via inhibition of the p38 MAPK-HSP27-F-actin pathway.

Anthrax Lethal Toxin Downregulates Claudin-5 Expression in Human Endothelial Tight Junctions

Vascular leakage pathologies such as pleural effusion and hemorrhage are hallmarks of anthrax pathogenesis. We previously reported that anthrax lethal toxin (LT), the major virulence factor of anthrax, reduces barrier function in cultured primary human microvascular endothelial cells. Here, we show that LT-induced barrier dysfunction is accompanied by the reduced expression of the endothelial tight junction (TJ) protein claudin-5 but no change in the expression of other TJ components occludin, ZO-1, ZO-2, or the adherens junction (AJ) protein VE-cadherin. The downregulation of claudin-5 correlated temporally and dose-dependently with the reduction of transendothelial electrical resistance. LT-induced loss of claudin-5 was independent of cell death and preceded the appearance of actin stress fibers and altered AJ morphology. Pharmacological inhibition of MEK-1/2, two kinases that are proteolytically inactivated by LT, showed a similar reduction in claudin-5 expression. We found that LT reduced claudin-5 mRNA levels but did not accelerate the rate of claudin-5 degradation. Mice challenged with LT also showed significant reduction in claudin-5 expression. Together, these findings support a possible role for LT disruption of endothelial TJs in the vascular leakage pathologies of anthrax.

In vitro modeling of inflammation‐induced blood‐brain barrier dysfunction in sickle cell disease

Patients with sickle cell disease (SCD) have a high incidence of cerebral vascular occlusion combined with inflammation. The properties of the blood‐brain barrier (BBB) in SCD are not characterized. We investigated structural and functional properties of SCD BBB using cerebrovascular endothelial cells (CVEC) from Berkeley SCD (BERK) and control C57BL/6 (WT) mice. BERK CVEC in primary cultures had normal growth potency and the ability to form cell contacts visualized by beta‐catenin and VE‐cadherin staining. BBB functions were tested by transendothelial electrical resistance (TEER) and by permeability to Alexa Fluor 488‐dextran in CVEC grown on transwell inserts. The baseline parameters of BBB functions were identical in WT and BERK CVEC. Pro‐inflammatory cytokine TNF‐alpha combined with cycloheximide (CHX) caused a time‐dependent (5–23 h) reduction in TEER and increased dextran permeability in both cell types. TNF/CHX‐induced BBB dysfunction was accompanied by loss of cell‐cell contacts, cell detachment, and by apoptosis. Antioxidant bilirubin conferred protection against TNF/CHX‐increased endothelial permeability, indicating that oxidative stress is a leading factor in inflammation‐induced BBB dysfunction in WT and SCD mice. Overall, CVEC from SCD mice maintain normal functional properties of BBB and are not hypersensitized to inflammation‐related injury. The study is supported by NIH/NINDS.

Lung Endothelial Injury Induced by HNA-3a Antibodies in TRALI

Abstract 40Transfusion related acute lung injury (TRALI) is a severe side-effect of blood transfusion characterized by the acute onset of non-cardiogenic pulmonary edema. Meanwhile, TRALI turned out to be the leading cause of transfusion related fatalities. Accumulated evidence demonstrates that antibodies against human neutrophil antigens (HNAs) play a major role in the pathomechanism of TRALI. Recent studies show that antibodies against the allelic isoform of choline transporter like protein 2 (CTL-2 Arg154 isoform; also known as HNA-3a) are associated with high TRALI mortality. The mechanism underlying this fatal reaction, however, is not clear. In this study, we aimed to identify the mechanism of fatal TRALI induced by HNA-3 antibody under in vitro as well as in vivo conditions.Analysis of mRNA by real-time PCR in different blood cells and endothelial cells (EC) revealed abundant copies of CTL-2 transcripts in ECs in comparison to neutrophils and platelets. This result was confirmed by immunochemical analysis using rabbit antibody specific for CTL-2 as well human antibodies against HNA-3a. In contrast to treatment of neutrophils, treatment of EC with HNA-3a antibodies, but not with antibodies against the antithetical allelic CTL-2 isoform (Gln154 isoform; HNA-3b), leads to significant production of reactive oxygen species (ROS). When HNA-3aa EC monolayers were treated with human anti-HNA-3a antibodies, a significant increase in albumin-FITC influx in transwell system was observed when compared to controls. In line with this observation, a strong reduction of transendothelial electrical resistance was measured. Microscopically, drastic stress fiber formation and gap formation was visible. Immunoblotting analysis of HNA-3a treated ECs showed a significant degradation of VE-cadherin. This observation indicates that anti-HNA-3a antibodies induce EC barrier disturbance via ROS-mediated destabilization of VE-cadherin in cell junctions. Accordingly, this antibody-mediated barrier leakage can be ameliorated by the vasoactive peptide adrenomedulin (ADM), which prevents cell destruction in response to oxidative stress.In an in vivo murine model of TRALI, with lipopolysaccharide primed C57BL/6 mice upon injection of HNA-3a antibodies a significant increase in lung weight and elevated concentration of albumin and number of neutrophils in the bronchoalveolar lavage was observed, indicating the formation of lung edema in these mice. Neutrophil depletion mitigated this effect in mice, but failed to prevent TRALI.Our data demonstrate the direct influence of transfused HNA-3a antibodies on endothelial barrier integrity in vitro as well as in vivo. This novel mechanism of TRALI may be helpful in defining prevention and treatment strategies in order to decrease transfusion mortality. Disclosures:No relevant conflicts of interest to declare.

