EDITORIAL FOCUS
Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton

J. H. Park, N. Okayama, D. Gute, A. Krsmanovic, H. Battarbee, and J. S. Alexander

Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of hypoxia/aglycemia on microvascular endothelial permeability were evaluated, and the second messenger systems and the cytoskeletal-junctional protein alterations in this response were also examined. Monolayers of human dermal microvascular endothelial cells on microcarrier beads were exposed to either thioglycolic acid (5 mM, an O2 chelator), glucose-free medium, or both stresses together. Permeability measurements were performed over a 90-min time course. Although neither hypoxia alone nor aglycemia alone increased endothelial permeability significantly, the combination of both increased significantly as early as 15 min. Intracellular Ca2+ measurements with fura 2-AM showed that hypoxia/aglycemia treatment increased Ca2+ influx. To determine the second messengers involved in increased permeability, monolayers were incubated for 30 min with the cytosolic Ca2+ scavenger 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mM), a classical protein kinase C (PKC) blocker, Gö-6976 (10 nM), a cGMP-dependent protein kinase (PKG) antagonist, KT-5823 (0.5 µM), or the mitogen-activated protein (MAP) kinase inhibitor PD-98059 (20 µM). Hypoxia/aglycemia-mediated permeability changes were blocked by chelating cell Ca2+, PKC blockade, PKG blockade, and by inhibiting p38 MAP kinase-1. Finally, changes in the binding of junctional proteins to the cytoskeleton under the same conditions were assessed. The concentrations of occludin and pan-reactive cadherin binding to the cytoskeleton were significantly decreased by only both stresses together. However, these effects were also blocked by pretreatment with TMB-8, Gö-6976, KT-5823 (not in occludin), and PD-98059. These data suggest that hypoxia/aglycemia-mediated endothelial permeability may occur through PKC, PKG, MAP kinase, and Ca2+ related to dissociation of cadherin-actin and occludin-actin junctional bonds.

thioglycollate; oxygen; glucose; microvasculature; junction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that tissue ischemia disrupts normal transport pathways for water and solutes (e.g., albumin) across the microvasculature, resulting in several forms of organ and tissue edema (32). The acute loss of endothelial barrier function under these circumstances is a significant cause of tissue pathology and loss of organ function (8). Even though accumulation of excess extracellular fluid and protein is a common manifestation of tissue injury, the sequence of molecular events that lead to increased permeability during ischemic injury is not known.

To assess how tissue deprivation of O2 and glucose during ischemia might contribute to increased permeability, we used several different methods from a previous established experiment. First, almost all in vitro studies have used nonhuman or human macrovascular endothelial cells, especially human umbilical vein endothelial cells (HUVEC; see Refs. 1, 13, 22, 23, 41). However, most pathophysiological events take place at the level of the microvasculature, which constitutes the vast majority of the human vascular compartment. Second, almost all in vitro studies of hypoxia have used a hypoxic chamber with an anoxic gas mixture (93% N2-5% CO2-2% H2; see Refs. 1, 22, 23, 37, 41). This system needs a long incubation time in this medium to produce an anoxic condition. Moreover, it is very difficult to handle the cells in anoxic chambers. We also tried a model of arrested flow to the cell column. However, we found that O2 was not appreciably used up by the cells in the column over even long periods of time (up to 60 min), and this treatment consistently failed to modify permeability. Here, to approximate ischemic conditions, we imposed aglycemia to hypoxia as an additional relevant stress. In the present study, we used cultured human dermal microvascular endothelial cells (HMEC-1) treated with 5 mM thioglycollate (O2 chelator) and aglycemia as a novel model of ischemia.

We and others have previously shown that junctional proteins, e.g., cadherins and occludin, play roles in endothelial junctional barrier function (5, 10, 25) and that endothelial barrier properties are related to the amount of junctional protein in the cells as well as their spatial organization (6, 9, 43). We hypothesized that the increased permeability associated with ischemic injury may represent an alteration in the structure and organization of the cytoskeletal junctional complexes.

The second messenger mechanisms regulating permeability are poorly understood. Several studies have identified cell Ca2+ and several protein kinases as important regulators of barrier function (30). Huang and Yuan (19) suggested that elevation of endothelial Ca2+ is an early event preceding nitric oxide (NO) synthesis, which may activate barrier changes. Wu et al. (45) have also shown that vascular endothelial growth factor (VEGF) modulates permeability by an NO-, guanylate cyclase-, and cGMP-dependent protein kinase (PKG)-dependent mechanism. Similarly, we also reported that the increased endothelial (HUVEC) permeability associated with vascular permeability factor/VEGF involves the loss of occludin organization apparently through the activation of extracellular signal-regulated kinase (ERK1/2; see Ref. 26). Protein kinase C (PKC) activation may also contribute to the regulation of endothelial barrier by regulating NO production or intracellular Ca2+ (19). On the basis of previous studies, we hypothesized that some of these second messengers may be involved in the hypoxia/aglycemia-mediated permeability change and investigated these roles by using their inhibitors.

