Human Endothelial Barrier Function Research Articles

Vitamin D Attenuates Endothelial Dysfunction in Uremic Rats and Maintains Human Endothelial Stability.

BackgroundDysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease (CKD). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD, not only by direct effects on the endothelium but also by an increment of α‐Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia‐induced endothelial damage and the extent to which this was dependent on increased α‐Klotho concentrations.Methods and ResultsIn a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial‐gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α‐Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real‐time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro‐permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial‐cadherin–based cell‐cell junctions and diminished F‐actin stress fiber organization, preventing the formation of endothelial intracellular gaps.ConclusionsOur results demonstrate that paricalcitol attenuates the CKD‐induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell‐cell interactions.

Stabilization of cell-cell junctions by active vitamin D ameliorates uraemia-induced loss of human endothelial barrier function.

Uraemia induces endothelial cell (EC) injury and impaired repair capacity, for which the underlying mechanism remains unclear. Active vitamin D (VD) may promote endothelial repair, however, the mechanism that mediates the effects of VD in chronic kidney disease are poorly understood. Thus, we investigated uraemia-induced endothelial damage and the protection against such damage by active VD. We applied electric cell-substrate impedance sensing (ECIS) to study real-time responses of human ECs exposed to pooled uraemic and non-uraemic plasma with or without the addition of active VD. The effects of indoxyl sulphate and p-cresol were tested in non-uraemic plasma. Structural changes for vascular endothelial (VE)-cadherin and F-actin were assessed by immunostaining and quantified. The exposure of ECs to uraemic media significantly decreased endothelial barrier function after 24 h. Cell migration after electrical wounding and recovery of the barrier after thrombin-induced loss of integrity were significantly impaired in uraemic-medium stimulated cells and cells exposed to indoxyl sulphate and p-cresol. This effect on ECIS was dependent on loss of cell-cell interaction. Mechanistically, we found that EC, exposed to uraemic media, displayed disrupted VE-cadherin interactions and F-actin reorganization. VD supplementation rescued both endothelial barrier function and cell-cell interactions in ECs exposed to uraemic media. These events were associated with an increment of VE-cadherin at intercellular junctions. Our data demonstrate a potentially clinically relevant mechanism for uraemia-induced endothelial damage. Furthermore, active VD rescued the uraemic medium-induced loss of cell-cell adhesion, revealing a novel role of active VD in preservation of endothelial integrity during uraemia.

Effects of Fe particle irradiation on human endothelial barrier structure and function

Space travel involves exposure to biologically effective heavy ion radiation and there is consequently a concern for possible degenerative disorders in humans. A significant target for radiation effects is the microvascular system, which is crucial to healthy functioning of the tissues. Its pathology is linked to disrupted endothelial barrier function and is not only a primary event in a range of degenerative diseases but also an important influencing factor in many others. Thus, an assessment of the effects of heavy ion radiation on endothelial barrier function would be useful for estimating the risks of space travel. This study was aimed at understanding the effects of high LET Fe particles (1 GeV/n) and is the first investigation of the effects of charged particles on the function of the human endothelial barrier. We used a set of established and novel endpoints to assess barrier function after exposure. These include, trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in 2D monolayers and in 3D tissue models), and permeability of molecules through the endothelial barrier. A dose of 0.50 Gy was sufficient to cause a progressive reduction in TEER measurements that were significant 48 hours after exposure. Concurrently, there were morphological changes and a 14% loss of cells from monolayers. Gaps also appeared in the normally continuous cell-border localization of the tight junction protein – ZO-1 but not the Platelet endothelial cell adhesion molecule (PECAM-1) in both monolayers and in 3D vessel models. Disruption of barrier function was confirmed by increased permeability to 3 kDa and 10 kDa dextran molecules. A dose of 0.25 Gy caused no detectible change in cell number, morphology, or TEER, but did cause barrier disruption since there were gaps in the cell border localization of ZO-1 and an increased permeability to 3 kDa dextran. These results indicate that Fe particles potently have impact on human endothelial barrier function and represent a risk for degenerative diseases in the space environment.

