Barrier Properties Of Endothelial Cells Research Articles

Interaction of microbial pathogens with host exocytic pathways.

Many microbial pathogens co-opt or perturb host membrane trafficking pathways. This review covers recent examples in which microbes interact with host exocytosis, the fusion of intracellular vesicles with the plasma membrane. The bacterial pathogens Listeria monocytogenes and Staphylococcus aureus subvert recycling endosomal pathways of exocytosis in order to induce their entry into human cells. By contrast, entry of the protozoan pathogen Trypanosoma cruzi or the virus adenovirus into host cells involves exploitation of lysosomal exocytosis. Toxins produced by Bacillus anthracis or Vibrio cholerae interfere with exocytosis pathways mediated by the GTPase Rab11 and the exocyst complex. By doing so, anthrax or cholera toxins impair recycling of cadherins to cell-cell junctions and disrupt the barrier properties of endothelial cells or intestinal epithelial cells, respectively. Uropathogenic Escherichia coli (UPEC) is expelled from bladder epithelial cells through two different exocytic routes that involve sensing of bacteria in vacuoles by host Toll-like receptor 4 (TLR4) or monitoring of the pH of lysosomes harbouring UPEC. The TLR4 pathway is mediated by multiple Rab GTPases and the exocyst, whereas the other pathway involves exocytosis of lysosomes. Expulsion of UPEC through these pathways is thought to benefit the host.

Growth-factor reduced Matrigel source influences stem cell derived brain microvascular endothelial cell barrier properties.

BackgroundPatient-derived induced pluripotent stem cells (iPSCs) are an innovative source as an in vitro model for neurological diseases. Recent studies have demonstrated the differentiation of brain microvascular endothelial cells (BMECs) from various stem cell sources, including iPSC lines. However, the impact of the culturing conditions used to maintain such stem cell pluripotency on their ability to differentiate into BMECs remains undocumented. In this study, we investigated the effect of different sources of Matrigel and stem cell maintenance medium on BMEC differentiation efficiency.MethodsThe IMR90-c4 iPSC line was maintained on mTeSR1 or in essential-8 (E-8) medium on growth factor-reduced (GFR) Matrigel from three different manufacturers. Cells were differentiated into BMECs following published protocols. The phenotype of BMEC monolayers was assessed by immunocytochemistry. Barrier function was assessed by transendothelial electrical resistance (TEER) and permeability to sodium fluorescein, whereas the presence of drug efflux pumps was assessed by uptake assay using fluorescent substrates.ResultsStem cell maintenance medium had little effect on the yield and barrier phenotype of IMR90-derived BMECs. The source of GFR-Matrigel used for the differentiation process significantly impacted the ability of IMR90-derived BMECs to form tight monolayers, as measured by TEER and fluorescein permeability. However, the Matrigel source had minimal effect on BMEC phenotype and drug efflux pump activity.ConclusionThis study supports the ability to differentiate BMECs from iPSCs grown in mTeSR1 or E-8 medium and also suggests that the origin of GFR-Matrigel has a marked inpact on BMEC barrier properties.

Blood cells and endothelial barrier function

The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction.

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium.

Glucocorticoids and endothelial cell barrier function

Glucocorticoids (GCs) are steroid hormones that have inflammatory and immunosuppressive effects on a wide variety of cells. They are used as therapy for inflammatory disease and as a common agent against edema. The blood brain barrier (BBB), comprising microvascular endothelial cells, serves as a permeability screen between the blood and the brain. As such, it maintains homeostasis of the central nervous system (CNS). In many CNS disorders, BBB integrity is compromised. GC treatment has been demonstrated to improve the tightness of the BBB. The responses and effects of GCs are mediated by the ubiquitous GC receptor (GR). Ligand-bound GR recognizes and binds to the GC response element located within the promoter region of target genes. Transactivation of certain target genes leads to improved barrier properties of endothelial cells. In this review, we deal with the role of GCs in endothelial cell barrier function. First, we describe the mechanisms of GC action at the molecular level. Next, we discuss the regulation of the BBB by GCs, with emphasis on genes targeted by GCs such as occludin, claudins and VE-cadherin. Finally, we present currently available GC therapeutic strategies and their limitations.

