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.

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.

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.

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.

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.

beta(2)-Integrin blockade driven by E-selectin promoter prevents neutrophil sequestration and lung injury in mice.

Interaction of CD11/CD18 beta(2) integrins on polymorphonuclear leukocytes (PMNs) with their counterreceptor, intercellular adhesion molecule-1, on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and migration across the pulmonary vascular endothelial barrier. Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated from the canine hookworm (Ancylostoma caninum), binds to the I domain of CD11a and CD11b and inhibits beta(2) integrin-dependent PMN adhesion. We describe a novel strategy using the endothelial cell-specific E-selectin promoter to induce NIF expression in an inflammation-specific manner in pulmonary vascular endothelial cells. A construct containing NIF cDNA driven by the inducible endothelial cell-specific E-selectin promoter (pESNIF) was transfected into human pulmonary artery endothelial cells (HPAECs). Lipopolysaccharide challenge (known to activate E-selectin) resulted in NIF mRNA and protein expression in transfected HPAECs. NIF expression induced by the E-selectin promoter prevented PMN adhesion to the activated HPAECs, whereas PMNs adhered avidly to activated HPAECs in the absence of NIF expression. To address the utility of this approach in conditionally preventing in vivo PMN sequestration, we injected mice intravenously with cationic liposomes containing the pESNIF construct. Analysis of lung tissue showed that intraperitoneal challenge of Escherichia coli resulted in NIF expression. Inflammation-specific NIF expression induced by the E-selectin promoter prevented lung PMN sequestration and vascular injury induced by E coli challenge. These studies suggest the feasibility of conditionally blocking beta(2) integrin function at sites where the endothelium is activated and thereby of locally preventing PMN activation and migration responses that lead to tissue inflammation.

G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells.

We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca(2+)](i). GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca(2+)](i) in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.

Regulator of G protein signaling RGS3T is localized to the nucleus and induces apoptosis.

RGS3 belongs to a family of the regulators of G protein signaling (RGS). We previously demonstrated that cytosolic RGS3 translocates to the membrane to inhibit G(q/11) signaling (Dulin, N. O., Sorokin, A., Reed, E., Elliott, S., Kehrl, J., and Dunn, M. J. (1999) Mol. Cell. Biol. 19, 714-723). This study examines the properties of a recently identified truncated variant termed RGS3T. Both RGS3 and RGS3T bound to endogenous Galpha(q/11) and inhibited endothelin-1-stimulated calcium mobilization and mitogen-activated protein kinase activity to a similar extent. However, unlike cytosolically localized RGS3, RGS3T was found predominantly in the nucleus and partially in the plasma membrane. Furthermore, RGS3T, but not RGS3, caused cell rounding and membrane blebbing. Finally, 44% of RGS3T-transfected cells underwent apoptosis after serum withdrawal, which was significantly higher than that of RGS3-transfected cells (7%). Peptide sequence analysis revealed two potential nuclear localization signal (NLS) sequences in RGS3T. Further truncation of the RGS3T N terminus containing putative NLSs resulted in a significant reduction of nuclear versus cytoplasmic staining of the protein. Moreover, this truncated RGS3T no longer induced apoptosis. In summary, RGS3 and its truncated variant RGS3T are similar in their ability to inhibit G(q/11) signaling but are different in their intracellular distribution. These data suggest that, in addition to being a GTPase-activating protein, RGS3T has other distinct functions in the nucleus of the cell.

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.

Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression.

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.

In vivo expression of neutrophil inhibitory factor via gene transfer prevents lipopolysaccharide-induced lung neutrophil infiltration and injury by a beta2 integrin-dependent mechanism.