Therefore, in this study, we investigated the effects of chemical hypoxia/aglycemia on endothelial barrier, the second messengers regulating barrier [especially intracellular Ca2+, PKG, PKC, and mitogen-activated protein (MAP) kinase], and how changes in cadherin-occludin-cytoskeleton structure control junctional permeability.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cell culture and antibodies. HMEC-1 were originated and provided by Dr. Francisco J. Candal (Centers for Disease Control and Prevention, Atlanta, GA). Cells were maintained in complete MCDB-131 medium (Sigma, St. Louis, MO) supplemented with 10% FCS (Hyclone, Logan, UT) in a 100% humidified atmosphere at 37°C with 7.5% CO2. Antibody to rabbit anti-pan-cadherin was purchased from Sigma. Antibodies to rabbit anti-occludin and anti-ZO-1 were obtained from Zymed Laboratories (San Francisco, CA). Anti-beta -catenin monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). As secondary antibodies, alkaline phosphatase-conjugated anti-rabbit and anti-mouse IgG (Sigma) and TRITC-conjugated anti-rabbit IgG (Zymed Laboratories) were used.

Analysis of O2 scavenging by thioglycollate. O2 concentration changes produced by thioglycollate were measured 30 min after dissolving different concentrations of thioglycolic acid (0-20 mM) in Hanks' balanced salt solution (HBSS). O2 tension was measured with a model 288 Blood Gas System (CIBA blood gas analyzer; Corning).

Cell viability test. Cell viability was determined by the trypan blue exclusion test (34). Confluent HMEC-1 monolayers in 96-well plates were exposed to HBSS (O2 tension, 179.7 mmHg; glucose concentration, 5.4 mM), glucose-free HBSS, HBSS-5 mM thioglycolic acid (O2 tension, 13.13 mmHg), or glucose-free HBSS-5 mM thioglycolic acid for 60 min. Monolayers were then returned to cell culture medium (glucose concentration, 5.4 mM) and stained with 0.4% trypan blue for 5 min immediately after changing medium and 24 h later. Blue-stained cells were scored as nonviable.

Microcarrier bead cultures. Cells were cultured on microcarrier beads as previously described (5, 16, 25). Briefly, cells were seeded on Cytodex-3 microcarrier beads (Pharmacia, Uppsala, Sweden) at a density of 2 × 104 cells/cm2, and attachment was achieved by intermittent stirring overnight. After this, microcarrier cultures were maintained at 60 rpm. Fifty percent of the medium was exchanged three times a week. Confluent cultures were used for assays between 7 and 14 days after seeding.