Role for A‐kinase anchoring proteins in cigarette smoke‐induced barrier dysfunction

The epithelial barrier is a protective layer against unwanted particles, present in cigarette smoke (CS), a risk factor for developing chronic obstructive pulmonary disease. cAMP, a second messenger, increases barrier properties in endothelial cells, but the role of cAMP in the epithelial barrier is not known. The cAMP effectors, PKA and Epac, may be involved, next to compartmentalization of cAMP signalling by A‐kinase anchoring proteins (AKAPs). Our hypothesis is that AKAPs are involved in CS‐induced effects on the maintenance of the barrier dysfunction. We studied the effect of cigarette smoke extract (CSE) on the barrier function by electrical cell‐substrate impedance sensing (ECIS) and by analyzing the main cell‐cell contact molecule E‐cadherin. CSE‐induced reduction of the functional epithelial barrier is independently of both PKA and Epac. Addition of Ht31 (AKAP‐inhibitor) prevented the effects of CSE on the barrier, strongly suggesting that compartmentalization of cAMP by AKAPs maintains the barrier properties. In addition CSE delocalizes E‐cadherin and AKAP9 from the membrane and reduces the expression of these proteins. Ht31 relocalizes and restores E‐cadherin and AKAP9 levels. Similar results on AKAP9 were obtained in primary epithelial cells from current smokers. In conclusion, AKAPs contribute to CSE‐induced disturbance of the epithelial barrier by translocation of E‐cadherin from the cell membrane. (NAF grant 3.2.09.034)

Contribution of a Single Host Genetic Locus to Mouse Adenovirus Type 1 Infection and Encephalitis

ABSTRACTSusceptibility to mouse adenovirus type 1 (MAV-1) is mouse strain dependent; susceptible mice die from hemorrhagic encephalomyelitis. The MAV-1 susceptibility quantitative trait locus Msq1 accounts for ~40% of the phenotypic (brain viral load) variance that occurs between resistant BALB/c and susceptible SJL mice after MAV-1 infection. Using an interval-specific congenic mouse strain (C.SJL-Msq1SJL), in which the SJL-derived allele Msq1SJL is present in a BALB/c background, we demonstrate that Msq1SJL controls the development of high brain viral titers in response to MAV-1 infection, yet does not account for the total extent of brain pathology or mortality in SJL mice. C.SJL-Msq1SJL mice had disruption of the blood-brain barrier and increased brain water content after MAV-1 infection, but these effects occurred later and were not as severe, respectively, as those noted in infected SJL mice. As expected, BALB/c mice showed minimal pathology in these assays. Infection of SJL- and C.SJL-Msq1SJL-derived primary mouse brain endothelial cells resulted in loss of barrier properties, whereas BALB/c-derived cells retained their barrier properties despite being equally capable of supporting MAV-1 infection. Finally, we provide evidence that organ pathology and inflammatory cell recruitment to the brain following MAV-1 infection were both influenced by Msq1. These results validate Msq1 as an important host factor in MAV-1 infection and refine the major role of the locus in development of MAV-1 encephalitis. They further suggest that additional host factors or gene interactions are involved in the mechanism of pathogenesis in MAV-1-infected SJL mice.