The binding of beta2 (CD18) integrins on PMN cell membrane to intercellular adhesion molecule (ICAM) counter-receptors on the surface of vascular endothelial cells mediates PMN adhesion to endothelial cells. Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm (Ancylostoma caninum), is a beta2 integrin antagonist that inhibits PMN adhesion to endothelial cells. We transferred the NIF gene into CD1 mouse lungs by intravenous injection of cationic liposomes to study the effects of in vivo NIF expression on LPS-induced lung PMN sequestration and the development of lung injury. RT-PCR and Northern blot analysis indicated the lung-selective expression of the NIF transgene, and immunocytochemistry showed prominent NIF expression in pulmonary microvessel endothelial cells. NIF staining was also observed in intraluminal leukocytes present in pulmonary microvessels. This may be the result of NIF binding to leukocytes after its secretion from the transduced lung cells, since there was no evidence of NIF gene expression in circulating leukocytes. Pulmonary vascular NIF expression abrogated the lung tissue PMN uptake and airspace migration of PMN and prevented lung vascular injury (as measured by the lung tissue uptake of [125I]labeled albumin) after the intraperitoneal LPS challenge (200 microg/mouse). Expression of a control protein, chloramphenicol acetyltransferase (CAT), by the same strategy, had no effect on these responses. In vitro studies showed that NIF prevented mouse PMN adhesion consistent with the inhibition of lung uptake after LPS challenge in NIF transgene-expressing mice. We conclude that pulmonary vascular expression of NIF, a specific beta2 integrin- binding protein, is a potentially useful gene transfer strategy in modulating the infiltration of PMN across the alveolar-capillary epithelial barrier and in preventing lung vascular endothelial injury.

Protein kinase C beta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells.

We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in human dermal microvascular endothelial cells (HMEC) exposed to alpha-thrombin or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC beta isozymes (PKC beta I and PKC beta II) by transducing cells with antisense cDNA of PKC beta I prevented the TPA-induced decrease in cell surface PAR-1 expression and restored approximately 60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, depletion of PKC beta isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKC beta isozymes, whereas the PKC beta-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

Site-specific thrombin receptor antibodies inhibit Ca2+ signaling and increased endothelial permeability.

Thrombin receptor is activated by thrombin-mediated cleavage of the receptor's NH2 terminus between Arg-41 and Ser-42, generating a new NH2 terminus that functions as a "tethered ligand" by binding to sites on the receptor. We prepared antibodies (Abs) directed against specific receptor domains to study the tethered ligand-receptor interactions required for signaling the increase in endothelial permeability to albumin. We used polyclonal Abs directed against the peptide sequences corresponding to the extracellular NH2 terminus [residues 70-99 (AbDD) and 1-160 (AbEE)] and extracellular loops 1 and 2 [residues 161-178 (AbL1) and 244-265 (AbL2)] of the seven-transmembrane thrombin receptor. Receptor activation was determined by measuring changes in cytosolic Ca2+ concentration ([Ca2+]i) in human dermal microvascular endothelial cells (HMEC) loaded with Ca(2+)-sensitive fura 2-acetoxymethyl ester dye. The transendothelial 125I-labeled albumin clearance rate (a measure of endothelial permeability) was determined across the confluent HMEC monolayers. AbEE (300 micrograms/ml), directed against the entire extracellular NH2-terminal extension, inhibited the thrombin-induced increases in [Ca2+]i and the endothelial 125I-albumin clearance rate (> 90% reduction in both responses). AbDD (300 micrograms/ml), directed against a sequence within the NH2-terminal extension, inhibited 70% of the thrombin-induced increase in [Ca2+]i and 60% of the increased 125I-albumin clearance rate. AbL2 (300 micrograms/ml) inhibited these responses by 70 and 80%, respectively. However, AbL1 (300 micrograms/ml) had no effect on either response. We conclude that NH2-terminal extension and loop 2 are critical sites for thrombin receptor activation in endothelial cells and thus lead to increased [Ca2+]i and transendothelial permeability to albumin.

Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway.

