Endothelial Cell Monolayer Permeability Research Articles

F-Actin cytoskeleton and sucrose permeability of immortalised rat brain microvascular endothelial cell monolayers: effects of cyclic AMP and astrocytic factors

The immortalised RBE4 cell line, derived from rat brain capillary endothelial cells, preserves many features of the in vivo brain endothelium, and hence is of interest as a potential in vitro model of the blood–brain barrier (BBB). This study reports the effects of elevated intracellular cAMP and factors released by astrocytes on the F-actin cytoskeleton and paracellular sucrose permeability of monolayers of RBE4 cells. RBE4 cells grown in control medium showed a marked increase in the F-actin staining at the cytoplasmic margin at confluence, which was not significantly enhanced by elevation of intracellular cAMP and/or addition of astrocyte-conditioned medium (ACM). The formation of the marginal band of F-actin was accompanied by an increase in the F-actin content of the RBE4 cells up to confluence, and a decline in F-actin content thereafter. Elevation of intracellular cAMP or co-culture above astrocytes significantly decreased the paracellular sucrose permeability of confluent RBE4 cell monolayers grown on collagen filters ( P<0.01 and P<0.001, respectively). Co-culture above astrocytes together with elevated cAMP also produced a significant decrease in the sucrose permeability of the monolayer ( P<0.01) but this was no greater than with astrocytes alone. These findings show that the RBE4 cell line may serve as a useful in vitro model for the study of brain endothelial cell physiology and agents which alter the permeability of the BBB.

Effect of tumor necrosis factor-alpha on the permeability of bovine brain microvessel endothelial cell monolayers

The administration of chemotherapy to patients with tumors of the central nervous system is often blocked by the blood-brain barrier. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that promotes vascular permeability in addition to its pro-inflammatory effects. However. no direct evidence exists as to whether TNF-alpha may increase permeability of the BBB. We evaluated the effect of TNF-alpha on the transport of cisplatin (COOP) or high molecular weight dextran labeled with fluorescein isothiocyanate (FITC-dextran) across bovine brain microvessel endothelial cell (BMEC) monolayers that was conducted on side-by-side diffusion chambers in vitro. The permeability coefficient for the transport of COOP across the untreated monolayer was 3.80 x 10-5 cm sec-1 at 30 minutes. After treating the BMEC monolayer with TNF-alpha (50 U ml-1 and 500 U ml-1) for 36 hours, the PC of COOP increased significantly to 8.94 x 10-5/ and 14.43 x 10-5 cm sec-1 respectively (p< 0.01). TNF-alpha had no effect on the transport of FITC-dextran across the BMEC monolayers. Electron microscopy showed that the tight junctions between the BMECs persisted even after treatment with TNF-alpha, whereas they had been partially disrupted following exposure to mannitol, 1600 mOsm kg-1• TNF-alpha selectively promoted the in vitro permeability of the blood-brain barrier to COOP without disrupting tight junctions. This system could be used as a model for experimental studies of chemotherapy. Findings suggested that the combined administration of TNF-alpha and COOP may be clinically useful. [Neural Res 1997; 19: 369-376]

Neutrophil-endothelial interactions in a cell-column model of the microvasculature: effects of fMLP.

The purpose of this study was to develop an in vitro model which, compared to stagnant two-chamber systems, is more analogous to the rheological conditions found in the vasculature and permits near simultaneous measurement of polymorphonuclear leukocytes (PMN) entrapment and endothelial, monolayer permeability. A previously described cell-column model of the vasculature was perfused with 2 x 10(6) PMN/ml at an average fluid velocity of 0.09 cm/s and an average estimated endothelial surface shear stress of 4.5 dyne/cm2 for a total of 60 min. PMN entrapment was estimated from counts of PMN in this recirculating system. Bovine endothelial permeability was estimated from an indicator dilution analysis of the three co-injected dyes: blue dextran (molecular weight [MW]) 2 x 10(6), sodium fluorescein (MW 342) and cyanocobalamin (MW 1355). Circulation of PMN through cell-columns did not activate PMN, although the number of circulating PMN decreased at a rate of 14% per 65 cm2 of endothelial surface in the first 15 min. Endothelial cell monolayer permeability did not change within 60 min. Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-5) M) activated PMN and significantly increased entrapment of circulating PMN to 27% per 65 cm2 in the first 15 min. Although the percentage of circulating PMN entrapped increased, endothelial monolayer permeability was not increased by either the addition of 10(-5)M fMLP or PMN + fMLP to the cell-column perfusate. Over the 60-min period studied, entrapment of PMN within a recirculating model of the microvasculature was not associated with an increase in endothelial permeability even after PMN stimulation with fMLP.