Tissue Plasminogen Activator Expression and Barrier Properties of Human Brain Microvascular Endothelial Cells

Background: Tissue plasminogen activator (tPA) regulates fibrinolysis and is routinely used as ischemic stroke pharmacotherapy. We hypothesized that brain microvascular tPA expression and barrier properties of endothelial cells are substantially related. Methods: Human brain microvascular endothelial cells were incubated with two agents known to modify cAMP pathways: forskolin and rolipram. We analyzed development of endothelial barrier properties, i.e., trans-endothelial electrical resistance (TEER), permeability of endothelial cell monolayer, expression of influx transporter glut-1 and endothelial tight junction molecules occludin and claudin-5, tPA antigen release, and levels of endothelial tPA mRNA. Results: Forskolin plus rolipram-treated endothelial cells showed increased TEER compared to controls (174±20% of control at day six, p<0.01), while permeability to albumin and 70kDa dextran was reduced (21±6.8% of control and 3.8±0.3% of control, respectively, p<0.001). In addition, occludin and claudin-5 protein were up-regulated, occludin mRNA was increased to 206±60% of control (p<0.05), glut-1 mRNA was increased to 196±68% of control (p<0.05), levels of tPA protein were reduced to 35±7.0% of control (p<0.001) after six days, and tPA mRNA was reduced to 32±7.7% of control (p<0.01). TPA and occludin mRNA levels were inversely associated (r=-0.68, p<0.05). Conclusions: In this in vitro model, barrier properties were strongly linked (by inverse association) with tPA expression of brain microvascular endothelial cells.

Bluetongue Virus and Double-Stranded RNA Increase Human Vascular Permeability: Role of p38 MAPK

Endothelial cell (EC) involvement in viral hemorrhagic fevers has been clearly established. However, virally activated mechanisms leading to endothelial activation and dysfunction are not well understood. Several different potential mechanisms such as direct viral infection, alterations in procoagulant/anticoagulant balance, and increased cytokine production have been suggested. We utilized a model of EC barrier dysfunction and vascular endothelial leakage to explore the effect of bluetongue virus (BTV), a hemorrhagic fever virus of ruminants, on human lung endothelial cell barrier properties. Infection of human lung EC with BTV induced a significant and dose-dependent decrease in trans-endothelial electrical resistance (TER). Furthermore, decreases in TER occurred in conjunction with cytoskeletal rearrangement, suggesting a direct mechanism for viral infection-mediated endothelial barrier disruption. Interestingly, double-stranded RNA (dsRNA) mimicked the effects of BTV on endothelial barrier properties. Both BTV- and dsRNA-induced endothelial barrier dysfunction was blocked by treatment with a pharmacological inhibitor of p38 MAPK. The induction of vascular permeability by dsRNA treatment or BTV infection was concomitent with induction of inflammatory cytokines. Taken together, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Collectively, our data suggest that inhibition of p38 MAPK may be a possible therapeutic approach to alter viral-induced acute hemorrhagic diseases.

Blood-brain barrier breakdown in septic encephalopathy and brain tumours.

Septic encephalopathy is associated with breakdown of the blood-brain barrier and cerebral oedema. These features are also common properties of brain tumours. Perimicrovessel oedema, disruption of associated astrocyte end feet and neuronal injury occur in a porcine model of acute septic encephalopathy. The adrenergic system has been implicated in the inflammatory response to sepsis and may play a role in controlling blood-brain barrier permeability, since the beta2-adrenoceptor agonist dopexamine inhibits perimicrovessel oedema formation whereas the alpha1-adrenoceptor agonist methoxamine provokes it. Electron microscopy revealed tight junction opening in high-grade astrocytoma microvessels. Expression of the tight junction protein occludin is reduced in these microvessels and this reduction is inversely correlated with the degree of cerebral oedema. Normal astrocytes secrete factors that induce barrier properties in endothelial cells, whereas high-grade astrocytomas secrete vascular endothelial growth factor, which stimulates angiogenesis, down regulates occludin and increases endothelial cell permeability. The water channel protein aquaporin-4 is normally expressed in astrocyte foot processes around cerebral microvessels. Its expression is massively up-regulated in high-grade astrocytoma and around metastatic adenocarcinoma. There is a significant correlation between aquaporin-4 expression and the degree of cerebral oedema, but it is not clear whether increased aquaporin-4 expression enhances oedema formation or clearance. These results suggest that the pathophysiology of brain oedema is multifactorial, but that there may be common processes operating regardless of the aetiology.