Acute effects of ionizing radiation on human endothelial barrier function

The human vasculature is critical to healthy functioning of the tissues of the body and a major factor in maintaining homeostasis is the endothelial barrier. In the brain, the blood–brain barrier (BBB) is highly specialized in order to sustain the neural tissue.Here, we have examined the effects of radiation on BBB models using a unique variety of endpoints to assess barrier function. These include trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in two-dimensional monolayers and in three-dimensional tissue models) and permeability of molecules through the endothelial barrier. Two culture conditions were used to represent conditions on the inside or the lumen of vessels and conditions on the outside or ablumenal side of vessels. For the lumen, cells were cultured in serum and growth factor containing media, and for the ablumenal side of vessels, cells were cultured in serum-free defined media.Initial experiments with gamma rays in serum-free conditions revealed a previously unknown acute effect involving cell detachment and the loss of the clinically relevant cell adhesion molecule—cell platelet endothelial adhesion molecule (PECAM)-1 [ 1]. Gamma radiation (5 Gy) induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the lower radiotherapy dose of 2 Gy. This dip in resistance correlated with the transient loss PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Loss in PECAM-1 occurred at least in part as detached microparticles. Redistribution of PECAM-1 microparticles was also seen in three-dimensional human tissue models. By 6 h, the remaining cells had migrated to reseal the barrier, coincident with TEER returning to control levels. Resealed monolayers contained fewer cells per unit area and their barrier function was weakened as corroborated by an increased permeability over 24 h. Because PECAM-1 is involved in barrier function and platelet aggregation, this effect is likely highly relevant to cancer radiotherapy using gamma rays.These studies were extended to include low linear energy transfer (LET) photons of X-rays and ion particles present in the space environment—low LET ion particles including high energy (1 GeV) protons and helium ions. X-rays under serum-free conditions also showed an acute response involving a dip in TEER at 3 h and the loss of PECAM-1 between cells as microparticles. Ion particles, however, did not show these effects of photons under serum-free conditions. Both protons and helium ions at doses up to 5 Gy did not produce this transient change in TEER or PECAM-1, although some longer term effects in TEER were noted.In the presence of serum and growth factors, however, all radiations tested showed short-term effects in TEER, that of a series of symmetrical peaks which diminished in size over several hours. For 1 GeV protons and helium ions, this effect could be fitted to an equation for under-damped oscillation, a pattern typical of a mechanism for timing of events in periodic processes. For gamma and X-rays, the underdamped oscillation was present but superimposed on a drop in resistance at 3 h similar to that seen in serum-free conditions.In conclusion, we have shown two acute effects of low LET radiation on the human endothelial barrier. First, a short-term effect of photons but not ion particles involving a single dip in TEER and the loss of PECAM-1 at 3 h after irradiation, and second an underdamped oscillation of TEER induced by both photons and ion particles that to date does not appear to be associated with the loss of PECAM-1 or any other junction molecules.

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.

Intracellular signalling involved in modulating human endothelial barrier function.

The endothelium dynamically regulates the extravasation of hormones, macromolecules and other solutes. In pathological conditions, endothelial hyperpermeability can be induced by vasoactive agents, which induce tiny leakage sites between the cells, and by cytokines, in particular vascular endothelial growth factor, which increase the exchange of plasma proteins by vesicles and intracellular pores. It is generally believed that the interaction of actin and non-muscle myosin in the periphery of the endothelial cell, and the destabilization of endothelial junctions, are required for endothelial hyperpermeability induced by vasoactive agents. Transient short-term hyperpermeability induced by histamine involves Ca2+/calmodulin-dependent activation of the myosin light chain (MLC) kinase. Prolonged elevated permeability induced by thrombin in addition involves activation of the small GTPase RhoA and Rho kinase, which inhibits dephosphorylation of MLC. It also involves the action of other protein kinases. Several mechanisms can increase endothelial barrier function, depending on the tissue affected and the cause of hyperpermeability. They include blockage of specific receptors, and elevation of cyclic AMP by agents such as beta2-adrenergic agents. Depending on the vascular bed, nitric oxide and cyclic GMP can counteract or aggravate endothelial hyperpermeability. Finally, inhibitors of RhoA activation and Rho kinase represent a potentially valuable group of agents with endothelial hyperpermeability-reducing properties.