Cell column methods. We used a chromatographic cell column filled with microvascular endothelial cell-covered microcarrier beads to measure changes in barrier function produced by experimental treatments (5, 16, 25). Permeability of the microvascular endothelial monolayers covering the beads was determined from a comparison of the elution curves of permeant versus nonpermeant tracers injected into the flow at the top of the column. Chromatographic cell columns were made from water-jacketed glass columns (0.65 cm diameter; Rainin, Emeryville, CA). Cell-covered beads were poured to a column height of ~2 cm, which provides 130 cm2 of endothelial cell culture surface (1 × 107 cells). Each column was equilibrated with HBSS with 15 mM HEPES (pH 7.4; Sigma). Perfusion through the column was maintained by a peristaltic pump (Minipuls II; Gilson, Middleton, WI) at 1 ml/min (chosen to approximate the gravity flow rate). A bolus of a flow tracer and monolayer permeant tracers was applied by a rotary injection valve (Rainin) by a 50-µl loop. The cell column and perfusate were kept at 37°C throughout the experiment. Multiple tracer indicator dilution analysis was used to assess cell layer permeability from the relative shapes of the elution profiles of tracers simultaneously applied to the top of a cell column. Blue dextran [molecular weight (MW) = 2 × 106] cannot penetrate the monolayer and follows the fluid phase, i.e., it is a flow tracer. Sodium fluorescein (MW = 376) and cyanocobalamin (MW = 1,355) were used as monolayer-permeant tracers. These tracers cross the cell layer at cell-cell junctions and equilibrate in the bead matrix. Importantly, none of these tracers can diffuse across cell membranes. Elution profiles were constructed from 66 samples of the column eluant. A fraction collector (model 203; Gilson) equipped with a drop counter was used to collect two drops of eluant per well for 66 wells in a 96-well plate. The absorbance of each of the 96 wells was read at 620, 540, and 492 nm on a plate reader (Titertek MCC 340) for blue dextran, cyanocobalamin, and sodium fluorescein, respectively, and was stored on a computer for analysis. The absorbances were used to calculate the fractional recovery per sample of each of the dyes. In this model, estimating permeability based on the column elution patterns of multiple tracers is an adaptation and extension of techniques used in vivo to assess capillary permeability (7, 39). To apply these techniques to these experiments, a mathematical model of tracer motion has been developed based on the physical picture of tracer behavior described above. In this previously described model (16), it is assumed that the elution profiles of cyanocobalamin and sodium fluorescein depend on the properties of the mobile phase of the column plus the paracellular permeability properties of the endothelial monolayer and the diffusive motion of the tracer within the microcarrier beads. In contrast to these permeant tracers, the elution of blue dextran depends only on the flow (mobile) phase properties of the column. Elution profile graphical results and the simple Crone extraction estimate of the permeability times surface area product are obtained virtually "on-line" using <50 s of initial data. This allows tracking of the experiment's progress. We have found that this simple estimate parallels the more complex modeling calculations based on analysis of the complete column elution patterns using the Sangren and Shepard (39) model, which were performed "off-line," after all data from each experiment were collected. A modified Marquardt iteration scheme was used to estimate the monolayer permeability that best approximates the experimental data by varying the two free model parameters permeability-surface area and tracer distribution volume. Best fit was determined by a computational algorithm that minimized the coefficient of variation between a computer-generated prediction of the permeant tracers elution profile and the experimental observed elution profile.

Treatment protocols. In all protocols, permeability measurements were made in three separate columns. Cell columns were initially perfused with "normal" perfusate (HBSS supplemented with 15 mM HEPES, pH 7.4). After equilibrating columns in this buffer for 15-30 min, measurements of baseline permeability were made. The column perfusate was then switched to either glucose-free HBSS, HBSS-5 mM thioglycolic acid, or glucose-free HBSS-5 mM thioglycolic acid, and permeability measurements were performed at 15-min intervals over 60 min. The perfusate was then switched back to normal perfusate (HBSS-15 mM HEPES without test agents) to determine recovery of permeability for an additional 30 min.

In a second series of experiments, the following inhibitors were added during only the 30-min baseline period to investigate the involvement of intracellular Ca2+, classical PKC (PKC-alpha , PKC-beta 1), PKG, and MAP kinase in hypoxia/aglycemia-mediated increased endothelial permeability: 1) an intracellular Ca2+ chelator, 0.1 mM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8; Sigma), 2) a classical PKC inhibitor, 10 nM Gö-6976 (Calbiochem, San Diego, CA), 3) a PKG inhibitor, 0.5 µM KT-5823 (Calbiochem), or 4) a specific MAP kinase-1 inhibitor, 20 µM PD-98059 (Biomol, Plymouth Meeting, PA). Monolayers were then exposed to hypoxia/aglycemia protocols as described, and permeability measurements were performed at 15, 30, 45, 60, 75, and 90 min.

Measurement of endothelial cytosolic Ca2+ in cultured monolayer. HMEC-1 were grown to confluence on the "floor" of 3.0-ml epifluorescence superfusion chambers (model TIS0420041B; Kent Scientific, Litchfield, CT). Before loading HMEC-1 monolayers with the Ca2+ chromophore, they were gently washed three times with 2.0 ml HBSS to remove medium phenol red. HMEC-1 were then fura 2 loaded for 15 min at 37°C using 5 µM of the cell-permeant form (fura 2-AM; Molecular Probes, Eugene, OR) dissolved in 0.1% DMSO (Sigma). Cells were subsequently washed three times with HBSS and incubated for 105 min to allow for hydrolysis of the AM. After hydrolysis, the chamber was again washed three times with HBSS and transferred to the stage of an Olympus IMT-2 microscope equipped with a heated stage insert (model TIS04201501; Kent Scientific). Fluorescence measurements were made at 35°C through an inverted epifluorescence microscope with a UV-F ×40 objective using a PTI model RM-D Microspectrofluorometer outfitted for photometric ratio fluorescence studies (Photon Technology International, Brunswick, NJ). Excitation wavelengths were chopped (10 Hz) at 340 and 380 nm, and emissions were monitored at 510 nm. Instrument components and data analysis were computer controlled by fluorescence software (FELIX). Changes in the emission ratio (340/380 nm) were taken as an index of changes in the intracellular Ca2+ concentration ([Ca2+]; see Ref. 12). Basal fluorescence intensities were first measured in HBSS. The medium was then changed to glucose-free HBSS-5 mM thioglycolic acid for 1 h and then back to HBSS for anther 30 min while periodically monitoring the fluorescence emissions. The data were averaged over a measurement period of 30 s and were compared with data from control experiments that were performed in the same condition.

