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

Chinnaswamy Tiruppathi*, Tabassum Naqvi*, Raudel Sandoval, Dolly Mehta, and Asrar B. Malik

Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Ca2+ 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 Ca2+ concentration ([Ca2+]i) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca2+]i lasting for 3 min, whereas TNF-alpha alone had no effect on [Ca2+]i. Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-alpha -exposed cells. In the absence of extracellular Ca2+, thrombin produced similar increases in [Ca2+]i in both control and TNF-alpha -exposed cells. In TNF-alpha -exposed cells, the thrombin-induced Ca2+ influx after intracellular Ca2+ store depletion was significantly greater and prolonged compared with control cells. Increased Ca2+ 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 Ca2+ influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.

store-operated calcium influx; Src tyrosine kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF)-alpha released during bacterial infection is a primary cause of the microvascular complications such as endothelial injury and tissue edema associated with sepsis (6, 17, 39). TNF-alpha increases the procoagulant activity by inducing the activation of coagulation factors such as thrombin and decreases the anticoagulant activity in the microvascular bed, resulting in fibrin deposition (5, 6, 13, 22, 26, 27). Because TNF-alpha generation can coexist with the activation of thrombin (6, 17, 39), the effects of these two mediators may be synergistic; however, it remains unclear whether TNF-alpha modulates thrombin-mediated responses such as leakiness of the microvascular endothelial barrier, and if so, by what mechanism.

Thrombin increases endothelial permeability to plasma proteins by inducing the formation of interendothelial gaps (10, 15, 20, 25, 34, 35, 37) as the direct result of signaling events activated by increased cytosolic Ca2+ concentration ([Ca2+]i) (4, 18, 19, 34, 35, 41). Thrombin acts on the endothelial cell surface proteinase-activated receptor-1 to elicit inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation, which increases [Ca2+]i by depleting Ca2+ stores and causing Ca2+ influx (18, 34, 35, 38). The rise in [Ca2+]i contributes to the mechanism of permeability increase at multiple levels. Increased [Ca2+]i is required for phosphorylation of myosin light chain by activation of Ca2+/calmodulin-dependent myosin light chain kinase, which, in turn, results in endothelial cell contracture and increased paracellular permeability (10, 15, 20, 25, 37). The increase in [Ca2+]i also induces vascular endothelial (VE)-cadherin junction disassembly by activation of the Ca2+-dependent protein kinase C-alpha isoform, which contributes to the increased endothelial permeability response (34). In addition, intracellular store Ca2+ depletion secondary to activation of Ins(1,4,5)P3 receptors is an essential requirement for activation of store-operated Ca2+channels and thus enables the influx of Ca2+ (14, 21, 30). Prevention of store-operated Ca2+ entry into endothelial cells reduced the thrombin-induced increase in transendothelial permeability (24, 29, 35), indicating an important role for store-operated Ca2+ channels in the mechanism of the response. Because Ca2+ signaling is a critical determinant of endothelial barrier dysfunction, in the present study, we addressed the effects of TNF-alpha in regulating the thrombin-activated Ca2+ influx and how this influences the endothelial permeability response. We show that TNF-alpha dramatically augments thrombin-induced Ca2+ influx in endothelial cells and that this response is sensitive to Src tyrosine kinase inhibition. Moreover, the increased Ca2+ influx induces protein tyrosine phosphorylation of adherens junctional proteins and increased actin stress fiber formation as well as augmenting the endothelial permeability response to thrombin. These results show that TNF-alpha activates Src tyrosine kinase and thereby induces Ca2+ influx in response to thrombin, resulting in the severe loss of endothelial barrier integrity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Human alpha -thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Endothelial growth medium (EBM-2) was obtained from Clonetics (San Diego, CA). Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced salt solution (HBSS), L-glutamine, phosphate-buffered saline (PBS), and trypsin were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was obtained from HyClone Laboratories (Logan, UT). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). Anti-c-Src polyclonal antibody (Ab), anti-v-Src monoclonal antibody (MAb), and protein A/G plus agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Src tyrosine kinase-specific inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) was purchased from Calbiochem (La Jolla, CA). An anti-phosphotyrosine MAb was from PharMingen (San Diego, CA). An Src kinase assay kit was obtained from Upstate Biotechnology (Lake Placid, NY). [gamma -32P]ATP was purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) obtained from Vec Technologies (Rensselaer, NY) were grown in EBM-2 supplemented with 10% FBS. Cells were cultured on tissue culture dishes coated with 0.1% gelatin and used between passages 4 and 8.