Effects of energy deprivation induced by fluorocitrate in immortalised rat brain microvessel endothelial cells

The effects of the mitochondrial aconitase inhibitor, fluorocitrate on the immortalised rat brain endothelial cell line (RBE4) were investigated. Treatment with different concentrations of fluorocitrate (0–1 mM) for 24 h induced a significant, concentration-dependent decrease in the MTT reduction (an index of mitochondrial function), intracellular ATP content, glucose consumption and lactate production by RBE4 cell monolayers but did not alter the glucose to lactate ratio at concentrations lower than 0.5 mM. At all concentrations, fluorocitrate induced a significant decrease in the protein content per well. Fluorocitrate treatment of confluent RBE4 cells induced a marked redistribution of the F-actin cytoskeleton from a characteristic marginal band to a more diffuse cytosolic pattern. This redistribution of the cytoskeleton coincided with a reduction in the total cellular F-actin content of the RBE4 cells at fluorocitrate concentrations greater than 0.5 mM. Treatment of confluent RBE4 cells with fluorocitrate had no significant effect on RBE4 cell monolayer permeability measured by FITC-dextran or [ 14C]sucrose. These results show that whilst energy deprivation following fluorocitrate treatment induces significant changes in the RBE4 cell F-actin cytoskeleton and cellular metabolism, it does not have any significant effect on endothelial cell monolayer permeability. These results demonstrate that profound toxic effects on endothelial cell structure and metabolism are not necessarily accompanied by changes in endothelial cell monolayer permeability.

Involvement of bradykinin B1 and B2 receptors in human PMN elastase release and increase in endothelial cell monolayer permeability

Bradykinin (BK) is a potent inflammatory mediator, which can release other inflammatory mediators by interacting with bradykinin B1 and B2 receptors. The role of kinins in regulating human PMN elastase release was studied. BK induced elastase release 5-fold over basal levels. Elastase release was inhibited by both B1 and B2 receptor antagonists. A specific B1 agonist des-Arg10-KD increased elastase release 4-fold. Since elastase has been implicated in vascular leak, the effect of BK on endothelial cell monolayer (EM) permeability was assessed. BK increased EM leak (I125flux) across the EM, whereas des-Arg10-KD was inactive. When co-cultured with human umbilical vein endothelial cells, des-Arg10-KD-treated PMNs increased EM leak by 35%. The elastase inhibitor AAVPK blocked des-Arg10-KD-induced leak by 80% suggesting that elastase is responsible for the increase in permeability. It is concluded that BK causes increased leak by inducing PMN elastase release via activation of both B1 and B2 receptors. BK blockade and elastase inhibition may be beneficial in inflammatory diseases such as ARDS which is characterized by increased lung permeability and both kinin and PMN activation are thought to participate.

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: A possible use of aortic endothelial cell for In vitro BBB model

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in l-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the l-glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.