Positively-charged microemulsion for improving the oral bioavailability of alendronate: in-vitro and in-vivo assessment

Alendronate is a poorly absorbed bisphosphonate with an oral bioavailability of 0.7%. In this study, a positively-charged microemulsion was prepared with the aim of improving the bioavailability of alendronate. The positively-charged microemulsion was evaluated for physical stability, cellular uptake and permeability enhancement on Caco-2 monolayers. The bioavailability of alendronate from the microemulsion was compared with the commercially available tablet (Fosmax) for beagle dogs. The 2.0, 0.4 and 0.2% positively-charged microemulsion, stable for 4 h after preparation, promoted alendronate transport across the Caco-2 cells by a factor of 194, 146,and 45.1, respectively, compared with the alendronate solution, though no significant cellular uptake enhancement of alendronate was observed. The permeability enhancement was parallel to the reduction in transendothelial electrical resistance, which indicated the microemulsion modulated the tight junctions and widened the paracellular pathway. In-vivo results showed that the microemulsion gave the highest alendronate plasma concentration at 502 ng/ml (C(max)) after 0.563 h (T(max)), while tablets gave a C(max) of 152 ng/ml after 0.750 h (T(max)). Furthermore, the AUC(0-∞) of alendronate from the microemulsion increased by 2.82-fold when compared with the tablets. Based on the results, the oral bioavailability of alendronate could be significantly improved by the positively-charged microemulsion, which opened the tight junctions and thus increased absorption through the paracellular route.

β‐adrenergic stimulation contributes to maintenance of endothelial barrier functions under resting conditions

Protective effects of cAMP on endothelial barrier properties are well established. We tested the hypothesis whether β‐adrenergic receptor‐induced cAMP increase is involved in maintenance of endothelial barrier function. Inhibition of β‐receptors by propranolol increased hydraulic conductivity in Ringer‐perfused rat postcapillary venules. Similarly, in human cultured microvascular endothelial cells (HDMEC), propranolol reduced transendothelial electrical resistance (TER) and cAMP levels, and induced intercellular gap formation. In contrast, activation of adrenergic receptors by epinephrine both increased cAMP and TER and resulted in linearized VE‐cadherin immunostaining. Forced formation of cAMP by adenylate cyclase activator forskolin but neither epinephrine nor phosphodiesterase inhibition by rolipram prevented propranolol‐induced TER reduction and VE‐cadherin reorganization. Taken together, our data indicate that β‐adrenergic receptor‐mediated generation of cAMP contributes to maintenance of endothelial barrier properties. Furthermore, in addition to epinephrine from systemic circulation locally generated β‐receptor agonists likely participate in this process. Thus, impaired endothelial barrier function may contribute to formation of lung and ankle edema observed in patients treated with β‐blockers. DFG SFB688, A4

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with Papp of 19.7±1.9×10−6 cm/s and 38.6±4.1×10−6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009

Lipopolysaccharide-enhanced transcellular transport of HIV-1 across the blood-brain barrier is mediated by the p38 mitogen-activated protein kinase pathway

Chronic systemic inflammation in the late stage of human immunodeficiency virus type-1 (HIV-1) infection could increase neuroinvasion of infected monocytes and cell-free virus, causing an aggravation of neurological disorders in AIDS patients. We previously showed that the peripheral administration of lipopolysaccharide (LPS) enhanced the uptake across the blood-brain barrier (BBB) of the HIV-1 viral protein gp120. Brain microvessel endothelial cells are targets of LPS. Here, we investigated whether the direct interaction between LPS and the BBB also affected HIV-1 transport using primary mouse brain microvessel endothelial cells (BMECs). LPS produced a dose (1–100 μg/mL)- and time (0.5–4 h)-dependent increase in HIV-1 transport and a decrease in transendothelial electrical resistance (TEER). Whereas indomethacin (cyclooxygenase inhibitor) and L-NAME (NO synthase inhibitor) did not affect the LPS-induced changes in HIV-1 transport or TEER, pentoxifylline (TNF-α inhibitor) attenuated the decrease in TEER induced by LPS, but not the LPS-induced increase in HIV-1 transport. LPS also increased the phosphorylation of p44/42 MAPK and p38 MAPK but not that of JNK. U0126 (p44/42 MAPK inhibitor) and SP600125 (JNK inhibitor) did not inhibit the LPS-induced increase in HIV-1 transport although U0126 attenuated the reduction in TEER. SB203580 (p38 MAPK inhibitor) inhibited the LPS-induced increase in HIV-1 transport without affecting TEER. Thus, LPS-enhanced HIV-1 transport is independent of changes in TEER and so is attributed to increased transcellular trafficking of HIV-1 across the BBB. These results show that LPS increases HIV-1 transcellular transport across the BBB by a pathway that is mediated by p38 MAPK phosphorylation in BMECs.