[Ca2+]i. The thrombin-induced increase in [Ca2+]i was measured with the Ca2+-sensitive fluorescent dye fura 2 (38). Cells were grown to confluence on 0.1% gelatin-coated 22-mm glass coverslips and then washed two times with serum-free medium and incubated for 2 h at 37°C in culture medium containing 1% FBS. They were washed two times with HBSS and then loaded with 3 µM fura 2-AM for 30 min at 37°C. After the loading, the cells were washed two times with HBSS. The cells were imaged with an Attoflor Ratio Vision digital fluorescence microscopy system (Atto Instruments, Rockville, MD) equipped with a Zeiss Axiovert S100 inverted microscope and F-Fluar ×40, 1.3 numerical aperture oil-immersion objective. Regions of interest in individual cells were marked and excited at 334 and 380 nm, with emission at 520 nm, at 5-s intervals. At the end of each experiment, 10 µM ionomycin was added to obtain the fluorescence of Ca2+-saturated fura 2 (high [Ca 2+]i) and 10 mM EGTA was added to obtain the fluorescence of free fura 2 (low [Ca2+]i). [Ca2+]i was calculated based on a dissociation constant of 225 nM with a two-point fit curve.

Ins(1,4,5)P3 generation. Ins(1,4,5)P3 generation in response to thrombin was measured by using a Biotrak D-myo-Ins(1,4,5)P3 3H assay kit from Amersham Pharmacia Biotech. Endothelial cells grown to confluence on 100-mm culture dishes were washed two times with medium containing 0.1% FBS and incubated for 2 h at 37°C in similar medium. After incubation, the cells were incubated with and without TNF-alpha (250 U/ml) for 2 h and then stimulated with thrombin (5 U/ml) for 15 and 30 s. The reaction was stopped by the addition of ice-cold 15% (vol/vol) trichloroacetic acid. The endothelial cells were scraped off the dishes, after which the cells were centrifuged for 15 min at 2,000 g. The supernatant was washed three times with 10 volumes of water-saturated diethyl ether and neutralized by titration to pH 7.5 with NaHCO3. Total Ins(1,4,5)P3 generation was determined following the protocol described by Amersham Pharmacia Biotech.

Immunoprecipitation and immunoblotting. HUVECs grown in 100-mm culture dishes were incubated with medium containing 1% FBS for 2 h at 37°C and exposed to TNF-alpha for 2 h before stimulation with thrombin. The cells were lysed with lysis buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 1 µg/ml of pepstatin) at 4°C for 30 min. The lysates were collected and centrifuged at 14,000 rpm at 4°C for 30 min. The clear supernatants were precleared with protein A/G agarose beads for 60 min at 4°C. The precleared supernatants were incubated with rabbit polyclonal anti-c-Src Ab at 4°C overnight with constant shaking. After incubation, protein A/G agarose beads were added, incubated for an additional hour at 4°C with constant shaking, and centrifuged at 14,000 rpm for 5 min. Beads were washed three times with ice-cold PBS containing 1 mM sodium orthovanadate and finally dissociated by boiling in SDS sample buffer for 5 min. The proteins were resolved by SDS-PAGE and transferred to Duralose membranes (Stratagene). The membranes were blocked with 5% nonfat dry milk in 10 mM Tris · HCl, pH 7.7, 150 mM NaCl, and 0.05% Tween 20 for 1 h at 22°C. The membranes were incubated with the indicated primary Abs (diluted in blocking buffer) at 22°C for 1 h. After three washes with wash buffer, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse Ab for 1 h. The protein bands were detected by enhanced chemiluminescence (Pierce Chemical, Rockford, IL).

Src kinase activity. Src activity was measured with the in vitro kinase assay kit from Upstate Biotechnology. Src was immunoprecipitated as described in Immunoprecipitation and immunoblotting, and the immunoprecipitate was washed three times with ice-cold PBS and suspended in kinase assay buffer (50 mM HEPES, pH 7.5, containing 0.1 mM EDTA and 0.015% polyoxyethylene glycol dodecyl ether). The final reaction volume (60 µl) contained 50 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.015% polyoxyethylene glycol dodecyl ether, 15 mM MgCl2, 1 mM Na3VO4, 150 µM ATP, and 33 µCi of [gamma -32P]ATP with and without 150 µM peptide substrate (KVEKIGEGTYGVVYK). The reaction was continued for 10 min at 30°C and terminated by adding 1% phosphoric acid. The samples were spotted on P81 filter paper and washed five times with 1% phosphoric acid and one time with methanol, and then the radioactivity associated with the filter was determined in a scintillation counter.

Phosphotyrosine immunostaining. HUVECs were grown to confluence on glass coverslips coated with 0.1% gelatin. The cells were washed three times with serum-free medium and kept for 2 h at 37°C in medium containing 1% FBS. Then the cells were primed with TNF-alpha (250 U/ml) in medium containing 1% FBS for 2 h at 37°C and then stimulated with thrombin. The cells were washed three times with ice-cold HBSS and fixed with 2% paraformaldehyde for 15 min at 22°C. The cells were then blocked with 1% bovine serum albumin in HBSS at 22°C for 60 min and then incubated with the anti-phosphotyrosine MAb (5 µg/ml) for 60 min at 22°C. The cells were washed two times with HBSS and incubated with rhodamine-labeled goat anti-mouse IgG (5 µg/ml) for 45 min at 22°C. After three washes with HBSS, the coverslips were mounted on glass slides with the ProLong antifade mounting medium kit (Molecular Probes). Total cell fluorescence was visualized with digital-imaging fluorescence microscopy as previously described (4).