HYPOXIA-INDUCED PROTEINS IN CULTURED ENDOTHELIAL CELLS † 280

Exposure of endothelial cells (EC) to hypoxia has been associated with increased neutrophil (PMN) adherence to EC and changes in EC monolayer permeability. We examined the possibility that these changes would be related to upregulation of specific proteins depending on the duration of hypoxia. EC were incubated in room air and 5% CO2 and grown to confluence in 6 well plates. The EC monolayers were exposed to hypoxia for varying periods of time(0, 12 and 24hrs) in a plexiglass chamber purged continuously with 5% CO2 and 95% N2. Control cells were exposed to normoxia in a similar manner and duration. At the specified time points the cells were solubilized with a Laemmli sample buffer, boiled and loaded onto a gel for electrophoresis. Cell extracts were analyzed by 12% SDS-PAGE. Equal amounts of protein were added per lane. The gels were silver stained for detection of protein bands. Conditioned media from all groups was analyzed in a similar manner. Cell viability during hypoxia was assessed by adherent cell counts and trypan blue exclusion. Hypoxia was associated with the appearance of three (3) new proteins, Mr 56-58kd, 45-46kd and 28-29kd. The upregulation appeared to increase over time with greater intensity at 24 hr than at 12 hrs. In addition, two (2) proteins appeared to be down regulated with increasing lengths of hypoxia-Mr 49-50kd and 30-32kd. The degree of downregulation was somewhat variable in appearance. Conditioned media did not show any new or upregulated proteins over time. Cell viability remained constant at >97% for all groups (including hypoxia). These data suggest that EC exposed to hypoxia upregulate a specific set of proteins that are unrelated to viability. These proteins may play a role in the observed responses of endothelial cells to hypoxia.

Astroglial cells inhibit the increasing permeability of brain endothelial cell monolayer following hypoxia/reoxygenation

Blood-brain barrier (BBB) is known to be structured with astroglial cells (AGs) and brain endothelial cells (BECs), and it has been proposed that these cells play different roles in the BBB. We cultivated AGs and BECs from infant rats (2-week-old), and these cells were cultured on the opposing side of collagen membrane to produce a co-culture model of the BBB in vitro. Permeability of the cell layer was evaluated by the electrical resistance through the membrane. To clarify the role of AGs in the BBB disruption following ischemia/reperfusion, electrical resistance of the co-culture model was compared to that of BEC monolayer following hypoxia/reoxygenation. The electrical resistance through BEC monolayer showed 55.5 ± 15.1% reduction at 4 h of hypoxia, and 93.3 ± 5.4% reduction at 8 h of hypoxia ( n = 8). However, the co-culture model showed attenuation of the reduction (24.8 ± 14.2%) at 4 h of hypoxia ( n = 8, P < 0.01), but not at 8 h of hypoxia (95.3 ± 5.0%). These results indicate that AGs reduce the increasing permeability of the BEC monolayer following short duration of hypoxia/reoxygenation. It is suggested that AGs may have a protective effect to the BBB disruption following ischemia/reperfusion.

Computation of brain-blood partitioning of organic solutes via free energy calculations.

The ratio of brain-blood partitioning, log(Cbrain/Cblood) (log BB), of a series of compounds that range from simple solutes to histamine H2 antagonists was correlated with computed solvation free energy in water (delta G degree W). The free energies were computed with the AMSOL 5.0 program using the AM1-SM2.1 solvation model. From a set of 55 compounds, a function was developed in which log BB was related to the free energy of solvation as follows: log BB = 0.054 delta G degree W + 0.43 (r = 0.82 and standard error = 0.41). This correlation provided successful prediction of brain-blood partitioning for compounds outside the training dataset. Furthermore, for a set of 10 drugs, delta G degree W correlated well with literature data for the permeability of endothelial cell monolayers from bovine brain microvessels. In neuroscience drug discovery, the use of computed solvation free energies to predict brain penetration provides a facile method for prioritizing synthetic targets.

Effects of rat hepatocytes on macromolecular permeability of bovine aortic endothelial cell monolayer.

The aim of this study was to examine whether the hyperpermeable structure of the liver endothelium in vivo is related to the interactions of hepatocytes in a culture system. The permeation of macromolecular FITC-labeled dextran (molecular weight 70,000) through a monolayer of bovine aortic endothelial cells (BAEC), cocultured with rat parenchymal hepatocytes (P-hep), was increased. When the BAEC were cocultured with nonparenchymal hepatocytes (N-hep), the permeability of the BAEC monolayer was not increased. However, when the BAEC were cocultured with a mixture of P-hep and N-hep (PN-hep), the BAEC monolayer was more permeable than when BAEC were cocultured with P-hep alone. The conditioned medium of P-hep did not alter the BAEC monolayer permeability, nor did the extracellular matrix of P-hep alter BAEC permeability. When the BAEC were cocultured with PN-hep, the F-actin content was not altered. These findings suggest that the interaction between hepatocytes and endothelial cells exerts an important effect on the hyperpermeable structure of the liver vessels in vivo.

