Transport across the endothelium: regulation of endothelial permeability.

An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways controlling endothelial barrier function have been identified. The transcellular pathway transports plasma proteins of the size of albumin or greater via the process of transcytosis in vesicle carriers originating from cell surface caveolae. Specific signalling cues are able to induce the internalisation of caveolae and their movement to the basal side of the endothelium. Caveolin-1, the primary structural protein required for the formation of caveolae, is also important in regulating vesicle trafficking through the cell by controlling the activity and localisation of signalling molecules that mediate vesicle fission, endocytosis, fusion and finally exocytosis. An important function of the transcytotic pathways is to regulate the delivery of albumin and immunoglobulins, thereby controlling tissue oncotic pressure and host-defence. The paracellular pathway induced during inflammation is formed by gaps between endothelial cells at the level of adherens and tight junctional complexes. Paracellular permeability is increased by second messenger signalling pathways involving Ca2+ influx via activation of store-operated channels, protein kinase Calpha (PKCalpha), and Rho kinase that together participate in the stimulation of myosin light chain phosphorylation, actin-myosin contraction, and disruption of the junctions. In this review of the field, we discuss the current understanding of the signalling pathways regulating paracellular and transcellular endothelial permeability.

Albumin uptake and transcytosis in endothelial cells in vivo induced by albumin-binding protein.

The 60-kDa endothelial cell surface albumin-binding glycoprotein (gp60) is postulated to be a docking site for albumin that mediates the uptake of albumin and its transport in cultured microvessel endothelial cells. In the present study, we used an isolated Krebs-perfused rat lung preparation to address the in vivo role of gp60 in mediating albumin uptake and transport. Addition of primary anti-gp60 antibody to the perfusate followed by the secondary antibody to cross-link gp60 increased the vessel wall (125)I-albumin permeability-surface area (PS) product 2.5-fold without affecting the capillary filtration coefficient (K(f,c;) a measure of liquid permeability). In contrast, EDTA (5 mM), which induces interendothelial gap formation, produced parallel increases in both K(f,c) and (125)I-albumin PS product. Increasing perfusate albumin concentration to >1 g/100 ml (EC(50) 1.2 g/100 ml) was sufficient to block (125)I-albumin PS product, indicating that the perfusate albumin competed with tracer albumin for transendothelial albumin transport. Cross-linking of gp60 in lungs perfused with saturating concentration of albumin resulted in a greater increase in (125)I-albumin PS product, indicating that gp60 function was capable of being modulated. These results show that activation of gp60 in pulmonary microvessels induces albumin uptake and its transport through a non-hydraulic pathway that fits with a model of albumin permeability via the transcellular pathway.

Reversible temperature-sensitive alterations in lung fluid balance.

Hypothermia is intentionally imposed during the harvesting of lungs for transplantation. The aim of this study was to investigate the fluid balance alterations in rat lung preparations exposed to hypothermic perfusion. Lowering perfusate temperature from 37 degrees C to values between 27 and 7 degrees C caused an immediate, marked pulmonary hypertension and vasoconstriction accompanied by rapid development of pulmonary edema (+1.15 g, or approximately 90%, gain in lung weight within 5 min). However, on rewarming, vasoconstriction was immediately reversed. Edema was resolved, but along a two-component time course: an immediate reduction of lung weight on rewarming (t 1/2 of 0.5 min) that mirrored the recovery of pulmonary artery pressure and vasoconstriction, and also a slower pressure-independent component of recovery (t 1/2 of 3.5 min). Ouabain (300 microM) markedly inhibited the lung's ability to recover from edema, indicating that fluid clearance from lung tissue was the result of activation of ouabain-sensitive (Na+,K+)-ATPase pump. Results could not be explained by vascular or airspace injury as lung sections from hypothermic lungs appeared normal. The findings indicate that hypothermia induces pulmonary edema formation, which can be rapidly cleared upon rewarming by activation of ouabain-sensitive (Na+,K+)-ATPase pump. Thus, impaired fluid clearance from lung extravascular spaces may be a critical factor limiting gas exchange in transplanted lungs exposed to hypothermia.

Transcriptional mechanisms of acute lung injury.

Acute lung injury occurs as a result of a cascade of cellular events initiated by either infectious or noninfectious inflammatory stimuli. An elevated level of proinflammatory mediators combined with a decreased expression of anti-inflammatory molecules is a critical component of lung inflammation. Expression of proinflammatory genes is regulated by transcriptional mechanisms. Nuclear factor-kappa B (NF-kappa B) is one critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of acute lung injury. Activation and regulation of NF-kappa B are tightly controlled by a complicated signaling cascade. In acute lung injury caused by infection of bacteria, Toll-like receptors play a central role in initiating the innate immune system and activating NF-kappa B. Anti-inflammatory cytokines such as interleukin-10 and interleukin-13 have been shown to suppress inflammatory processes through inhibiting NF-kappa B activation. NF-kappa B can interact with other transcription factors, and these interactions thereby lead to greater transcriptional selectivity. Modification of transcription is likely to be a logical therapeutic target for acute lung injury.