In a separate experiment, HMEC-1 were pretreated with 1 mM LaCl3, a nonselective Ca2+ channel blocker, for 30 min to elucidate whether the source of hypoxia/aglycemia-mediated Ca2+ increase is due to Ca2+ influx from the extracellular space or Ca2+ release from intracellular stores, and 10 µM histamine was added to the superfusion bath to examine if it is able to elevate the intracellular Ca2+ in HMEC-1. At the end of all experiments, the effects of 5 µM Br-A-23187 (a relatively specific Ca2+ ionophore) on the fluorescence ratio were recorded as a positive control.

Assay of binding of junctional proteins to the cytoskeleton. For this assay, HMEC-1 were cultured in 12-well plates. Confluent monolayers were washed gently with HBSS and then exposed to HBSS, glucose-free HBSS, HBSS-5 mM thioglycolic acid, or glucose-free HBSS-5 mM thioglycolic acid for 60 min. The medium was removed, and 1 ml of extraction buffer [PBS + 1% Triton X-100 + 1 mM phenylmethylsulfonyl fluoride (PMSF)] was added to each well for 5 min. After removal of extraction buffer, cells were solubilized with 0.5 ml of PBS plus 1% SDS plus 1 mM PMSF and collected in an Eppendorf tube. Each sample was then sonicated with a Vibra Cell Tissue Disruptor (Sonics & Materials, Danbury, CT), and 3 µl of each sample were spotted onto nitrocellulose membranes. After blocking (5% milk in PBS) for 2 h at 25°C, membranes were incubated with 1:1,000 diluted primary antibody for 1 h and washed with 0.1% milk/PBS three times for 5 min during each wash. Secondary antibodies of goat anti-rabbit IgG alkaline phosphatase and sheep anti-mouse IgG alkaline phosphatase (1:2,000; Sigma) were incubated with the membranes for 1 h. Last, membranes were washed three times with 0.1% milk-PBS for 5 min each and developed by using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma) colorimetric reagents. The densities of dots were measured using an HP ScanJet IIcx flat-bed scanner and Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). We prepared a protein standard for measurement of the applied protein at each spot and measured this using a protein-specific gold dye (Auro Dye forte; Amersham, Buckinghamshire, UK). We spotted the same samples on nitrocellulose, reacted the blots for the proteins we wished to assess, and normalized the density of the spot to the protein standard.

In a second series of experiments, confluent monolayers were incubated for 30 min with 0.1 mM TMB-8, 10 nM Gö-6976, 0.5 µM KT-5823, or 20 µM PD-98059 to examine the effect of these inhibitors on changes in cadherin-occludin-cytoskeleton structure in response to hypoxia/aglycemia. After removal of pretreatment media, monolayers were exposed to glucose-free HBSS-5 mM thioglycolic acid for 1 h. Assay of binding of pan-reactive cadherin and occludin to the cytoskeleton was performed as described above.

Statistical analysis. Unless otherwise indicated, all results are expressed as means ± SE. We evaluated changes in permeability over time by one-way ANOVA; post hoc comparison at various time points with control (in Fig. 2) or glucosefree-5 mM thioglycollate treated values (in Fig. 3 and 4) used Bonferroni's post hoc test. Comparisons of intracellular Ca2+ change between control and treatment groups were made with an unpaired t-test. We also used a one-way ANOVA with Bonferroni's post hoc test to determine statistical significance in an assay of binding of junctional proteins to the cytoskeleton. Calculations were performed using Graphpad Instat software (San Diego, CA). P values <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

O2 tension as a function of thioglycolic acid concentration. The O2 tension in the media treated with different concentrations of thioglycolic acid showed a thioglycollate concentration-dependent decrease. The PO2 measured in samples of 5 mM thioglycollate-treated medium and of medium without thioglycollate were found to be 13.13 ± 2.97 and 179 ± 0.4 mmHg, respectively (Fig. 1). This O2-chelating effect of thioglycolic acid persisted for at least 24 h, and no change in the pH of media was observed at any thioglycollate concentration.