Viable “Haemophilus somnus” Induces Myosin Light-Chain Kinase-Dependent Decrease in Brain Endothelial Cell Monolayer Resistance

"Haemophilus somnus" causes thrombotic meningoencephalitis in cattle. Our laboratory has previously reported that H. somnus has the ability to adhere to, but not invade, bovine brain endothelial cells (BBEC) in vitro. The goal of this study was to determine if H. somnus alters brain endothelial cell monolayer integrity in vitro, in a manner that would be expected to contribute to inflammation of the central nervous system (CNS). Monolayer integrity was monitored by measuring transendothelial electrical resistance (TEER) and albumin flux. BBEC incubated with H. somnus underwent rapid cytoskeletal rearrangement, significant increases in albumin flux, and reductions in TEER. Decreased monolayer TEER was preceded by phosphorylation of the myosin regulatory light chain and was partially dependent on tumor necrosis factor alpha and myosin light-chain kinase but not interleukin-1beta. Neither heat-killed H. somnus, formalin-fixed H. somnus, nor purified lipooligosaccharide altered monolayer integrity within a 2-h incubation period, whereas conditioned medium from H. somnus-treated BBEC caused a modest reduction in TEER. The data from this study support the hypothesis that viable H. somnus alters integrity of the blood-brain barrier by promoting contraction of BBEC and increasing paracellular permeability of the CNS vasculature.

Hypoxia Disrupts the Barrier Function of Neural Blood Vessels through Changes in the Expression of Claudin-5 in Endothelial Cells

The mechanisms underlying the hypoxia-induced disruption of the barrier function of neural vasculature were analyzed with reference to the expression of claudin-5, a component of tight junctions between neural endothelial cells. The movement of claudin-5 from the cytoplasm to the plasma membrane of cultured confluent brain-derived endothelial (bEND.3) cells was closely correlated with the increase in the transendothelial electrical resistance. Inhibition of the expression of claudin-5 by RNAi resulted in a reduction of transendothelial electrical resistance, indicating a critical role of claudin-5 in the barrier property. Hypoxia (1% O(2)) altered the location of claudin-5 in the plasma membrane and the level of claudin-5 protein in bEND.3 cells, and these changes were accompanied by a decrease in the transendothelial electrical resistance. In vivo the claudin-5 molecules were expressed under normoxia in the plasma membrane of retinal microvascular endothelial cells but were significantly reduced under hypoxic conditions. Tracer experiments revealed that the barrier function of hypoxic retinal vasculature with depressed claudin-5 expression was selectively disrupted against small molecules, which is very similar to the phenotype of claudin-5-deficient mice. These in vitro and in vivo data indicate that claudin-5 is a target molecule of hypoxia leading to the disruption of the barrier function of neural vasculature.

Thr 207 of claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP

We have recently shown that cyclic AMP (cAMP) increases claudin-5 immunoreactivity along cell boundaries and could promote phosphorylation of claudin-5 on threonine residues in porcine blood–brain barrier (BBB) endothelial cells via a protein kinase A (PKA)-dependent pathway ( Exp. Cell Res. 290 [2003] 275). Along this line, we identified a putative phosphorylation site for PKA at Thr 207 in the intracytoplasmic carboxyl terminal domain of claudin-5. To clarify the biological significance of this site in regulation of endothelial barrier functions, we established rat lung endothelial (RLE) cells expressing doxycycline (Dox)-inducible wild-type claudin-5 and a mutant with a substitution of Ala for Thr 207 (CL5T207A). We show that induction of wild-type claudin-5 is sufficient to reconstitute the paracellular barrier against inulin (5 kDa), but not mannitol (182 Da), in leaky RLE cells. By contrast, the barrier against both molecules was induced in the mutant cells. We also demonstrate that, upon cAMP treatment, Thr 207 of claudin-5 is involved in enhancement of claudin-5 immunoreactive signals along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based endothelial barrier against mannitol, but not inulin. cAMP decreased the claudin-5-based endothelial barrier, strongly suggesting that other tight-junction molecule(s) are required to elevate endothelial barrier functions in response to cAMP.

Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells.

Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.

Biphasic effects of 17-beta-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells.

Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction function are an early and key event during the vascular spread of cancer and inflammation. This study sought to determine the role of estrogen in the regulation of tight junctions and expression of molecules making tight junctions in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17-beta-estradiol induced a concentration- and time-dependent biphasic effect on tight junction. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17-beta-estradiol at lower concentrations (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 min and the second drop occurring 48 h after 17-beta-estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17-beta-estradiol, in line with changes of TER. This study shows that 17-beta-estradiol can induce concentration- and time-related biphasic effects on tight junction functions expression of occludin in endothelial cells and that this perturbation of tight junction functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.

Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.

Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 microM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.