Synergistic effects of tumor necrosis factor-alpha and thrombin in increasing endothelial permeability.

Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-alpha, we determined the effects of TNF-alpha on the mechanism of thrombin-induced increase in endothelial permeability. We assessed Ca(2+) signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-alpha for 2 h, thrombin produced a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca(2+)](i) lasting for 3 min, whereas TNF-alpha alone had no effect on [Ca(2+)](i.) Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-alpha-exposed cells. In the absence of extracellular Ca(2+), thrombin produced similar increases in [Ca(2+)](i) in both control and TNF-alpha-exposed cells. In TNF-alpha-exposed cells, the thrombin-induced Ca(2+) influx after intracellular Ca(2+) store depletion was significantly greater and prolonged compared with control cells. Increased Ca(2+) entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PP1. After TNF-alpha exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-alpha stimulation of endothelial cells results in amplification of the thrombin-induced Ca(2+) influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.

Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice.

The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood. We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1. PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage. Lung tissue PMN number increased by 6-fold within 1 h after i.p. Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control). PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h. Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times. In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E. coli challenge. Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration. The increased pulmonary microvascular permeability induced by E. coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.

Protein kinase C-delta regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase.

The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that protein kinase C-delta (PKC-delta) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-delta and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-delta inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-delta. Inhibition of PKC-delta or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-kappaB activity and resultant ICAM-1 expression. The effects of PKC-delta inhibition were secondary to inhibition of IKKbeta activation and of subsequent NF-kappaB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IkappaBalpha degradation without affecting the DNA binding function of nuclear NF-kappaB. Thus, PKC-delta signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKbeta, which mediates NF-kappaB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-kappaB p65 (RelA).

Ca(2+) signalling and PKCalpha activate increased endothelial permeability by disassembly of VE-cadherin junctions.

1. The role of intracellular Ca(2+) mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) in both cases was due to release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+). 2. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial (125)I-albumin permeability. Thapsigargin induced activation of PKCalpha and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCalpha activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC(20)), whereas thrombin induced MLC(20) phosphorylation consistent with formation of actin stress fibres. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca(2+)](i) elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca(2+) signalling and activation of PKCalpha in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Evidence for the role of alveolar epithelial gp60 in active transalveolar albumin transport in the rat lung.

1. Transcytosis of albumin, involving the 60 kDa albumin-binding glycoprotein, gp60, was studied in cultured type II alveolar epithelial cells obtained from rat lungs. 2. Type II cells internalized the interfacial fluorescent dye RH 414, which marks for plasmalemma vesicles. Fluorescent forms of albumin and anti-gp60 antibody colocalized in the same plasmalemma vesicles. 3. Antibody (100 microg ml(-1)) cross-linking of gp60 for brief periods (15 min) markedly stimulated vesicular uptake of fluorescently tagged albumin. The caveolar disrupting agent, filipin (10 nM), abolished the stimulated internalization of albumin. 4. The vast majority of plasmalemmal vesicles carrying albumin also immunostained for caveolin-1; however, lysosomes did not stain for caveolin-1. Filipin depleted the epithelial cells of the caveolin-1-positive, albumin-transporting plasmalemma vesicles. 5. Prolonged (> 1 h) stimulation of type II cells with cross-linking anti-gp60 antibody produced loss of cell-surface gp60 and abolished endocytic albumin uptake. 6. Transalveolar transport of albumin was also studied in the isogravimetric rat lung preparation perfused at 37 degrees C. (125)I-labelled albumin was instilled into distal airspaces of lungs, and the resulting (125)I-labelled albumin efflux into the vascular perfusate was determined. 7. Unlabelled albumin (studied over a range of 0-10 g (100 instilled ml)(-1)) inhibited 40 % of the transport of labelled albumin ((5.7 +/- 0.4) x 10(5) counts (instilled ml)(-1)) with an IC(50) value of 0.34 g (100 ml)(-1). 8. Filipin blocked the displacement-sensitive component of (125)I-labelled albumin transport, but had no effect on the transport of the paracellular tracer (3)[H]mannitol. 9. Displacement-sensitive (125)I-labelled albumin transport had a significantly greater Q(10) (27-37 degrees C) than the non-displaceable component. 10. Cross-linking of gp60 by antibody instillation stimulated only the displacement-sensitive (125)I-labelled albumin transalveolar transport in intact rat lungs. 11. To estimate the transport capacity of the displacement-sensitive system, the percentage of instilled (125)I-labelled albumin counts remaining in lung tissue was compared in lungs treated with instillates containing either 0.05 g (100 ml)(-1) unlabelled albumin or 5 g (100 ml)(-1) unlabelled albumin. Approximately 25 % of instilled (125)I-labelled albumin was cleared from the lung preparations per hour by the displacement-sensitive transport pathway. This component was blocked by filipin.