Distribution of actin stress fibers. HUVECs were grown to confluence on gelatin-coated glass coverslips. The cells were washed three times with HBSS and fixed with 4% paraformaldehyde for 20 min at 22°C and then permeabilized with 0.1% Triton X-100 for 30 min at 22°C. The cells were stained with Alexa 568-phalloidin for 30 min at 22°C, washed two times with HBSS, mounted with ProLong antifade mounting medium, and viewed under a digital fluorescence microscope (Diapshot 200, Nikon Instruments, Fair Lawn, NJ).

Transendothelial cell electrical resistance. The thrombin-induced endothelial cell contractile response was measured as previously described (37). HUVECs were seeded on a gelatin-coated gold electrode (5.0 × 10-4 cm2) and grown to confluence. The small electrode and the larger counterelectrode were connected to a phase-sensitive lock-in amplifier. An approximate constant current of 1 µA was supplied by a 1-V, 4,000-Hz AC signal connected serially to a 1-MOmega resistor between the small electrode and the larger counterelectrode. The voltage between the small electrode and the large electrode was monitored by lock-in amplifier, stored, and processed with a personal computer. The same computer controlled the output of the amplifier and switched the measurement to different electrodes in the course of an experiment. Before the experiment, the confluent endothelial monolayer was kept in medium containing 1% FBS for 2 h. The cells were incubated with and without TNF-alpha for 2 h, and then the thrombin-induced change in resistance of the endothelial monolayer was measured. In some experiments, the TNF-alpha -induced change in resistance was measured directly. The data are presented as resistance normalized to its value at time 0 as previously described (3, 4, 37).

Transendothelial 125I-albumin permeability. Permeability of 125I-albumin across the HUVEC monolayer was determined with Costar Transwell units as previously described (34, 35). This system measures transendothelial flux (luminal to abluminal) of tracer macromolecules in the absence of hydrostatic and oncotic pressure gradients. The system consists of luminal and abluminal compartments separated by a polycarbonate filter (0.4-µm pore size, 6.5-mm diameter). The luminal (upper) side of filters was coated with gelatin. HUVECs were seeded at 105 cells/filter and grown for 4-5 days to attain confluence. The cells were washed and incubated with medium containing 1% FBS for 2 h before TNF-alpha exposure for 2 h at 37°C. HUVEC monolayers were then transferred to 10 mM HEPES-DMEM, pH 7.4, containing 5 mg/ml of bovine serum albumin. Both the luminal and abluminal compartments contained the same medium at volumes of 0.2 and 1.0 ml, respectively. Thrombin (5 U/ml) was added to the upper chamber containing tracer 125I-albumin (5 × 106 counts · min-1 · ml-1). After the addition of thrombin, 0.05-ml samples from the lower compartment were collected at 10-min intervals for 90 min for determination of the transendothelial clearance rate of 125I-albumin as previously described (34, 35).

Statistical analysis. Statistical comparisons were made with two-tailed Student's t-test. Values are reported as means ± SE. Differences in mean values between and among groups were measured by one-way analysis of variance with Bonferroni correction. Values were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha augments the thrombin-induced increase in [Ca2+]i. We exposed HUVECs to varying concentrations of TNF-alpha (0.5-3 ng/ml, i.e., 50-300 U/ml) for 2 h and measured the transendothelial monolayer electrical resistance to assess endothelial monolayer integrity. We observed no significant changes in transendothelial monolayer electrical resistance compared with that in the control HUVEC monolayer. To study whether TNF-alpha exposure produces a synergistic effect on thrombin-activated responses in HUVECs, we first measured the changes in [Ca2+]i. We exposed TNF-alpha (250 U/ml) for 30 and 60 min and 2 h and then measured the thrombin-induced increase in [Ca2+]i. In control cells, thrombin increased the [Ca2+]i (peak value of 1,070 ± 75 nM) followed by a gradual decline to the basal level (67 ± 5 nM) within 3 min after thrombin challenge (Fig. 1A, Table 1). TNF-alpha exposure up to 60 min had no significant effect on the thrombin-induced increase in [Ca2+]i (Table 1). Also, TNF-alpha alone had no significant effect on [Ca2+]i (Fig. 1B, inset). However, in cells pretreated with TNF-alpha for 2 h, thrombin produced an initial peak increase in the [Ca2+]i (peak value of 1,085 ± 78 nM; Fig. 1B, Table 1), but the value remained elevated (380 ± 25 nM 3 min after thrombin addition) and reached the basal level ~10 min after thrombin challenge (Fig. 1B). Thus TNF-alpha exposure for 2 h markedly amplified the thrombin-induced increase in [Ca2+]i in HUVECs. Further experiments were carried out exposing HUVECs with TNF-alpha for 2 h to study its synergistic effects with thrombin.