Platelet-activating factor and angiogenesis.

Platelet-activating factor (PAF) is a phospholipid autacoid with a spectrum of diverse and potent biological properties relevant for the development of inflammatory reaction and septic shock1. Recently, PAF has been also implicated in the embryogenesis and cell differentiation2,3. Researchers believe that PAF is a mediator of cell to cell communication which may function both as intercellular and intracellular messenger. Since PAF is produced after appropriate stimulation by many cell types, including most inflammatory cells, it has been implicated in the pathogenesis of a variety of diseases4. Moreover, human endothelial cells (EC) were found to produce PAF after stimulation by several inflammatory mediators5,6. Many cell types that synthesized PAF, including EC, are also target for PAF7,8 and express PAF-binding sites on their surface9. PAF, indeed, directly stimulates the endothelium, as it enhances the permeability of EC monolayer and induces changes of the cell cytoskeleton, leading to cell retraction and formation of intercellular gaps8. Moreover, it was observed that, in sub-confluent EC cultures, the redistribution of cytoskeleton was characteristic of cells undergoing translational movements8. These observations prompted us to evaluate whether PAF may stimulate the migration of EC in vitro.

血液・脳関門機能とアストロサイト

The basis for the blood-brain barrier in mammals is the selective transport properties of brain capillary endothelium, including the elaborate system of tight intercellular occluding junctions that occur between apposed membrane faces of these cells. This unique specialization of brain capillary endothelial cells appears late in development and has been postulated to be under the inductive influence of astrocytes in the central nervous system. To examine this astrocytic contribution to endothelial cell monolayer permeability, we employed the cocultures of bovine endothelial cells (aortic or brain capillary endothelial cells, BBEC or BAEC) with astrocytes in a double chamber system. In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes. The obtained results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.

Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities

Thrombin-induced cultured bovine endothelial cell (EC) gap formation and albumin permeability is initiated by contraction, which is dependent upon myosin light chain kinase-mediated myosin light chain (MLC) phosphorylation. MLC are then rapidly dephosphorylated (J. G. N. Garcia, H. W. Davis, and C. E. Patterson, J. Cell. Physiol. 163: 510-522, 1995), suggesting a role for MLC dephosphorylation in regulation of EC barrier function. Therefore, we studied the effect of semiselective protein phosphatase (PPase) inhibitors, calyculin A and okadaic acid, on MLC phosphorylation status, myosin-associated PPase activity, and EC monolayer permeability. Calyculin A (0.1-10 nM), but not okadaic acid (1-100 nM) produced significant dose-dependent enhancement of both MLC phosphorylation (three- to four-fold) and EC permeability (eightfold). EC homogenates were utilized to assess Ser/Thr PPase activities using either [32P]phosphorylase A or 32P-labeled skeletal MLC as substrates. Calyculin A at 5 nM (sufficient to inhibit type 1 and type 2A PPase) produced approximately 95% inhibition of all EC PPase activity against both substrates, whereas 2 nM okadaic acid (selective for PPase 2A) only partially inhibited EC PPase activity (40-60%). Fractionation of EC homogenates produced a supernatant fraction containing < 10% of total myosin and a pellet fraction with > 90% of total myosin. PPase activity in the myosin-enriched pellet was insensitive to 2 nM okadaic acid (0% inhibition) but sensitive to 5 nM calyculin (> 95% inhibition). Immunoreactive PPase 1 was present in both fractions, whereas PPase 2A was present only in the myosin-depleted fraction. We conclude that a type 1 myosin-associated PPase is involved in regulation of EC contractility and barrier function.

Arg-Gly-Asp peptide increases endothelial hydraulic conductivity: comparison with thrombin response.