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Fig. 1.   O2 tension decreases as a function of thioglycolic acid concentrations. Thioglycollate was dissolved at concentrations between 0.01 and 20 mM, and the O2 concentration was determined after 30 min.

Effect of hypoxia and aglycemia on the permeability of endothelial cells on microcarrier beads. Endothelial monolayers on microcarrier beads exposed to hypoxia (5 mM thioglycollate), aglycemia, or hypoxia plus aglycemia exhibited permeability changes in a time-dependent manner. Neither hypoxia alone nor aglycemia alone increased endothelial permeability significantly (60 min permeability, 152 ± 11 and 135 ± 5% of baseline, respectively). However, the combination of both increased significantly after as early as 15 min (15 min, 144 ± 2%; 60 min, 189 ± 18% of baseline; Fig. 2). After removal of thioglycollate and glucose-free medium and replacement with HBSS, permeability did not reverse rapidly (Fig. 2). Interestingly, these effects of hypoxia/aglycemia on permeability were significantly attenuated by pretreatment with intracellular Ca2+ chelation (with TMB-8, 0.1 mM), classical PKC inhibition (with Gö-6976, 10 nM; Fig. 3), PKG inhibition (with KT-5823, 0.5 µM), or MAP kinase inhibition (with PD-98059, 20 µM; Fig. 4).


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Fig. 2.   Effect of hypoxia/aglycemia on endothelial permeability. Human dermal microvascular endothelial cell (HMEC)-1 monolayers were treated with Hanks' balanced salt solution (HBSS; control; n = 3 experiments), glucose-free HBSS [G(-); n = 6], HBSS-5 mM thioglycolic acid (TG; n = 6), or glucose-free HBSS-5 mM thioglycolic acid [G(-)/TG; n = 6] for 60 min and then were treated with HBSS for 30 min. Permeability was expressed as fold increase over baseline. Neither hypoxia alone nor aglycemia alone changed endothelial permeability significantly. However, the combination of both increased permeability significantly (as early as 15 min) and showed an additive effect on permeability. Values are means ± SE. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control.



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Fig. 3.   Effect of intracellular Ca2+ chelation [with 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8)] and "classical" protein kinase C [PKC (PKC-alpha , PKC-beta 1)] inhibition (with Gö-6978) on increased endothelial permeability induced by hypoxia/aglycemia. HMEC-1 monolayers were pretreated with 0.1 mM TMB-8 [TMB-8 + G(-)/TG; n = 4] or 10 nM Gö-6976 [Gö-6976 + G(-)/TG; n = 3] in HBSS during 30-min baseline period. Cells were exposed to glucose-free HBSS-5 mM thioglycolic acid for 60 min and switched to HBSS for 30 min. Permeability was expressed as fold increase over baseline. Chelating intracellular Ca2+ or blocking classical PKC significantly attenuated the increased permeability due to hypoxia/aglycemia. Values are means ± SE. * P < 0.05 and ** P < 0.01, Gö-6978 + G(-)/ TG vs. G(-)/TG; # P < 0.05 and ### P < 0.001, TMB-8 + G(-)/TG vs. G(-)/TG.



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Fig. 4.   Effect of mitogen-activated protein (MAP) kinase and cGMP-dependent protein kinase (PKG) inhibition on increased endothelial permeability induced by hypoxia/aglycemia. HMEC-1 monolayers were pretreated with 20 µM PD-98059 [PD-98059 + G(-)/TG; n = 3] or 0.5 µM KT-5823 [KT-5823 + G(-)/TG; n = 6] in HBSS during 30-min baseline period. Cells were exposed to glucose-free HBSS-5 mM thioglycolic acid for 60 min and switched to HBSS for 30 min. Permeability was expressed as fold increase over baseline. MAP kinase inhibition (with PD-98059) and PKG inhibition (with KT-5823) significantly attenuated the increased permeability due to hypoxia/aglycemia. Values are means ± SE. * P < 0.05, ** P < 0.01, and *** P < 0.001, KT-5823 + G(-)/TG vs. G(-)/TG; # P < 0.05 and ### P < 0.001, PD-98059 + G(-)/TG vs. G(-)/TG.

Effect of thioglycolic acid on cell viability. Trypan blue staining and general cell appearance indicated that there were no significant changes in viability of cells between groups and the times of trypan blue staining, and no (<1%) cells were trypan blue stained or detached.