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Fig. 1.   Effects of tumor necrosis factor (TNF)-alpha exposure on thrombin (Thr)-induced increase in cytosolic Ca2+ concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). HUVECs grown to confluence on glass coverslips were washed 2 times with medium containing 1% fetal bovine serum (FBS) and incubated with the same medium for 2 h at 37°C. Cells were then exposed to TNF-alpha (250 U/ml) for 2 h at 37°C. After incubation, cells were washed, loaded with 3 µM fura 2-AM in Hanks' balanced salt solution (HBSS) for 30 min at 37°C, and then stimulated with thrombin (5 U/ml) to measure changes in [Ca2+]i.. A: control cells. B: cells exposed to TNF-alpha . B, inset: TNF-alpha (250 U/ml) was added to fura 2-AM-loaded cells to measure change in [Ca2+]i. Arrows, times at which thrombin was added. Results are means of a representative experiment; n = 40-60 cells/experiment. Experiment was repeated 4 times. The results are summarized in Table 1. Other details are described in METHODS.


                              
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Table 1.   Effects of TNF-alpha exposure on thrombin-induced increase in [Ca2+]i in HUVECs

TNF-alpha has no effect on thrombin-induced Ins(1,4,5)P3 generation. To address whether the effects of TNF-alpha on thrombin-induced increase in [Ca2+]i are the result of differences in the production of Ins(1,4,5)P3, we measured thrombin-induced Ins(1,4,5)P3 generation in control and TNF-alpha -exposed cells. Basal Ins(1,4,5)P3 levels were not significantly changed in these two groups (Table 2). Moreover, thrombin exposure produced similar increases in Ins(1,4,5)P3 concentration in both control and TNF-alpha -pretreated cells (Table 2), indicating that the increased Ins(1,4,5)P3 production does not account for the greater rise in [Ca2+]i.

                              
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Table 2.   Effects of TNF-alpha on thrombin-induced inositol 1,4,5-trisphosphate generation

TNF-alpha amplifies thrombin-induced Ca2+ influx. Because thrombin-induced Ins(1,4,5)P3 generation was not affected by TNF-alpha exposure, we measured the thrombin response in the absence of extracellular Ca2+ to assess Ca2+ influx. In the absence of extracellular Ca2+, thrombin produced an initial peak increase in [Ca2+]i that rapidly returned to the baseline value in control cells (Fig. 2A, Table 2). The addition of Ca2+ to the extracellular medium after thrombin-induced Ca2+ store depletion caused Ca2+ entry; the value peaked at 615 ± 55 nM and returned to the basal level as in control cells (Fig. 2A, Table 2). In TNF-alpha -primed cells in the absence of extracellular Ca2+, thrombin produced an initial peak increase in [Ca2+]i similar to that in control cells; however, the addition of Ca2+ to the extracellular medium after store depletion produced a significantly greater increase in the [Ca2+]i (peak value of 948 ± 70 nM; Fig. 2B, Table 3), and the elevated [Ca2+]i persisted for a longer period compared with the control value (Fig. 2, A vs. B). The addition of Ca2+ in the extracellular medium in control or TNF-alpha -exposed cells alone did not induce Ca2+ influx (data not shown). These results suggest that thrombin-induced intracellular store depletion induces Ca2+ entry in HUVECs and, moreover, that TNF-alpha exposure augmented the Ca2+ influx in response to thrombin.


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Fig. 2.   Effects of TNF-alpha exposure on thrombin-induced Ca2+ entry in HUVECs. HUVECs grown to confluence on glass coverslips were incubated with and without TNF-alpha (250 U/ml) for 2 h in medium containing 1% FBS and then loaded with fura 2-AM for 30 min at 37°C. Cells were washed 2 times, placed in Ca2+- and Mg2+-free HBSS, and then stimulated with thrombin (5 U/ml). After return of [Ca2+]i to baseline levels, cells were stimulated with CaCl2 to induce Ca2+ influx. Src kinase inhibitor PP1 (10 µM) was incubated with cells 30 min before measurement of the change in [Ca2+]i and was present during the entire Ca2+ measurement experiment. Arrows, times at which thrombin or Ca2+ was added. A and C: control cells. B and D: cells exposed to TNF-alpha . C and D: cells treated with PP1. Data are from a representative experiment. Results from 4-5 experiments are summarized in Table 2. Other details are described in METHODS and Fig. 1.