The contribution of integrin receptors to the regulation of endothelial permeability was studied using cultured bovine pulmonary microvascular endothelial cell (BPMVEC) monolayers by the measurement of hydraulic conductivity (Lp). Treatment of monolayers with a peptide containing the sequence Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) (0.85 mM) to compete for the RGD sequence of extracellular matrix (ECM) proteins increased endothelial Lp threefold, whereas the control peptide Gly-Arg-Gly-Glu-Ser-Pro had no effect on Lp. This action of GRGDSP on Lp was not significantly altered by dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 0.5 mM). Endothelial Lp increased twofold when the monolayers were challenged with alpha-thrombin (5 x 10(-8) M for 10 min), and this response was completely reversed by DBcAMP. The strength of adhesion of endothelial cells was estimated by evaluating the ability of endothelial cells to remain attached to ECM after treating the monolayers with 0.05% trypsin plus 0.5 mM EDTA. Exposure of the monolayers to either GRGDSP or alpha-thrombin significantly reduced the strength of adhesion to the ECM. DBcAMP prevented the antiadhesive effect of alpha-thrombin but not that of GRGDSP. Treatment of the monolayers with either alpha-thrombin or GRGDSP caused formation of intercellular gaps, but only the thrombin-induced intercellular gaps were accompanied by reorganization of actin filaments. These results indicate that integrin binding to ECM proteins regulates an important determinant of endothelial permeability and that alpha-thrombin and GRGDSP increase endothelial cell monolayer permeability by different mechanisms.

Soluble CD 14 in serum mediates LPS-induced increase in permeability of bovine pulmonary arterial endothelial cell monolayers in vitro

Lipopolysaccharide (LPS) is a mediator of septic shock and acute respiratory distress syndrome (ARDS), conditions which are characterized by high-permeability pulmonary edema. LPS increases endothelial permeability both directly and indirectly via the pro-inflammatory cytokines produced by monocytes and macrophages. We investigated the role of soluble CD 14 in serum in the increased endothelial permeability induced by LPS. Bovine pulmonary artery endothelial cells were grown to confluence on a microporous filter and the 125I-albumin clearance rate across the monolayer was determined. Even a high concentration of LPS (1 μg/ml) did not increase endothelial permeability under a serum-free condition. In the presence of more than 3% normal human serum, LPS increased endothelial permeability. The presence of neutralizing anti-CD14 monoclonal antibody eliminated the serum-dependent effect of LPS. The addition of recombinant sCD14 completely replaced the requirement for serum. LPS-binding protein (LBP) did not enhance the rsCD14-mediated LPS effect, and anti-LBP antibody did not attenuate the serumdependent LPS effect. These findings suggest that sCD14 in serum mediates the permeability-increasing effect of LPS on endothelial cells but that LBP is not necessary for this effect.

Tumor-conditioned medium increases macromolecular permeability of endothelial cell monolayer

Tumor-conditioned medium increases macromolecular permeability of endothelial cell monolayer

Tumor-conditioned medium increases macromolecular permeability of endothelial cell monolayer

The permeation of macromolecular FITC-labeled dextran (molecular weight 70 000) through bovine aortic endothelial cells (BAEC) monolayer, which were cultured for 5 days with conditioned medium prepared from mouse melanoma B16, was increased. However, when BAEC, which were cultured with normal medium until confluent, were treated with B16 conditioned medium (B16-CM) for 30 min, the permeability did not increase. The B16-CM also increased the permeability of the endothelial monolayers of bovine veins and the human umbilical vein, but did not increase that of the epithelial monolayer. The B16-CM did not alter the distribution or content of F-actin on the BAEC. BAEC cultured in the presence of B16-CM for 5 days were detached from the dish, and then seeded into a chamber at one-fifth of confluent cell density. After 5 days of culture in normal medium, the BAEC were grown to confluence and their permeability was increased. These findings suggest that B16-CM increased the endothelial permeability irreversibly without the decrease of F-actin, and that soluble factor(s) which were secreted from the tumor cells participate in the construction of the hyperpermeable structure of tumor vessels in vivo.

