Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function

The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but it also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells that line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerated de novo assembly of endothelial cell-cell junctions and increased the barrier function of endothelial monolayers. In contrast, depressing Rap1 activity by expressing Rap1GAP led to disassembly of these junctions and increased their permeability. We also demonstrate that endogenous Rap1 was rapidly activated at early stages of junctional assembly, confirming the involvement of Rap1 during junctional assembly. Intriguingly, elevating Rap1 activity selectively within endothelial cells decreased leukocyte transendothelial migration, whereas inhibiting Rap1 activity by expression of Rap1GAP increased leukocyte transendothelial migration, providing physiological relevance to our hypothesis that Rap1 augments barrier function of inter-endothelial cell junctions. Furthermore, these results suggest that Rap1 may be a novel therapeutic target for clinical conditions in which an inappropriate inflammatory response leads to disease.     

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.     

Mechanisms regulating endothelial cell barrier function

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.     

Pertussis toxin transiently affects barrier integrity, organelle organization and transmigration of monocytes in a human brain microvascular endothelial cell barrier model

Encephalopathies and neurological disorders are sometimes associated with respiratory tract infections caused by Bordetella pertussis. For these complications to occur cerebral barriers have to be compromised. Therefore, the influence of pertussis toxin (PT), a decisive virulence determinant of B. pertussis, on endothelial barrier integrity was investigated. Human brain microvascular endothelial cells cultured on Transwell filter devices were used as model for the blood brain barrier. PT, but not its B-oligomer, induced a reduction of the transendothelial resistance and enhanced the permeability for the protein marker horseradish peroxidase. Moreover, transmigration of human monocytes was also elevated suggesting a PT-associated enhancement of the diapedesis of blood leucocytes. Uptake and trafficking of PT was followed by electron microscopy via clathrin-coated pits and accumulation in lysosomes and microvesicular bodies. The breach in barrier integrity was accompanied by a transient disintegration of Golgi structures. Interestingly, PT-induced effects were only transient and restoration of barrier function was observed after 24 h. In summary, intoxication by PT causes a transient destruction of the cellular organization in human brain-derived endothelial cells resulting in a transient disruption of barrier functions. We suggest that these findings reflect early steps in the development of neurological disorders associated with pertussis disease.     

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01–0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis. © 1995 Wiley-Liss, Inc.     

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.     

Initial steps of metastasis: cell invasion and endothelial transmigration

Metastasis is the leading cause of cancer mortality. The metastatic cascade represents a multi-step process which includes local tumor cell invasion, entry into the vasculature followed by the exit of carcinoma cells from the circulation and colonization at the distal sites. At the earliest stage of successful cancer cell dissemination, the primary cancer adapts the secondary site of tumor colonization involving the tumor-stroma crosstalk. The migration and plasticity of cancer cells as well as the surrounding environment such as stromal and endothelial cells are mandatory. Consequently, the mechanisms of cell movement are of utmost relevance for targeted intervention of which three different types have been reported. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of movement and intravasate the blood or lymph vasculature. Intravasation by the interaction of tumor cells with the vascular endothelium is mechanistically poorly understood. Changes in the epithelial plasticity enable carcinoma cells to switch between these types of motility. The types of migration may change depending on the intervention thereby increasing the velocity and aggressiveness of invading cancer cells. Interference with collective or mesenchymal cell invasion by targeting integrin expression or metalloproteinase activity, respectively, resulted in an amoeboid cell phenotype as the ultimate exit strategy of cancer cells. There are little mechanistic details reported in vivo showing that the amoeboid behavior can be either reversed or efficiently inhibited. Future concepts of metastasis intervention must simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate. Beyond the targeting of cell movements, the adhesion of cancer cells to the stroma in heterotypic circulating tumor cell emboli is of paramount relevance for anti-metastatic therapy.     

Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function

Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP(1,2) has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP(1,2) were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and delta-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-alpha), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not delta-peptide, induced a recovery of endothelial barrier function following treatment with TNF-alpha. Our results demonstrate that Ebola virus GP(1,2) in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.     

Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function

A spectrum of cholesterol oxidation derivatives (oxysterols) is generated in food products exposed to heat or radiation in the presence of oxygen. One of these derivatives (cholestan-3 beta,5 alpha,6 beta-triol) was shown to compromise the selective barrier function of cultured vascular endothelial cell monolayers, an action that may initiate atherosclerotic lesion formation. This study sought to investigate the relationship of cholesterol synthesis inhibition by several naturally occurring oxysterols to depression of vascular endothelial cell monolayer barrier function, determined as an increase in albumin transfer across cultured endothelial monolayers. All oxysterols tested caused a variable time- and dose-dependent elevation in trans-endothelial albumin transfer, and they were also able to inhibit cholesterol biosynthesis to varying degrees. Pure cholesterol was without effect on both counts. The correlation between the increase in albumin transfer related to oxysterol exposure and the ability of oxysterols to suppress cholesterol biosynthesis was, however, poor. Moreover, mevinolin, a water-soluble competitive inhibitor of cholesterol synthesis, reduced the rate of cholesterol synthesis to 0.9% of control but did not significantly increase albumin transfer. Cholestan-3 beta,5 alpha,6 beta-triol caused a 660% elevation in albumin transfer while cholesterol synthesis remained at 11% of control. We conclude that changes in endothelial barrier function caused by exposure to the oxysterols examined, but not pure cholesterol, are probably related to factors other than the well-known action of cholesterol biosynthesis inhibition. These findings may have implications in the development of atherosclerosis.     