                              
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Table 3.   TNF-alpha augments thrombin-induced Ca2+ influx in HUVECs

Previous studies have shown that tyrosine kinase inhibitors such as genistein prevented bradykinin-stimulated Ca2+ influx in fibroblasts (2) and endothelial cells (7). To study the possible role of Src tyrosine kinase activation in mediating Ca2+ entry in HUVECs, we determined the effect of the Src family tyrosine kinase inhibitor PP1 on the thrombin-induced increase in [Ca2+]i. HUVECs were incubated with 10 µM PP1 for 30 min and then challenged with thrombin. PP1 inhibited ~70% of the thrombin-induced Ca2+entry in control cells (Fig. 2, A vs. C, Table 3) as well as in TNF-alpha exposed cells (Fig. 2, B vs. D, Table 3), without affecting the initial peak increase in [Ca2+]i. Increasing the PP1 concentration had no additional effect in reducing the Ca2+ entry in HUVECs (data not shown).

TNF-alpha increases Src activity in endothelial cells. Because the Src tyrosine kinase-specific inhibitor prevented the store depletion-mediated Ca2+ entry in both control and TNF-alpha -exposed cells, we measured the autophosphorylation of Src to assess Src activation. The cell lysates were immunoprecipitated with anti-Src Ab and blotted with anti-phosphotyrosine MAb (see METHODS). TNF-alpha or thrombin treatment alone increased tyrosine phosphorylation of Src by approximately twofold (Fig. 3A). The addition of thrombin to TNF-alpha -exposed cells further increased tyrosine phosphorylation of Src (Fig. 3A). Because tyrosine phosphorylation of Src can either activate (Tyr416 phosphorylation) or inhibit (Tyr527 phosphorylation) Src activity (12), we measured Src activity with a peptide substrate (see METHODS). HUVECs were incubated with TNF-alpha (250 U/ml) for 2 h and lysed, and the cell lysates were immunoprecipitated with anti-Src Ab. The immunoprecipitated proteins were used for the in vitro Src kinase assay. Thrombin increased the phosphorylation of the peptide substrate by approximately twofold over the basal value (Fig. 3B). TNF-alpha treatment alone increased the phosphorylation of the peptide substrate by approximately twofold over the control value (Fig. 3B). The addition of thrombin to TNF-alpha -exposed cells further increased phosphorylation of the peptide substrate (Fig. 3B).


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Fig. 3.   A: TNF-alpha exposure induces tyrosine phosphorylation of Src in HUVECs. HUVECs grown to confluence on 100-mm dishes were washed 2 times with serum-free medium, incubated for 2 h at 37°C in medium containing 1% FBS, and then exposed to TNF-alpha for 2 h at 37°C. Cells were then stimulated with thrombin (5 U/ml) for 2 min, washed 2 times with ice-cold PBS, and lysed, and cell lysates were immunoprecipitated with anti-Src antibody. Other experimental details are described in METHODS. Precipitated proteins were resolved by SDS-PAGE, transferred to Duralose membranes, and blotted with either anti-Src (a) or anti-phosphotyrosine (PY) monoclonal antibody (MAb; b). IB, immunoblot. Data are representative of 3 independent sets of experiments. B: TNF-alpha exposure induces Src kinase activity in HUVECs. HUVECs grown to confluence in 100-mm dishes were washed 2 times with serum-free medium, incubated for 2 h at 37°C in medium containing 1% FBS for 2 h, and then exposed to TNF-alpha for 2 h at 37°C. Cells were then stimulated with thrombin (5 U/ml) for 2 min, washed 2 times with ice-cold PBS, and lysed, and cell lysates were immunoprecipitated with anti-Src polyclonal antibody. Immunoprecipitated proteins were used for in vitro phosphorylation of peptide substrate (see details in METHODS). cpm, Counts/min. Values are means ± SE. The experiment was repeated 4 times. Significantly different compared with control cells (not stimulated with either thrombin or TNF-alpha ): * P < 0.05; ** P < 0.001.

TNF-alpha increases thrombin-induced tyrosine phosphorylation of adherens junction proteins. We next determined the thrombin-induced tyrosine phosphorylation of adherens junction proteins in control and TNF-alpha -exposed cells. Thrombin markedly increased tyrosine phosphorylation of junction proteins along with the formation of intercellular gaps in control cells (Fig. 4, A vs. C). TNF-alpha alone slightly increased tyrosine phosphorylation of junctional proteins but failed to induce gap formation (Fig. 4E). Stimulation of TNF-alpha -exposed cells with thrombin produced a greater increase in tyrosine phosphorylation of junctional proteins than thrombin alone and greater interendothelial gap formation over thrombin alone (Fig. 4, G vs. C). The Src tyrosine kinase inhibitor PP1 prevented thrombin-induced tyrosine phosphorylation in both control and TNF-alpha -primed cells (Fig. 4, B, D, F, and H). Furthermore, PP1 treatment significantly reduced the size of the interendothelial gaps in response to thrombin (Fig. 4, B, D, F, and H).