Intracellular Ca2+ change in response to hypoxia/aglycemia. Changes in the emission ratio of fura 2 were used to assess relative changes in the HMEC-1 intracellular [Ca2+] (12). The average basal HMEC-1 fura 2 ratio when incubated with HBSS was found to be 0.56 ± 0.04 in cultured monolayers. When the medium was switched to glucose-free HBSS-5 mM thioglycolic acid, the fura 2 ratio increased by 12. 43 ± 0.30% (P < 0.01, n = 3) and 22. 43 ± 1.67% (P < 0.01, n = 3) after 30 and 60 min, respectively. However, 30 min after switching back to HBSS, this ratio returned to a level that was similar to that seen after 30 min of hypoxia/aglycemia (Fig. 5A). These effects of hypoxia/aglycemia were completely abolished by a 30-min preincubation with 1 mM LaCl3, which shows that the observed Ca2+ increase is mainly due to a Ca2+ influx from the extracellular space (Fig. 5B). In contrast to hypoxia/aglycemia, acute exposure of cells to histamine (10 µM) resulted in a very rapid peak in the fluorescence ratio, which doubled within 1 min (Fig. 5B, inset). As a control, 5 µM Br-A-23187 was used to equilibrate the [Ca2+] across the HMEC-1 cell membrane. Br-A-23187 reliably produced two- to threefold increases in signal, consistent with a specific increase in the intracellular [Ca2+] (Fig. 5A).



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Fig. 5.   Effect of hypoxia/aglycemia, Br-A-23187, LaCl3, and histamine on intracellular Ca2+ concentration in HMEC-1. A: original trace of the 340-to-380 nm (340/380) ratio of fluorescence of fura 2-loaded HMEC-1 that were exposed to glucose-free HBSS-5 mM thioglycolic acid for 60 min and then switched to HBSS for 30 min and the ratio under the influence of 5 µM Br-A-23187. Gradual increase in intracellular Ca2+ produced by hypoxia/aglycemia can be seen. Exposure to Br-A-23187 (positive control) produced a large increase in intracellular Ca2+. B: 340/380 ratio under the influence of 1 mM LaCl3 and of 10 µM histamine (inset). Increase in Ca2+ produced by hypoxia/aglycemia was blocked by 1 mM LaCl3. In inset, HMEC-1 monolayers increased intracellular Ca2+ upon challenge with 10 µM histamine.

Changes in cadherin-occludin-cytoskeleton structure. Because nonbinding, junctionally dissociated proteins were removed after treatment with our extraction buffer, proteins that left over monolayers are either part of the Triton-insoluble cytoskeleton or a protein tightly bound to it. We measured this binding by comparison of the densitometry of our blots under different treatment conditions. Figure 6 shows that junctional protein binding to the cytoskeleton was not changed significantly by hypoxia alone, or by aglycemia alone. However, when endothelial monolayers were exposed to hypoxia/aglycemia together, the concentrations of "cytoskeleton-associated" occludin and pan-reactive cadherin were decreased significantly. Under these conditions, the concentrations of cytoskeleton-associated ZO-1 and beta -catenin did not change (Fig. 6). Interestingly, these effects of hypoxia/aglycemia on dissociation of pan-reactive cadherin and occludin from the cytoskeleton were blocked by pretreatment with 0.1 mM TMB-8, 10 nM Gö-6976 (Fig. 7A), 0.5 µM KT-5823 (not in occludin), or 20 µM PD-98059 (Fig. 7B).


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Fig. 6.   Dot-blotting analysis for cytoskeleton-associated pan-cadherin, occludin, beta -catenin, and ZO-1 in HMEC-1 treated with HBSS (control), glucose-free HBSS [G(-)], HBSS-5 mM thioglycolic acid (TG), or glucose-free HBSS-5 mM thioglycolic acid [G(-)/TG] for 60 min. Junctional protein binding to the cytoskeleton was not changed significantly by hypoxia alone or by aglycemia alone. However, the concentrations of occludin and pan-cadherin binding to the cytoskeleton decreased significantly after hypoxia/aglycemia treatment. beta -Catenin and ZO-1 did not change their association with cytoskeleton under the same condition. Each value represents mean ± SE, n = 3. *** P < 0.001 compared with control.