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin.

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.

Sex steroidal regulation of vessel permeability associated with vessel endothelial cadherin (V-cadherin)

In an attempt to understand the roles of cadherins in the placenta, mRNA expression and biological function of cadherins in 3A(tPA-30-1) cells (derived from human term placenta and transformed by SV40), and in HUV-EC-C cells (derived from the endothelial cells in human umbilical cord) were studied under the influence of sex steroids. Estradiol transiently decreased the endothelial cell barrier properties (ECBP) of HUV-EC-C cells, and progesterone reversed the changes induced by estradiol. However, neither estradiol nor progesterone demonstrated any effect on cell aggregation of either 3A(tPA-30-1) or HUV-EC-C cells. Estradiol transiently decreased the level of V-cadherin and its mRNA in HUV-EC-C cells, and progesterone reversed the level decreased by estradiol. However, neither estradiol nor progesterone demonstrated any effect on the level of E-cadherin mRNA in 3A(tPA-30-1) cells. Therefore, a sex steroidal role for placental development and function related to cadherins seems to focus on the endothelial cells, plausibly via vessel permeability for the utilization of placental products.

Astrocyte-conditioned medium induces blood-brain barrier properties in endothelial cells.

1. We evaluated the induction of endothelial barrier functions by the type 1 astrocyte-derived supernatant in culture using horseradish peroxidase (HRP) as a parameter. 2. A gel filtration fraction (molecular weight 16-20 kDa) of type 1 astrocyte-derived supernatant reduced plasmalemmal vesicles and induced tight junctions in endothelial cells. 3. In addition, this fraction promoted the uptake of [3H]-glucosamine by endothelial cells. This cell activity disappeared after heating at 95 degrees C for 10 min and was sensitive to trypsin. 4. These results suggest that a type 1 astrocyte-derived protein factor(s) promotes proteoglycan synthesis with a resultant increase in charge selectivity in endothelial cells and plays an important role in the induction of blood-brain barrier functions.

Maturation of the blood–retina barrier in the developing pecten oculi of the chicken

The major interest in the development of the blood–brain barrier and its underlying induction mechanisms is given by the crucial role they play in the maturation of the central nervous system in general. Whilst it is believed that it is the microenvironment in the brain that destines the endothelial cells to become committed to barrier properties, the analysis of the multitude of factors probably responsible for this commitment is extremely difficult. Therefore, in a previous study, we inaugurated the pecten oculi of the avian eye as a relatively simple in vivo model of the blood–brain barrier [Gerhardt, S. et al., Cell Tissue Res., 285 (1996) 91–100]. In the present study, we demonstrate data on the development of the pecten which allow us to understand better the commitment of barrier properties in endothelial cells in an environment which is considerably less complex than that realized in the brain. The pecten is built up by mainly two cell types, the pigmented glial cells and the endothelial cells. The pigmented cells, which are believed to originate from the retinal pigment epithelium, lose their tight junctions in the microenvironment of the vitreous body, whereas the endothelial cells, which originate from the permeable choroidal vessels, gain tight junctions and other barrier properties in the microenvironment of the vitreous body. On embryonic day 7 (E7), tight and gap junctions between epithelial-like glial cells line the vitreal border of the developing pecten. By E16, these junctions disappear, and the endothelial cells gradually acquire barrier characteristics (continuous and P-face associated tight junctions, no extravasation of lanthanum nitrate, and the exclusive expression of the glucose transporter isoform 1 and the barrier specific antigen HT7 in their luminal and abluminal membranes). The results are discussed considering the switch from an epithelial (glial) to an endothelial barrier.

The blood-brain barrier: cellular basis.