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Fig. 4.   Effects of TNF-alpha exposure on thrombin-induced tyrosine phosphorylation of junctional proteins in HUVECs. HUVECs grown to confluence on glass coverslips were washed 2 times with serum-free medium and then incubated with 1% FBS-containing medium for 2 h at 37°C. The cells were then exposed to TNF-alpha for 2 h at 37°C in medium containing 1% FBS. In some experiments, cells were preincubated with PP1 (10 µM) for 30 min before addition of either TNF-alpha (250 U/ml for 2 h) or thrombin (5 U/ml for 10 min). Cells were washed, fixed, permeabilized, and incubated with anti-phosphotyrosine MAb (5 µg/ml) for 60 min at 22°C. The anti-phosphotyrosine MAb binding was detected by incubation with rhodamine-labeled goat anti-mouse IgG (5 µg/ml) for 45 min. Cells were washed, and fluorescence was visualized with a digital-imaging fluorescence microscope. A: control cells. B: PP1-treated control cells. C: thrombin-treated cells. D: PP1 + thrombin-treated cells. E: TNF-alpha -treated cells. F: PP1 + TNF-alpha -treated cells. G: TNF-alpha  + thrombin-treated cells. H: PP1 + TNF-alpha  + thrombin-treated cells. Arrows, interendothelial gap formation. The experiment was repeated 3 times with similar results. Bar, 20 µm.

TNF-alpha augments thrombin-induced stress fiber formation. Because TNF-alpha priming enhanced Ca2+ entry in response to thrombin, we measured actin stress fiber formation in control and TNF-alpha -treated cells. In control as well as in TNF-alpha (250 U/ml for 2 h)-exposed cells, only peripheral actin bands were observed (Fig. 5). In control cells, thrombin increased the actin stress fiber formation within 5 min, and actin filamentous changes returned to normal within 2 h after thrombin challenge (Fig. 5, top). In TNF-alpha -exposed cells, thrombin induced a marked increase in stress fiber formation (Fig. 5, bottom) and prolonged the recovery time compared with control cells (Fig. 5, bottom vs. top).


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Fig. 5.   Effects of TNF-alpha exposure on thrombin-induced actin stress fiber formation. HUVECs were grown to confluence on gelatin-coated glass coverslips. Cells were washed and incubated with 1% FBS-containing medium for 2 h at 37°C and then incubated with and without TNF-alpha for 2 h. Cells were then challenged with thrombin (5 U/ml) for the indicated times, washed with HBSS, fixed with 1% paraformaldehyde for 15 min at 22°C, and permeabilized with 0.1% Triton X-100 for 30 min at 22°C. Cells were stained with Alexa 568-phalloidin, washed 2 times with HBSS, mounted with ProLong antifade mounting medium, and viewed with a digital fluorescence microscope. Experiment was repeated 3 times with similar results.

TNF-alpha augments thrombin-induced transendothelial resistance and transendothelial albumin permeability. We measured the thrombin-induced changes in transendothelial monolayer electrical resistance and transendothelial albumin clearance. Exposure of HUVECs to TNF-alpha (250 U/ml) alone for 2 h had no significant effect on transendothelial monolayer resistance (Fig. 6A). Thrombin produced an ~40% maximum decrease in transendothelial monolayer electrical resistance and resistance returned to normal within 2 h after thrombin challenge (Fig. 6). In TNF-alpha -primed cells, thrombin produced an ~40% maximum decrease in transendothelial monolayer electrical resistance, but the monolayer resistance recovery to the basal level was markedly delayed (Fig. 6). The Src kinase inhibitor PP1 prevented the delayed recovery of endothelial monolayer resistance in TNF-alpha -primed cells (Fig. 6). PP1 had no significant effect on the initial drop in resistance (i.e., 30 min after thrombin challenge) induced by thrombin (Fig. 6) in either control or TNF-alpha -primed HUVECs. These results indicate that Src activation-mediated Ca2+ influx prolonged the thrombin-induced endothelial contractile response, consistent with the greater actin stress fiber formation.


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Fig. 6.   Effects of TNF-alpha on thrombin-induced change in transendothelial monolayer electrical resistance. A: HUVECs were grown to confluence on gold electrodes (see METHODS). Before experiments, cells were washed 2 times with serum-free medium and incubated in medium containing 1% FBS for 2 h at 37°C. The cells were exposed to TNF-alpha (250 U/ml) for 2 h and challenged with thrombin (5 U/ml) to measure changes in transendothelial electrical resistance. In some electrodes, TNF-alpha (250 U/ml) was added directly, and changes in transendothelial electrical resistance were measured. In some experiments, cells were preincubated with PP1 (10 µM) for 30 min before addition of either thrombin or TNF-alpha (arrow). Results are from a representative experiment. PP1 alone had no effect on transendothelial monolayer electrical resistance (data not shown). B: thrombin-induced maximum decrease in resistance was determined as percent decrease at 30 and 90 min. Values are means ± SE from 5 experiments. * P < 0.05 compared with thrombin alone-treated control cells at 90 min.