G alpha 16 couples chemoattractant receptors to NF-kappa B activation.

The guanine nucleotide-binding regulatory protein alpha-subunit, Galpha(16), is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from Galpha(16) coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a kappaB-driven luciferase reporter, Galpha(16), and the formyl peptide receptor responded to fMLP with a approximately 7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of IkappaBalpha and elevation of nuclear kappaB-DNA binding activity, indicating activation of NF-kappaB. In contrast to Galpha(16), expression of Galpha(q), Galpha(13), and Galpha(i2) resulted in a marginal increase in kappaB luciferase activity. A GTPase-deficient, constitutively active Galpha(16) mutant (Q212L) could replace agonist stimulation for activation of NF-kappaB. Furthermore, expression of Galpha(16) (Q212L) markedly enhanced TNF-alpha-induced kappaB reporter activity. The Galpha(16)-mediated NF-kappaB activation was paralleled by an increase in phospholipase C-beta activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca(2+). The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKCalpha enhanced the effect of Galpha(16), and a dominant negative PKCalpha partially blocked Galpha(16)-mediated NF-kappaB activation. In addition to formyl peptide receptor, Galpha(16) also enhanced NF-kappaB activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of Galpha(16) in transcriptional regulation downstream of chemoattractant receptors.

Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function.

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-alpha with Rho. Activated PKC-alpha but not PKC-epsilon induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.

Decreased oxidant buffering impairs NF-kappaB activation and ICAM-1 transcription in endothelial cells.

The DNA binding activity of the transcription factor, NF-kappaB, is regulated by the phosphorylation and degradation of its inhibitory protein, IkappaB, and post-translational modification involving redox reaction of a cysteine residue (Cys62) of NF-kappaB. We addressed the role of the redox state of endothelial cells in modulating TNFalpha-induced NF-kappaB activity. The effects of TNFalpha on DNA-binding activity of NF-kappaB and expression of mRNA encoding ICAM-1 (an NF-kappaB-activated gene) were studied in human pulmonary artery endothelial (HPAE) cells under basal conditions and after decreasing the intracellular glutathione (GSH) concentration. HPAE cells were treated with buthionine sulfoximine (BSO) (16 h), an inhibitor of GSH synthesis, which caused concentration-dependent decreases in GSH concentration. Stimulation of control cells with TNFalpha resulted in reactive oxygen species (ROS) generation and activation of NF-kappaB binding to the ICAM-1 promoter and ICAM-1 transcription. However, stimulation of GSH-depleted cells with TNFalpha resulted in ROS accumulation secondary to the decreased ROS buffering capacity, and marked impairment of NF-kappaB-binding activity and ICAM-1 mRNA expression. Exposure of BSO-treated cells to the reducing agent dithiothreitol (DTT) before TNFalpha treatment or supplementation of nuclear extract (isolated after TNFalpha challenge of BSO-treated cells) with DTT significantly augmented the effect of TNFalpha on NF-kappaB-binding activity and ICAM-1 mRNA expression. Thus the oxidative modification of NF-kappaB secondary to the loss of ROS buffering capacity may regulate NF-kappaB binding to ICAM-1 promoter, and thereby ICAM-1 transcription in endothelial cells.

Requirement for Ca2+ signaling in the mechanism of thrombin-induced increase in endothelial permeability.

We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40-50% decrease in transendothelial monolayer electrical resistance and a twofold increase in 125I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular Ca2+ concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. La3+ reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in 125I-albumin permeability in HUVECs. Because the absence of Ca2+ signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular Ca2+ concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased 125I-albumin permeability. These results indicate that Ca2+ influx and activation of Ca2+-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability.

Evidence of transcellular permeability pathway in microvessels.