Homocysteine in microvascular endothelial cell barrier permeability

Redox stress activates the endothelium and upregulates matrix metalloproteinases (MMPs), which degrade the matrix and lead to blood-endothelial barrier leakage. Interestingly, elevated levels of plasma homocysteine (Hcy) are associated with vascular dementia, seizure, stroke, and Alzheimer disease. Hcy competes with the γ-aminobutyric acid (GABA)-A/B receptors and behave like an excitatory neurotransmitter. GABA stimulates the inhibitory neurotransmitter GABA-A/B receptor and decreases arterial blood pressure. However, the neural mechanisms of microvascular remodeling in hyperhomocysteinemia are unclear. This review addresses the idea that Hcy induces microvascular permeability by attenuating the GABA-A/B receptors and increasing redox stress, which activates a disintegrin and metalloproteinase that suppresses tissue inhibitors of metalloproteinase. This process causes disruption of the matrix in the blood-brain barrier. Understanding the mechanism of Hcy-mediated changes in permeability of the blood-brain barrier and extracellular matrix that can alter the neuronal environment in cerebral-vascular dementia is of great importance in developing treatments for this disease.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

Serotonin,norepinephrine, and histamine mediation of endothelial cell barrier function in vitro

The effects of serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE), and histamine on endothelial cell barrier function were examined in vitro. Bovine aortic endothelial (BAE) cells grown to confluence on microcarriers formed a measurable barrier to the passage of a trypan blue dye-bovine serum albumin conjugate (TB-BSA) from the culture medium into the microcarrier matrix. Vascular smooth muscle (VSM) cells or Swiss 3T3 fibroblasts impeded TB-BSA diffusion only 42% and 56%, respectively, relative to BAE cells. These results suggest that barrier formation may be an endothelial cell-specific phenomenon. Treatment of BAE cells with histamine was associated with 2-to 3-fold increases in the rate of TB-BSA diffusion. In contrast, treatment with 5-HT or NE at concentrations ranging from normal to pathophysiological circulating plasma levels significantly impeded TB-BSA diffusion by up to 43% and 33%, respectively, relative to untreated controls. The barrier-modulating effects of the vasoactive amines were dose-dependent, cell-specific, and in some cases appear to be receptor-mediated. These results are consistent with previous reports that histamine increases vascular permeability in part by affecting diffusion between endothelial cells; they support the hypothesis that 5-HT and NE contribute to the maintenance of the endothelial barrier in vivo.     

Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca(2+)-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca(2+)- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.     

Instrumental noise estimates stabilize and quantify endothelial cell micro-impedance barrier function parameter estimates

A novel transcellular micro-impedance biosensor, referred to as the electric cell-substrate impedance sensor or ECIS, has become increasingly applied to the study and quantification of endothelial cell physiology. In principle, frequency dependent impedance measurements obtained from this sensor can be used to estimate the cell–cell and cell–matrix impedance components of endothelial cell barrier function based on simple geometric models. Few studies, however, have examined the numerical optimization of these barrier function parameters and established their error bounds. This study, therefore, illustrates the implementation of a multi-response Levenberg–Marquardt algorithm that includes instrumental noise estimates and applies it to frequency dependent porcine pulmonary artery endothelial cell impedance measurements. The stability of cell–cell, cell–matrix and membrane impedance parameter estimates based on this approach is carefully examined, and several forms of parameter instability and refinement illustrated. Including frequency dependent noise variance estimates in the numerical optimization reduced the parameter value dependence on the frequency range of measured impedances. The increased stability provided by a multi-response non-linear fit over one-dimensional algorithms indicated that both real and imaginary data should be used in the parameter optimization. Error estimates based on single fits and Monte Carlo simulations showed that the model barrier parameters were often highly correlated with each other. Independently resolving the different parameters can, therefore, present a challenge to the experimentalist and demand the use of non-linear multivariate statistical methods when comparing different sets of parameters.     

Impact of extracellular RNA on endothelial barrier function

Different types of high and low molecular weight extracellular RNA (eRNA) are liberated from cells upon conditions of tissue damage or vascular diseases and have been demonstrated in vivo and in vitro to influence the integrity and barrier function of the vascular endothelium. Among the types of self eRNA studied in this respect, ribosomal RNA appears to engage cytokines to promote hyperpermeability, while counteracting RNase1 serves as a potent vessel-protective factor. Different microRNAs may change the expression program of endothelial cells with consequences for cellular contacts and stability. Non-self viral RNAs are recognized by Toll-like receptors that transmit intracellular inflammation signals to disturb the vascular barrier function, largely in connection with infectious diseases. Although derived from the same nucleotide building blocks, the various forms of eRNA exhibit a multitude of molecular interactions with the endothelium that may drastically change its phenotypical characteristics. The impact of eRNA on vascular integrity in health and disease is summarized in this concise review.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

Role of microtubule cytoskeleton in regulation of endothelial barrier function

Cytoplasmic microtubules are an obligatory component of the cytoskeleton of all types of cells. Microtubules are involved in many cellular processes including directed transport of vesicles and signaling molecules and changes in cell shape during its spreading, polarization, and movement. The intracellular organization of the system of microtubules and their dynamic properties are different in different types of cells because they play a key role in the implementation of a variety of cell and tissue functions, including the regulation of the endothelial barrier function. This review presents an overview of current studies on the properties of endothelial microtubules, their interaction with other components of the cytoskeleton and cell adhesion structures, and the role of microtubules in the regulation of the endothelial barrier function.     

Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.