We measured transendothelial albumin clearance to assess endothelial barrier function. Thrombin alone increased transendothelial 125I-albumin clearance twofold over the basal value (Fig. 7). TNF-alpha alone did not increase albumin clearance; however, the addition of thrombin to TNF-alpha -exposed monolayers increased albumin clearance three- to fourfold over the basal value (Fig. 7).


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Fig. 7.   Effects of TNF-alpha on thrombin-induced increase in transendothelial 125I-albumin permeability. HUVECs were grown to confluence on microporous filters and incubated with TNF-alpha (250 U/ml) for 2 h at 37°C and then challenged with thrombin (5 U/ml) to measure the albumin clearance rate. Other details are described in METHODS. Values are means ± SE. Experiment was repeated 4 times in triplicate. Significantly different from control group: * P < 0.05; ** P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thrombin is a procoagulant as well as a potent proinflammatory mediator that increases vascular endothelial permeability (10, 15, 20, 25, 31, 34, 35). Thrombin by binding to its receptor, proteinase-activated receptor-1, in endothelial cells increases [Ca2+]i. This is the result of the generation of Ins(1,4,5)P3 and the release of intracellular Ca2+ as well as of the influx of Ca2+ (18, 34, 35, 38). Recent studies (14, 21, 30) indicate that agonist-dependent intracellular store Ca2+ depletion is directly coupled to Ca2+ influx to rapidly refill the intracellular Ca2+ stores. Ca2+ influx secondary to intracellular store depletion (i.e., "capacitative Ca2+ entry") is mediated by activation of store-operated Ca2+ channels localized in the plasma membrane (14). The rise in intracellular Ca2+ signaling induced by thrombin is critically involved in the mechanism of the increase in endothelial permeability (18, 34, 35, 38). Sandoval et al. (35) and others (24, 29) have shown that thrombin-induced Ca2+ influx is, in fact, an essential requirement for the increase in endothelial permeability. In the present study, we addressed the possibility that another proinflammatory mediator, TNF-alpha , generated during sepsis (6, 17, 39), may synergistically affect the thrombin-induced increase in endothelial permeability by pathways that activate Ca2+ entry. To address the actions of TNF-alpha and thrombin, we exposed endothelial cells to TNF-alpha for defined periods and studied the thrombin-induced alterations in Ca2+ signaling and endothelial barrier function.

We observed that thrombin challenge of endothelial cells in the presence of nominal extracellular medium Ca2+ (1.26 mM) produced a characteristic initial peak increase in the [Ca2+]i followed by a decline to basal levels. However, in endothelial cells exposed to TNF-alpha , thrombin produced a similar initial peak increase in [Ca2+]i, but surprisingly, the concentration remained elevated for up to 10 min. TNF-alpha alone had no effect on the endothelial [Ca2+]i. Because the thrombin-induced Ca2+ influx is functionally coupled to intracellular store Ca2+depletion, we also measured thrombin-induced Ins(1,4,5)P3 generation in control and TNF-alpha -primed cells. Ins(1,4,5)P3 generation activated by thrombin was not different in these cells, suggesting that the sustained increase in [Ca2+]i due to a TNF-alpha "priming effect" was not the result of Ins(1,4,5)P3-mediated intracellular store depletion. Because Ins(1,4,5)P3 generation occurs secondary to the activation of thrombin receptors (18, 35, 38), the finding of similar thrombin-induced Ins(1,4,5)P3 generation after TNF-alpha also suggests that the differential Ca2+ signaling response cannot be explained by increased thrombin receptor activation in the TNF-alpha -primed endothelial cells.

Because Src tyrosine kinase activation has been implicated in the mechanism of Ca2+ influx after activation of G protein-coupled receptors (2), we assessed this possibility using the Src inhibitor PP1. In the absence of Ca2+ in the extracellular medium, thrombin produced an initial peak increase in [Ca2+]i that was not different between control and TNF-alpha -primed cells. The addition of extracellular Ca2+ after store depletion induced by thrombin, however, produced a significantly greater rise in the increase in [Ca2+]i in the TNF-alpha -exposed endothelial cells. Importantly, this increase was prevented by PP1, suggesting that the thrombin-induced Ca2+ influx is regulated by Src activation. We also showed that TNF-alpha exposure of endothelial cells amplified Src kinase activity after thrombin challenge; thus the increased Ca2+ influx in response to thrombin in the TNF-alpha -primed cells may reflect the Src activation.