Intracellular signalling involved in modulating human endothelial barrier function

The endothelium actively regulates the extravasation of solutes, hormones and macromolecules. In pathological conditions endothelial hyperpermeability can be induced by vasoactive agents that induce tiny leakage sites between the cells, and by cytokines, in particular vascular endothelial growth factor, that increase exchange of plasma proteins by vesicles and intracellular pores. It is generally believed that the interaction of actin and nonmuscle myosin in the periphery of the endothelial cell is required for endothelial hyperpermeability induced by vasoactive agents. Histamine causes a transient increase in endothelial permeability which normalises within seversal minutes. By H1‐receptor activation it raises the cytoplasmic Ca2+ concentration and stimulates the Ca2+/calmodulin dependent activation of the myosin light chain (MLC) kinase. The phophorylated MLC promotes actin‐non–muscle–myosin interaction in the endothelial cell. Thrombin induces a prolonged hyperpermeability in human endothelial monolayers. In addition to the Ca2± dependent activation of MLC kinase, a sensitisation of the cell occurs by activation of RhoA and Rho kinase. Furthermore, protein tyrosine kinase activity contributes to the thrombin‐induced hyperpermeability.In most types of endothelia the barrier function of endothelial cells is improved both in vivo and in vitro by agents that increase the cellular cyclic AMP concentration. Catecholamines contribute to the enforcement of the endothelial barrier function by activation of (2‐adrenergic receptors and increasing the endothelial cyclic AMP content. Nitric oxide and cyclic GMP can also counteract thrombin‐induced endothelial hyperpermeability in specific types of vessels while in other types they contribute to endothelial hyperpermeability. In human aorta endothelial cells NO induced by vasoactive agents increases the generation of cGMP, which activates cGMP‐dependent protein kinase‐I. This activity counteracts the effect on endothelial permeability by reducing the elevation of Ca2+ in the cell. Furthermore inhibitors of RhoA formation and Rho kinase represent a potentially valuable additional group of agents with endothelial hyperpermeability reducing properties. They not only reduce thrombin‐induced endothelial permeability in vitro but also reduced the interaction of leukocytes with the endothelial cells and accompanying vascular leakage in vivo.

Neutrophil-derived Glutamate Regulates Vascular Endothelial Barrier Function

Endothelial barrier function is altered by the release of soluble polymorphonuclear leukocyte (PMN)-derived mediators during inflammatory states. However, endogenous pathways to describe such changes are only recently appreciated. Using an in vitro endothelial paracellular permeability model, cell-free supernatants from formylmethionylleucylphenylalanine-stimulated PMNs were observed to significantly alter endothelial permeability. Biophysical and biochemical analysis of PMN supernatants identified PMN-derived glutamate in modulating endothelial permeability. Furthermore, novel expression of metabotropic glutamate receptor 1 (mGluR1), mGluR4, and mGluR5 by human brain and dermal microvascular endothelial cells was demonstrated by reverse transcription-PCR, in situ hybridization, immunofluorescence, and Western blot analysis. Treatment of human brain endothelia with glutamate or selective, mGluR group I or III agonists resulted in a time-dependent loss of phosphorylated vasodilator-stimulated phosphoprotein (VASP) and significantly increased endothelial permeability. Glutamate-induced decreases in brain endothelial barrier function and phosphorylated VASP were significantly attenuated by pretreatment of human brain endothelia with selective mGluR antagonists. These observations were extended to an in vivo hypoxic mouse model in which pretreatment with mGluR antagonists significantly decreased fluorescein isothiocyanate-dextran flux across the blood-brain barrier. We conclude that activated human PMNs release glutamate and that endothelial expression of group I or III mGluRs function to decrease human brain endothelial VASP phosphorylation and barrier function. These results identify a novel pathway by which PMN-derived glutamate may regulate human endothelial barrier function.