Increased capillary permeability induced by human follicular fluid: a hypothesis for an ovarian origin of the hyperstimulation syndrome

To examine the effect of follicular fluid (FF) and peritoneal fluid (PF) from patients undergoing assisted reproductive technology procedures on endothelial barrier function. This was determined in vitro by measuring the permeability of filter-grown bovine aortic endothelial cell monolayers to a permeability marker. Endothelial cells obtained from bovine thoracic arotas were treated with collagenase solution and plated on millicell filters, on which they formed confluent monolayers. Flux rate was determined at 60 minutes by measuring the radioactive tracer (3H mannitol) permeating from the apical to the basolateral part of the filter. Fifty-eight samples of FF and PF, both from stimulated and natural cycles were analyzed and grouped according to the number of eggs retrieved. Follicular fluid and PF samples from natural cycles were used as controls. There was an augmentation in the permeability rate of both FF and PF from patients undergoing controlled ovarian hyperstimulation (COH) who responded with an increasing number of eggs compared with controls (51% and 39%, respectively). When analyzing samples from patients who responded with a low number of oocytes, no significant increase was observed. It is known that in OHSS, the increase in capillary permeability is related to the administration of gonadotropins, and is believed to be mediated by a vasoactive substance of ovarian origin. In this study, FF and PF from patients undergoing COH showed a significant increase in the permeability rate through endothelial cells in vitro. Based on these findings, it could be hypothesized that if the same events took place in vivo, the isolation of this factor from ovarian source could be of significant importance to elucidate the pathogenesis of OHSS.

HIV-1 infection alters monocyte interactions with human microvascular endothelial cells.

HIV infection of monocytes resulted in twofold elevation of adhesion molecule LFA-1 (both alpha L/CD11a and beta 2/CD18 subunits) and LFA-3 (CD58), with no apparent increase in LFA-2 (CD2) or various beta 1-integrins. Homotypic aggregation of monocytes was evident 2 h after exposure to virus and was inhibited by mAbs to both the alpha L- and beta 2-subunits of LFA-1. HIV-infected monocytes also showed a marked increase in adherence to human capillary endothelial cell monolayers derived from brain, lung, and skin. This adherence was inhibited by mAb to either LFA-1 subunit and by mAb to the counter-receptor intercellular adhesion molecule-1. Cocultivation of HIV-infected monocytes with endothelial cells increased permeability of endothelial cell monolayers to 125I albumin in transwell assay systems. The increased endothelial permeability induced by HIV-infected monocytes was associated with a substantial disruption of the endothelial cell monolayer. Morphologic disruption was not a direct toxic effect on endothelial cells, but appeared to be secondary to changes in endothelial cell-cell or cell-matrix interactions. Northern blot analysis showed increased expression of gelatinase B (92-kDa gelatinase), tissue inhibitor of metalloproteinase TIMP-1, and TIMP-2 in the HIV-infected monocytes. Consistent with these Northern analyses, secretion of gelatinase activity in culture fluids of HIV-infected monocytes was also increased and was dependent on the stage of virus replication. Incubation of HIV-infected monocytes with the proteinase inhibitors TIMP-1 and TIMP-2 inhibited the increased permeability of endothelial cell monolayers to 125I albumin. These results suggest possible mechanisms for extravasation of HIV-infected monocytes through vascular endothelium into tissue in early stages of HIV disease.

Increased permeability of bovine aortic endothelial cell monolayers in response to a thromboxane A2-mimetic.

The present study was designed to investigate the ability of thromboxane to modulate the clearance rate of 125I-albumin through bovine aortic endothelial cell (BAEC) monolayer grown on polycarbonate micropore membrane. Stimulation of BAEC with the TXA2 mimetic U44069 (10(-8), 10(-7) and 10(-6) M) elicited a dose-dependent increase of labeled albumin passage across BAEC monolayers. This effect was markedly reduced by the TXA2 antagonist L655240 (10(-7) and 10(-6) M). Our results suggest that TXA2 may modulate the permeability of endothelial cells directly through activation of specific receptors.