The mechanism by which Src can regulate Ca2+ influx occurring secondary to store depletion is unclear. One possibility is that Src phosphorylates plasma membrane transient receptor potential (Trp) channels expressed in endothelial cells (9, 23), which mediate the Ca2+ influx during Ins(1,4,5)P3-sensitive intracellular store depletion (21, 30). Although Src was activated in TNF-alpha -exposed cells, there was no change in [Ca2+]i without thrombin stimulation, suggesting that the greater Ca2+ influx mediated by thrombin in TNF-alpha -primed cells is coupled to intracellular store depletion. These results are consistent with Src-induced activation of Trp-like channels and thus could account for the increased Ca2+ influx observed in the TNF-alpha -exposed endothelial cells. The mechanism may be analogous to the Src-dependent tyrosine phosphorylation of Ca2+-sensitive K+ channels, resulting in increased channel activity (16).

To address the functional significance of the greatly amplified Ca2+ influx after TNF-alpha exposure of endothelial cells, we assessed tyrosine phosphorylation of adherens junctional proteins implicated in the mechanism of increased endothelial permeability (8, 36, 40). Thrombin increased the tyrosine phosphorylation of junctional proteins and the formation of interendothelial gaps that are characteristically associated with the loss of barrier function (10, 15, 20, 25, 31, 37). In the present study, we showed that the addition of thrombin to TNF-alpha -primed cells markedly augmented both tyrosine phosphorylation and gap formation. Moreover, PP1 prevented these alterations. Because PP1 treatment had no effect on the initial peak increase in [Ca2+]i resulting from intracellular store depletion, whereas it inhibited the Ca2+ influx component, the results suggest that the tyrosine phosphorylation of adherens junction proteins is dependent on the augmented Ca2+ influx.

We measured the transendothelial monolayer electrical resistance [an assessment of endothelial cell shape change induced by Ca2+ signaling (37)] and transendothelial albumin permeability, a direct measure of endothelial barrier function (20, 34, 35, 41). Ca2+ signaling is critical in the mechanism of thrombin-induced myosin light chain phosphorylation and subsequent actomysin cross bridging (which induces actin stress fiber formation) (11, 34, 35, 41). In control cells, thrombin markedly increased stress fibers within 5 min, and the cells recovered within 2 h after thrombin exposure. TNF-alpha alone failed to increase stress fibers; however, thrombin-induced actin stress fiber formation was markedly enhanced after TNF-alpha exposure, and the cells failed to recover even 2 h after thrombin challenge. Thus the prolonged intracellular Ca2+ elevation may account for this atypical alteration of stress fiber formation in the TNF-alpha -primed cells. The changes in transendothelial electrical resistance paralleled the actin stress fiber formation. The addition of thrombin to control monolayers produced a decrease in endothelial monolayer resistance, and the value returned to the normal range within 2 h after the addition of thrombin. However, in TNF-alpha -primed cells, thrombin produced a similar maximum decrease in resistance, but the endothelial cell monolayer resistance recovery time was significantly prolonged. Moreover, the thrombin-induced increase in transendothelial 125I-albumin clearance in TNF-alpha -primed cells was also greater. PP1 prevented these responses, demonstrating the importance of Src in mediating the loss of endothelial barrier function after TNF-alpha priming. These results suggest that the Src activation-dependent Ca2+ influx is an important factor signaling endothelial barrier dysfunction.

The mechanism of Src activation in TNF-alpha -primed cells in the absence of thrombin is unclear. Src may be activated by reactive oxygen species (ROS) (1, 28). Moreover, ROS production in response to TNF-alpha exposure occurs in endothelial cells (32, 33). In the present study, we observed that Src activation occurred in HUVECs 2 h after TNF-alpha exposure. This time frame is consistent with the H2O2-induced Src activation in cardiac myocytes (28). Thus it is possible that TNF-alpha -mediated ROS generation (1, 28, 32, 33) in endothelial cells induces Src activation, and this, in turn, is responsible for augmentation of Ca2+ influx on challenge with thrombin.

In summary, we studied the synergistic effects of TNF-alpha on the thrombin-induced increase in [Ca2+]i and endothelial barrier function. Exposure of endothelial cells to TNF-alpha increased the activation of Src tyrosine kinase and markedly increased thrombin-induced Ca2+ influx. These results suggest that the generation of thrombin and TNF-alpha during sepsis can synergistically activate Ca2+ influx to cause endothelial barrier dysfunction. Thus drugs designed to interfere with Src-activated Ca2+ influx in endothelial cells may be useful in treating inflammation and tissue injury associated with sepsis.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of General Medical Sciences Grant GM-58531 and National Heart, Lung, and Blood Institute Grants HL-45638, P01-HL-60678, and T32-HL-07829.


    FOOTNOTES

* Chinnaswamy Tiruppathi and Tabassum Naqvi contributed equally to this work.

Address for reprint requests and other correspondence: C. Tiruppathi, Dept. of Pharmacology (M/C 868), College of Medicine, The Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612 (E-mail: tiruc{at}uic.edu).

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. Section 1734 solely to indicate this fact.

Received 21 March 2001; accepted in final form 4 June 2001.


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