Increase In Transendothelial Resistance Research Articles

Selective expression of claudin-5 in thymic endothelial cells regulates the blood-thymus barrier and T-cell export.

T-cell development depends on the thymic microenvironment, in which endothelial cells (ECs) play a vital role. Interestingly, vascular permeability of the thymic cortex is lower than in other organs, suggesting the existence of a blood-thymus barrier (BTB). On the other hand, blood-borne molecules and dendritic cells bearing self-antigens are accessible to the medulla, facilitating central tolerance induction, and continuous T-precursor immigration and mature thymocyte egress occur through the vessels at the cortico-medullary junction (CMJ). We found that claudin-5 (Cld5), a membrane protein of tight junctions, was expressed in essentially all ECs of the cortical vasculatures, whereas approximately half of the ECs of the medulla and CMJ lacked Cld5 expression. An intravenously (i.v.) injected biotin tracer hardly penetrated cortical Cld5+ vessels, but it leaked into the medullary parenchyma through Cld5- vessels. Cld5 expression in an EC cell line caused a remarkable increase in trans-endothelial resistance in vitro, and the biotin tracer leaked from the cortical vasculatures in Cldn5-/- mice. Furthermore, i.v.-injected sphingosine-1 phosphate distributed selectively into the medulla through the Cld5- vessels, probably ensuring the egress of CD3high mature thymocytes from Cld5- vessels at the CMJ. These results suggest that distinct Cld5 expression profiles in the cortex and medulla may control the BTB and the T-cell gateway to blood circulation, respectively.

TRPV4-Mediated Regulation of the Blood Brain Barrier Is Abolished During Inflammation.

Blood-brain barrier (BBB) dysfunction is critically involved in determining the extent of several central nervous systems (CNS) pathologies and here in particular neuroinflammatory conditions. Inhibiting BBB breakdown could reduce the level of vasogenic edema and the number of immune cells invading the CNS, thereby counteracting neuronal injury. Transient receptor potential (TRP) channels have an important role as environmental sensors and constitute attractive therapeutic targets that are involved in calcium homeostasis during pathologies of the CNS. Transient receptor potential vanilloid 4 (TRPV4) is a calcium permeable, non-selective cation channel highly expressed in endothelial cells. As it is involved in the regulation of the blood brain barrier permeability and consequently cerebral edema formation, we anticipated a regulatory role of TRPV4 in CNS inflammation and subsequent neuronal damage. Here, we detected an increase in transendothelial resistance in mouse brain microvascular endothelial cells (MbMECs) after treatment with a selective TRPV4 inhibitor. However, this effect was abolished after the addition of IFNγ and TNFα indicating that inflammatory conditions override TRPV4-mediated permeability. Accordingly, we did not observe a protection of Trpv4-deficient mice when compared to wildtype controls in a preclinical model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), and no differences in infarct sizes following transient middle cerebral artery occlusion (tMCAO), the experimental stroke model, which leads to an acute postischemic inflammatory response. Furthermore, Evans Blue injections did not show differences in alterations of the blood brain barrier (BBB) permeability between genotypes in both animal models. Together, TRPV4 does not regulate brain microvascular endothelial permeability under inflammation.

Sox18 Preserves the Pulmonary Endothelial Barrier Under Conditions of Increased Shear Stress

Shear stress secondary to increased pulmonary blood flow (PBF) is elevated in some children born with congenital cardiac abnormalities. However, the majority of these patients do not develop pulmonary edema, despite high levels of permeability inducing factors. Previous studies have suggested that laminar fluid shear stress can enhance pulmonary vascular barrier integrity. However, little is known about the mechanisms by which this occurs. Using microarray analysis, we have previously shown that Sox18, a transcription factor involved in blood vessel development and endothelial barrier integrity, is up-regulated in an ovine model of congenital heart disease with increased PBF (shunt). By subjecting ovine pulmonary arterial endothelial cells (PAEC) to laminar flow (20 dyn/cm(2) ), we identified an increase in trans-endothelial resistance (TER) across the PAEC monolayer that correlated with an increase in Sox18 expression. Further, the TER was also enhanced when Sox18 was over-expressed and attenuated when Sox18 expression was reduced, suggesting that Sox18 maintains the endothelial barrier integrity in response to shear stress. Further, we found that shear stress up-regulates the cellular tight junction protein, Claudin-5, in a Sox18 dependent manner, and Claudin-5 depletion abolished the Sox18 mediated increase in TER in response to shear stress. Finally, utilizing peripheral lung tissue of 4 week old shunt lambs with increased PBF, we found that both Sox18 and Claudin-5 mRNA and protein levels were elevated. In conclusion, these novel findings suggest that increased laminar flow protects endothelial barrier function via Sox18 dependent up-regulation of Claudin-5 expression.