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Fig. 7.   Dot-blotting analysis of the effects of TMB-8, Gö-6976, KT-5823, and PD-98059 on the dissociation of pan-reactive cadherin and occludin from the cytoskeleton induced by hypoxia/aglycemia. Before exposure to glucose-free HBSS-5 mM thioglycolic acid for 60 min, HMEC-1 monolayers were incubated for 30 min with 0.1 mM TMB-8, 10 nM Gö-6976, 0.5 µM KT-5823, or 20 µM PD-98059. Dissociation of occludin and pan-cadherin from the cytoskeleton was prevented by classical PKC inhibition and by intracellular Ca2+ chelation (A). Occludin dissociation was partly blocked by MAP kinase, but not by PKG inhibition. Pan-cadherin dissociation was blocked by MAP kinase and by PKG inhibition (B). Each value represents mean ± SE, n = 3. *** P < 0.001, G(-)/TG vs. control; ### P < 0.001 vs. G(-)/TG.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study established that a chemical ischemia (using thioglycolic acid and aglycemia) is a useful model for studying the effects of ischemia on microvascular endothelial barrier. Because thioglycollate produces a rapid, nontoxic, and controllable hypoxic environment, thioglycollate models may permit comparison of several different levels of ischemia with and without other challenges. Most previous ischemia studies have focused on the effects of O2 depletion alone on endothelial functions (13, 22, 23). However, because ischemia is an interruption of both the O2 and nutrient supply, we added glucose depletion to this model as an additional relevant stress.

We found that solute permeability was slightly increased by hypoxia or by glucose deprivation separately. However, together, hypoxia and aglycemia produced large changes in solute permeability (60 min permeability, 152 ± 11, 135 ± 5, and 189 ± 18% of baseline, respectively). These findings are consistent with a report by Abbruscato and Davis (1) that hypoxia alone needed long exposures (48 h) to result in a significant increase in permeability of bovine brain microvascular endothelial cells to [14C]sucrose. Statistically significant changes in basal permeability were observed between 3 and 6 h of both hypoxia and aglycemia. Rasio et al. (37) also reported a three- to fourfold increased permeability in the eel rete mirabile capillaries after metabolic depletion with cyanide and iodoacetate in a glucose-free medium. We found that permeability did not rapidly reverse after the restoration of O2 and glucose. This result is at least consistent with other reports showing that reoxygenation increases the microvascular permeability (22, 23).

Hypoxic damage to endothelial cells may be made worse under aglycemia because of rapid ATP depletion (1). Depletion of high energy metabolites (e.g., glucose) during tissue ischemia may alter several cell second messenger systems that alter barrier function, that is, ATP depletion causes Ca2+ influx into the cell (40), and Ca2+ ionophore promotes transendothelial flux of water and proteins (17, 38). Other studies have demonstrated that reduced ATP generation results in restructuring and loss of actin filaments in endothelial cells (1, 37, 44). We found that hypoxia/aglycemia produced a modest and slow increase in intracellular Ca2+, and the effects on Ca2+ flux were completely prevented by a 30-min preincubation with LaCl3, a nonspecific Ca2+ channel blocker. Also, the microvascular endothelial hyperpermeability induced by hypoxia/aglycemia was blocked by pretreatment with the cell Ca2+ chelator TMB-8 (0.1 mM). Abbruscato and Davis (1) also reported that the L-type Ca2+ channel antagonist nifedipine (10 nM) and the blocker of a nonspecific cation channel, SKF-96365 (100 nM), significantly reduced the permeability change induced by 6 h of hypoxia and aglycemia. These data suggest that ambient glucose and O2 are necessary to maintain low intracellular Ca2+ levels, which are elevated early in several forms of hyperpermeability (13, 19).

Histamine, a typical physiological mediator that produces leaky sites in postcapillary venules during inflammation, has been shown to promote albumin diffusion by stimulating Ca2+ influx into endothelial cells and affecting their integrity (38). Ikeda et al. (21) revealed that histamine produces a rapid peak of intracellular Ca2+ influx in cultured human mucosal microvascular endothelial cells. We also observed similar responses to histamine in HMEC-1.

Under normal circumstances, the vascular endothelium forms a tightly regulated barrier whose permeability can be either increased or decreased by vasoactive mediators (e.g., H2O2, bradykinin, thrombin, epinephrine, histamine, and adenosine; see Refs. 11, 14, 15, 19, 30, 35, 38). Several papers reported that barrier is also reversibly modified by exposure to second messengers that mobilize these barrier-regulating agents (e.g., cAMP, cGMP, PKC, Ca2+, NO ·) and may be related to actin and junctional protein reorganization (4, 27).