We investigated the function of gp60, an endothelial cell membrane 60-kDa albumin-binding protein localized in caveolae, and the mechanism of its activation in regulating endothelial permeability of albumin. Gp60 organization on the bovine pulmonary microvessel endothelial cell (BPMVEC) surface was punctate as shown by immunofluorescence using an anti-gp60 antibody (Ab) conjugated with bisfunctional, N-hydroxysuccinimidyl fluorophore (Cy3). Addition of a secondary Ab to anti-gp60 Ab-treated BPMVEC induced cross-linking of gp60 as evident by increased size of fluorescent particles and cell surface gp60 clustering. Gp60 cross-linking also produced 2-3-fold increases in the endothelial cell uptake and the luminal to abluminal permeability of 125I-albumin as well as the fluid-phase tracer, horseradish peroxidase. The increased transendothelial permeability of macromolecules was the result of transcytosis as it was not associated with an increase in the paracellular pathway. Incubation of anti-gp60 Ab with BPMVEC at 37 degrees C caused internalization of gp60, and thereby reduced the uptake of the macromolecules. Activation of gp60 by either albumin (the gp60 ligand) or gp60 cross-linking induced the phosphorylation of both gp60 and caveolin-1 (the major structural caveolar protein) on tyrosine residues. Gp60 activation also phosphorylated the Src family tyrosine kinases pp60(c-Src) and Fyn. The activated pp60(c-Src) and Fyn co-immunoprecipitated with caveolin-1 in BPMVEC membrane. Protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, prevented gp60-activated macromolecule uptake and transcytosis in a concentration-dependent manner, indicating the functional significance of the PTK pathway in activating albumin transcytosis. These findings indicate that activation of gp60 stimulates the Src PTK signaling pathway, and thus regulates the transcytosis of albumin across the endothelial cell monolayer.

Tumor necrosis factor decreases thrombin receptor expression in endothelial cells.

We examined the effects of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF alpha) on the expression of proteolytically activated thrombin receptor (PATR) in human umbilical vein endothelial cells (HUVEC). PATR mRNA and protein levels were measured in confluent HUVEC monolayers after challenge with TNF alpha. Northern analysis indicated that TNF alpha treatment resulted in 2- to 3-fold decrease in PATR mRNA in a time- and dose-dependent manner. PATR mRNA level returned to the control level within 6 hr. The nuclear run-on assay indicated that the decreased mRNA signal was due to reduction in the transcription rate. Immunoblotting experiments indicated that the decrease in expression of PATR protein followed in time the decrease in mRNA; the lowest level of protein expression was achieved at 22 hr after TNF alpha treatment. PATR protein returned to basal value within 40 hr after TNA alpha challenge. To assess alterations in endothelial cell function after TNF alpha treatment, we measured thrombin-induced increase in cytosolic Ca2+ ([Ca2+]i) and the cell shape change (measured by decrease in electrical impedance of endothelial cell monolayer). In HUVEC treated with TNF alpha (100 U/ml for 22 hr), the rise in [Ca2+]i after thrombin challenge was approximately 2-fold less than in control thrombin-treated cells. The decrease in electrical impedance of HUVEC monolayers in response to thrombin after TNF alpha treatment was also significantly reduced. However, the rise in [Ca2+]i in response to histamine was not altered by TNF alpha pretreatment. In conclusion, TNF alpha exposure of endothelial cells decreased both mRNA and protein expression of PATR, which explain the decreased activation of thrombin generated signals after the TNF alpha exposure.

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.

Growth characteristics of pulmonary artery smooth muscle cells from fawn-hooded rats.

We compared the proliferative rates of vascular smooth muscle cells (VSMC) from pulmonary arteries of pulmonary hypertensive fawn-hooded rats (FHR) with VSMC from normotensive Sprague-Dawley rats (SDR). VSMC from FHR grew at increased rates and reached higher densities at all serum concentrations studied (5-20%) than the VSMC from SDR. The VSMC from FHR also responded to epidermal growth factor (EGF) at low serum concentrations, as evidenced by significantly greater DNA synthetic rates, than the control VSMC. The increased growth in these cells could be due to increased number and/or affinity of EGF receptors because of the higher specific binding of 125I-EGF to the VSMC from FHR. The VSMC from FHR and SDR were equally sensitive to the antiproliferative effects of heparin, suggesting that the heparin-sensitive pathways are not altered in the VSMC from FHR. These results suggest that the development of pulmonary hypertension in FHR may be related to the higher proliferative capacity of the pulmonary VSMC, which may be coupled to increased activity of the EGF receptors on these cells.