Perfusion experiments with horseradish peroxidase have established that the morphological substrate of the blood-brain barrier is represented by microvascular endothelial cells. They are characterized by complexly arranged tight junctions and a very low rate of transcytotic vesicular transport. They express transport enzymes, carrier systems and brain endothelial cell-specific molecules of unknown function not expressed by any other endothelial cell population. These blood-brain barrier properties are not intrinsic to these cells but are inducible by the surrounding brain tissue. Type I astrocytes injected into the anterior eye chamber of the rat or onto the chick chorioallantoic membrane are able to induce a host-derived angiogenesis and some blood-brain barrier properties in endothelial cells of non-neural origin. Recently we have shown that this cellular interaction is due to the secretion of a soluble astrocyte derived factor(s). Astrocytes are also implicated in the maintenance, functional regulation and the repair of the blood-brain barrier. Complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, pericytes, nerve endings, microglial cells and the extracellular fluid, take place and are required for the proper functioning of the blood-brain barrier, which in addition is regionally different as reflected by endothelial cell heterogeneity.

Induction of the blood-brain barrier specific HT7 and neurothelin epitopes in endothelial cells of the chick chorioallantoic vessels by a soluble factor derived from astrocytes

Some blood-brain barrier properties of microvascular endothelial cells have been shown to be inducible by astrocytes. We tested the hypothesis that this cellular interaction is mediated by a soluble factor(s). Chick chorioallantoic vessels in ovo were constantly exposed to astrocyte-conditioned medium. We found that endothelial cells exposed to astrocyte-derived factors, but not to glioma- or endothelial cell-derived factors, expressed the HT7 antigen and neurothelin, two specific markers of the blood-brain barrier phenotype. These results indicate that a soluble factor(s) secreted by astrocytes is capable to induce specific blood-brain barrier properties in endothelial cells of non-neural origin.

Effect of platelet activating factor on leukocyte-endothelial cell interactions

The proinflammatory effects of platelet activating factor (PAF) that impact upon tissue inflammation were studied in vitro using the adherence of human neutrophils to endothelium and the increase in macromolecule permeability of endothelial monolayers. PAF produced both a time- and dose-dependent increase in neutrophil -endothelial cell adhesion. The adhesion promoting properties observed were primarily due to an effect of PAF on endothelium and not on neutrophils and was independent of endothelial cell cyclooxygenase products. The PAF receptor antagonist kadsurenone inhibited the adhesion response suggesting endothelial surface PAF receptors are involved in these responses. Whereas PAF alone did not alter endothelial cell barrier properties,leukocyte activation (neutrophil an platelets) with PAF produced significant increases in 125I-albumin clearance across endothelial monolayers. These studies suggest that PAF has potent proinflammatory effects and that it can play a significant role in the endothelial response to injury.

Astrocytes induce blood-brain barrier properties in endothelial cells.

The highly impermeable tight junctions between endothelial cells forming the capillaries and venules in the central nervous system (CNS) of higher vertebrates are thought to be responsible for the blood-brain barrier that impedes the passive diffusion of solutes from the blood into the extracellular space of the CNS. The ability of CNS endothelial cells to form a blood-brain barrier is not intrinsic to these cells but instead is induced by the CNS environment: Stewart and Wiley demonstrated that when avascular tissue from 3-day-old quail brain is transplanted into the coelomic cavity of chick embryos, the chick endothelial cells that vascularize the quail brain grafts form a competent blood-brain barrier; on the other hand, when avascular embryonic quail coelomic grafts are transplanted into embryonic chick brain, the chick endothelial cells that invade the mesenchymal tissue grafts form leaky capillaries and venules. It is, however, not known which cells in the CNS are responsible for inducing endothelial cells to form the tight junctions characteristic of the blood-brain barrier. Astrocytes are the most likely candidates since their processes form endfeet that collectively surround CNS microvessels. In this report we provide direct evidence that astrocytes are capable of inducing blood-brain barrier properties in non-neural endothelial cells in vivo.