Some reports showed that intracellular Ca2+ changes precede and may activate NO synthase (NOS) activity to modulate the endothelial barrier function (13, 24, 29, 33). Huang and Yuan (19) suggested two main messages in endothelial permeability involving NO. In the first, agonists like histamine stimulate phospholipase C to yield inositol polyphosphates (inositol trisphosphate, inositol tetrakisphosphate, etc.), which mobilize stored and extracellular Ca2+, triggering NO production. NO stimulates guanylate cyclase to produce cGMP, a potent activator of PKG. The NO-cGMP-PKG system may regulate barrier by modulating structure and/or function of proteins in the endothelial cytoskeleton or junction or by controlling cell-matrix contact. The second leg is via PKC. PKC may increase permeability by directly acting on the endothelial structural proteins or by indirectly modifying NOS activity (19). Although we did not directly measure NO or cGMP production by hypoxia/aglycemia, we did demonstrate that the effects of hypoxia plus aglycemia on barrier function were partially blocked by pretreatment with the PKG inhibitor KT-5823. Similarly, the PKC inhibitor Gö-6976 also blocked these permeability changes. The data support a scheme in which interactions between the Ca2+-NOS-PKG and the PKC pathways may play important roles in the control of permeability during ischemia.

Parenti et al. (36) extended these findings and suggested that NO and cGMP contributed to the VEGF-dependent ERK1/2 activation. Huot et al. (20) have also shown that peroxide administration causes an early increase in ERK1/2 activity, a more prolonged increase in p38 MAP kinase activation, and rearrangement of the actin cytoskeleton. This rearrangement involved activation of p38 MAP kinase, MAP kinase-activated protein kinase-2/3, and heat shock protein 27. Our data also show that hypoxia/aglycemia-induced hyperpermeability was significantly attenuated by pretreatment with a specific inhibitor of MAP kinase-1, PD-98059. Clearly, MAP kinases appear to also be important signal intermediates in several models of microvascular permeability, including ischemia.

The literature now suggests that microvascular endothelial permeability is regulated through 1) receptor activation, 2) second messenger mobilization, 3) cytoskeleton, and 4) junction reorganization (14, 15, 30, 31, 42). Although all of these models assume that interactions of the cytoskeleton with the cell membrane modify cell shape, apposition, and barrier function, the specific role of individual components of the cytoskeleton and the junction remains undefined. The actin cytoskeleton interacts with both the cadherins and the occludins, two classes of cell-cell adhesion molecules present in the adherens and occludens junctions that promote cell apposition and barrier (5, 10, 25). However, it is not understood how junctional protein organization or cytoskeleton binding regulates barrier function.

Recent evidence (2, 3, 18, 28) suggests that beta -catenin may serve a critical function in controlling cadherin function, that is, cadherins that are not associated with the actin cytoskeleton (through alpha - and beta -catenins) cannot support normal cadherin cell-cell homotypic binding (28). We observed that junctional protein binding to the cytoskeleton was not changed significantly by hypoxia alone, or by aglycemia alone. However, the concentrations of occludin and pan-reactive cadherin binding to the cytoskeleton were both significantly decreased when monolayers were treated with hypoxia and aglycemia together. Importantly, the concentrations of ZO-1 and beta -catenin did not show such changes. The effects of hypoxia/aglycemia on dissociation of pan-reactive cadherin and occludin from the cytoskeleton were blocked by pretreatment with TMB-8, Gö-6976, KT-5823, or PD-98059. These altered cadherin/catenin and occludin/ZO-1/ZO-2 binding interactions may be consistent with diminished junctional adhesion and increased endothelial permeability.

We have previously demonstrated that interference with the normal binding capacity of cadherins increased endothelial permeability in vitro (5). Therefore, our data here suggest that second messenger-regulated cytoskeleton binding interactions may control junction apposition and permeability. These data may also indicate that the quantity of functionally active (i.e., "bound") junctional protein (cadherin or occludin) is a central determinant of barrier.

In conclusion, we have shown that chemical hypoxia/aglycemia, a novel model of ischemia, increases microvascular endothelial permeability through elevation of Ca2+ and activation of PKG, PKC, and MAP kinase, which may control cadherin-occludin-cytoskeleton binding. Therapies that control these specific second messengers or stabilize the bonds between junctional proteins and the cytoskeleton may provide novel means of controlling inflammation.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HL-47615 and DK-43785.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. S. Alexander, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (E-mail: jalexa{at}lsumc.edu).

Received 22 March 1999; accepted in final form 2 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(6):C1066-C1074
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