We used video fluorescence microscopy of the vascular bed in the cremaster muscle of rat and mouse to study the transfer of plasmalemma vesicles (caveolae) across the microvessel barrier in situ. The water-soluble styryl pyridinium dye RH414, which adsorbs to and fluoresces at the membrane-water interface, was used as a marker for vesicular traffic through endothelial cells. Fluorescein isothiocyanate (FITC), similar in molecular size to the styryl pyridinium probe, was used to mark for dye transfer by the paracellular pathway. Transcellular dye flux was determined by comparing the fluorescence intensities of RH414 and FITC on either side of the vessel wall (i.e., in microvessel lumen and in muscle tissue at various distances from the microvessel wall). We observed that RH414 accumulated in the interstitium more rapidly than FITC. We next studied the role of the 60-kDa albumin-binding glycoprotein gp60, hypothesized to activate transcellular permeability, in stimulating the transcellular vesicle traffic. Introduction of anti-gp60 antibody into the microvessel to cross-link and activate gp60 markedly increased the transvascular flux of RH414. Control isotype-matched antibody had no effect on the RH414 flux. The sterol-binding agent filipin, which disassembles caveolae, inhibited the RH414 flux induced by gp60 cross-linking. The transfer of styryl pyridinium dyes in intact microvessels suggests that plasmalemmal membrane traffic across the skeletal muscle microvessel barrier is a constitutively active process. The results indicate that the gp60-dependent pathway is important in regulating endothelial permeability in situ via a transcellular mechanism.

Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice.

We investigated the function of proteinase-activated receptor-1 (PAR-1) in the regulation of pulmonary microvascular permeability in response to thrombin challenge using PAR-1 knockout mice (-/-). Lungs were isolated and perfused with albumin (5 g/100 ml)-Krebs solution at constant flow (2 ml/min). Lung wet weight and pulmonary artery pressure (P(pa)) were continuously monitored. We determined the capillary filtration coefficient (K(fc)) and (125)I-labeled albumin (BSA) permeability-surface area product (PS) to assess changes in pulmonary microvessel permeability to liquid and albumin, respectively. Normal and PAR-1-null lung preparations received in the perfusate: 1) thrombin or 2) selective PAR-1 agonist peptide (TFLLRNPNDK-NH(2)). In control PAR-1 (+/+) mouse lungs, (125)I-albumin PS and K(fc) were significantly increased over baseline (by approximately 7- and 1.5-fold, respectively) within 20 min of alpha-thrombin (100 nM) challenge. PAR-1 agonist peptide (5 microM) gave similar results, whereas control peptide (5 microM; FTLLRNPNDK-NH(2)) was ineffective. At relatively high concentrations, thrombin (500 nM) or PAR-1 agonist peptide (10 microM) also induced increases in P(pa) and lung wet weight. All effects of thrombin (100 or 500 nM) or PAR-1 agonist peptide (5 or 10 microM) were prevented in PAR-1-null lung preparations. Baseline measures of microvessel permeability and P(pa) in the PAR-1-null preparations were indistinguishable from those in normal lungs. Moreover, PAR-1-null preparations gave normal vasoconstrictor response to thromboxane analog, U-46619 (100 nM). The results indicate that the PAR-1 receptor is critical in mediating the permeability-increasing and vasoconstrictor effects of thrombin in pulmonary microvessels.

Reversibility of increased microvessel permeability in response to VE-cadherin disassembly.

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.

Protein kinase C-zeta mediates TNF-alpha-induced ICAM-1 gene transcription in endothelial cells.

We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-alpha (TNF-alpha)-induced oxidant generation in endothelial cells, a requirement for nuclear factor-kappaB (NF-kappaB) activation and intercellular adhesion molecule-1 (ICAM-1) gene transcription. Depletion of the conventional (c) and novel (n) PKC isozymes following 24 h exposure of human pulmonary artery endothelial (HPAE) cells with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM), failed to prevent TNF-alpha-induced oxidant generation. In contrast, inhibition of PKC-zeta synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-alpha stimulation. We observed that PKC-zeta also induced the TNF-alpha-induced NF-kappaB binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We showed that expression of the dominant negative mutant of PKC-zeta prevented the TNF-alpha-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-zeta augmented the response. These data imply a critical role for the PKC-zeta isozyme in regulating TNF-alpha-induced oxidant generation and in signaling the activation of NF-kappaB and ICAM-1 transcription in endothelial cells.

Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway.

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein G(i), and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial (125)I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11-amino acid G(alphai) carboxyl-terminal peptide inhibited endothelial (125)I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of G(alphai) with the caveolin-1, whereas dn-Src inhibited G(alphai) binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream G(i)-coupled Src kinase signaling pathway.

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.

Activation of NF-kappaB induced by H(2)O(2) and TNF-alpha and its effects on ICAM-1 expression in endothelial cells.

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappaB in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H(2)O(2) and TNF-alpha in mediating activation of NF-kappaB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H(2)O(2) added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H(2)O(2) also failed to induce NF-kappaB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappaB-driven gene expression approximately 10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H(2)O(2)- and TNF-alpha-induced NF-kappaB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappaB p65 in HMEC-1 cells, whereas H(2)O(2) had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappaB to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappaB p65 and confer transcriptional competency to NF-kappaB.