Iloprost suppresses LPS‐induced lung injury and inflammation via Rac1‐dependent mechanism

Prostacyclin exhibits protective effects on vascular endothelium and enhances endothelial barrier, as reflected by rapid increase in transendothelial resistance. This study investigated for the first time protective effects of stable prostacyclin analog iloprost using in vitro and in vivo models of lung injury and inflammation induced by bacterial wall lypopolysacharide (LPS). Iloprost attenuated disruption of endothelial monolayer and suppressed activation of inflammatory signaling, including p38 mitogen activated protein kinase, NFkB pathway, Rho signaling, attenuated ICAM1 expression, cytokine release, neutrophil adhesion and migration after LPS challenge. In vivo, iloprost exhibited potent inhibition of LPS‐induced lung injury and vascular leak, as detected by suppression of protein and neutrophil accumulation in bronchoalveolar lavage fluid and reduced myeloperoxidase activation, ICAM‐1 expression, and Evans blue extravasation in the lungs. Inhibition of Rac1 activity using pharmacologic inhibitors or siRNA‐mediated knockdown abolished barrier‐protective and anti‐inflammatory effects of iloprost in vitro and in vivo. We conclude that iloprost‐induced elevation of intracellular cAMP triggers Rac signaling, which attenuates LPS‐induced inflammatory pathways of vascular permeability.Funding: NHLBI HL87823, HL076259, and HL058064

Growth factor- and heparin-dependent regulation of constitutive and agonist-mediated human endothelial barrier function

We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.

Endothelial phenotype of the materno–fetal barrier

The terminal villous capillaries of the human placenta are the sites of maximal materno‐fetal exchange. These capillaries are highly angiogenic and are formed in the last trimester of pregnancy by looped angiogenesis from existing microvessels in the intermediate villi. They represent an ‘activated’ phenotype by possessing adherens junctions which contain VE‐cadherin (– and (– catenin but lack plakoglobin, the component of well differentiated junctions. The tight junctions present here show negative immunoreactivity to occludin and claudin‐1. The conduit vessels further upstream in the stem villi have the full complement of adherens and tight junctional molecules. VEGF and angiopoietin‐2 is present in terminal villi whilst angiopoietin‐1 appears to be a predominant feature of stem villi. The phenotype of junctions present in nascent vessels of the first trimester placentae was similar to the angiogenic terminal villous capillaries. The predominant growth factor here was VEGF, localised to all villi. Isolation of endothelial cells from last trimester placenta microvessels (HPMEC) demonstrated that these cells are capable of differential expression and dynamic regulation of cell‐cell junctions. Cells grown in the presence of ECGS showed a reduction in the level and junctional localisation of plakoglobin and occludin, with associated increased permeability to macromolecules. These adhesion molecules were up‐regulated and targeted back to cell‐cell contact regions by growing HPMEC in the presence of cAMP enhancing agents. This resulted in a 1.7 times increase in transendothelial resistance whilst proliferation of HPMEC cells was inhibited by elevated cAMP. HUVEC cells isolated from the umbilical cords of these placentae contain the full repertoire of AJ molecules in normal culture conditions with no change in composition or resistance under elevated cAMP. The differential expression of junctional adhesion molecules along the placental vascular tree and their in vitro plasticity suggests there is a physiological relevance of junctional regulation in the placenta. The activated junctional phenotype of the newly formed terminal microvessels allows them to participate in increased growth, proliferation and solute exchange in the last trimester when fetal demand is at its greatest. Furthemore, this necessary junctional immaturity of new/angiogenic human vessels appear to be at the expense of barrier function.Funded by the Wellcome Trust.

Cyclic AMP and Acidic Fibroblast Growth Factor Have Opposing Effects on Tight and Adherens Junctions in Microvascular Endothelial Cells in Vitro

Endothelial adherens junctions (AJ) and tight junctions (TJ) are important determinants of vascular permeability and cell morphology. Here, we investigate their regulation, in primary human placental microvascular endothelial cell (HPMEC) cultures, by either aFGF plus heparin (ECGS) or elevated cAMP. The proliferation of HPMEC was weakly stimulated by ECGS, while cAMP was inhibitory. ECGS had little effect on transendothelial resistance (TER), but increased macromolecular permeability, whereas cAMP induced a twofold increase in TER and reduced macromolecular permeability. Ultrastructurally, ECGS-treated HPMEC exhibited an “activated” phenotype typified by proliferating cells, with poorly organized cell–cell junctions, whereas cAMP-treated cells appeared quiescent and markedly flattened with extended paracellular junctions, resembling endothelium in situ. The expression and localization of junctional molecules, F-actin, and junctional phosphotyrosine were examined by confocal microscopy and immunoblotting. Junctional molecules in ECGS-treated cells were less organized at lateral membranes than in control cells, whereas in cAMP-treated cells, they were highly localized at continuous contacts. These differences correlated with the intensity of junctional phosphotyrosine, being lowest with cAMP treatment. In the AJ of ECGS-treated and control cells, β-catenin predominated but in cAMP-treated cells, γ-catenin/plakoglobin was enriched. In addition, cAMP upregulated junctional expression of VE-cadherin and PECAM-1 and increased the levels of the TJ molecules occludin and ZO−1. The expression levels of junctional components, and their tyrosine phosphorylation, play an important role in dynamic regulation of endothelial cell–cell junctions.