Several endothelial functions are altered during HIV infection including endothelial cell adhesion molecule expression, leukocyte adhesion, and microvascular integrity. The mechanisms by which HIV enters and disseminates throughout the body are now fairly well understood; manifestations of HIV infection often do not correlate directly with viral titers, but rather reflect the effects of several factors released following HIV infection. It is known that HIV appears in the circulation early after infection, commonly in CD4 T-cells and cells of the monocyte lineage [1,2]. Later, HIV infected macrophages are detected throughout many organ systems . Initially, studies suggested HIV infection of endothelial cells was a possible mode of HIV tissue transmission [4,5]; however, HIV rarely, if ever, infects endothelial cells [6,7]. Thus, HIV infection of tissues is probably mediated by transmission of HIV or HIV infected cells across the endothelial barrier.
Many effects of HIV infection are not mediated by lytic propagation of viral particles in cells, but by the activity of secreted viral factors. For example, Tat-1, a virus encoded transactivating protein plays a critical role in replication of HIV-1 [8–13]. In addition to its transactivation function in viral replication, Tat exerts several effects on non-infected cells, e.g., as an extracellular ligand which modulates endothelial proliferation. The observations that Tat is secreted by acutely infected cells , and that anti-Tat antibodies are frequently detected in the serum of AIDS patients are consistent with a role for extracellular Tat in infected individuals , independent of HIV. Tat may modulate endothelial proliferation by promoting secretion of growth regulatory cytokines such as interleukin-6  or by activating the FLK-1 receptor . Both FLK-1 receptor binding and interleukin-6 might also influence vascular integrity and increase endothelial monolayer permeability [18,19].
The molecular mechanism(s) responsible for the multiple effects of Tat remain unknown. Since HIV infection is associated with diminished vascular integrity, Tat-1 might also modulate the endothelial barrier. This might involve Tat binding to the FLK-1 receptor and altered tyrosine kinase activity  or altered gene transcription transcription and translation. Tat helps to activate the HIV promoter , and also represses transcription of several genes through effects on Sp-1 like elements [10,11]. Tat also influences elongation factor activity  and also modulates expression of endothelial cell adhesion molecules . Therefore Tat receptor binding, uptake in cells  or modulation of transcription/translation could contribute to this loss of endothelial integrity.
In this study, we show that recombinant Tat protein increases endothelial monolayer permeability in a time and concentration-dependent manner. We determined the signal pathways involved in Tat-mediated permeability, and examined how Tat redistributes tyrosine-phosphorylated junction proteins. Lastly, we investigated the molecular weights of Tat-phosphorylated proteins as a function of time. Our data indicate that at least some detrimental effects of HIV, e.g., vascular permeability, may be mediated by Tat through mitogen activated protein (MAP)/tyrosine kinase pathways. These findings suggest that some pathological effects of HIV on the vasculature might be treated using inhibitors of these signal pathways.
Materials and methods
The procedures used to obtain human endothelial cells were approved by the Institutional Review Board for Human Research at the Louisiana State University Health Science Center. Each subject provided written consent and was paid for participating in the study.
Purification of HIV Tat protein
Purification of recombinant Tat from a glutathione S transferase (GST)–Tat fusion vector was essentially as described previously . In brief, the pure Tat protein (40 ng/μl) was cleaved from a GST–Tat fusion vector by thrombin digestion and flash frozen in liquid nitrogen in a buffer containing 7.6 mM dithiothreitol, 33 mM Tris–HCl, pH 7.4 and 20% glycerol. A similar extract from cells expressing the non-fusion GST protein was used as a control. Tat activity was measured by light emission in a HeLa line into which an HIV-LTR driven luciferase reporter had previously been electroporated. Purified Tat was added to HeLa cells, and light emission measured and used as an index of Tat activity. Only Tat preparations that consistently gave multiple-fold increase in luciferase activity in the reporter assay were used in our studies.
Endothelial cell culture
Human umbilical vein endothelial cells (HUVEC) were harvested from umbilical cords by collagenase treatment as described previously . The cells were cultured in endothelial cell growth medium (Clonetics, San Diego, California, USA) supplemented with 10% calf serum (Hyclone, Logan, Maryland, USA), heparin sodium (10 IU/ml; Sigma Chemical Co. St. Louis, Missouri, USA), antibiotics (100 IU/ml penicillin, 100 mg/ml streptomycin, and 0.125 mg amphotericin B), and endothelial cell growth factor (80 ng/ml; Biomedical Technologies, Stoughton, Massachusetts, USA). All other tissue culture reagents were obtained from Gibco (Grand Island, New York). The cell cultures were incubated at 37°C in a 100% humidified atmosphere with 5% CO2 and expanded by brief trypsinization (0.25% trypsin in phosphate buffered saline) containing 0.02 % EDTA. Primary passage HUVEC were seeded onto fibronectin coated (25 μg/ml) tissue culture plates and used when confluent. Culture medium was replaced every second day. Only first-passage cultures were used in these studies. Cells were identified as endothelial cells by their cobblestone appearance at confluence, and by positive labeling with (i) acetylated low density lipoprotein labeled with Dil-Ac-LDL (Biomedical Technologies) and (ii) mouse anti-human factor VIII (Calbiochem, San Diego, California, USA).
Endothelial electrical resistance
HUVEC were plated on 8-μm pore size tissue culture inserts (Becton Dickinson, Franklin Lakes, New Jersey, USA) coated with fibronectin (25 μg/ml) at approximately 75% confluency. The resistance across the monolayer was measured using WPI (New Haven, Connecticut, USA) EVOM volt–ohm meter with `chopstick' electrodes. The value obtained from a blank insert coated with fibronectin was subtracted to give the net resistance, which was multiplied by the membrane area to give the resistance in area-corrected units. Values were expressed as Ω/cm2, taking into account the surface area of the filter (0.38 cm2).
At 100% confluence, trans-monolayer electrical resistance was measured. When resistance was stable (at > 12 Ω/cm2), the culture medium from the upper (apical) compartment of the monolayer was removed, and replaced with medium containing Tat (1, 10, 100, 150 or 500 ng/ml) or control medium containing 19 nM dithiothreitol, 0.05% glycerol and 6.25 pg/ml (the buffer that Tat is usually dissolved in).
We measured endothelial permeability in response to Tat exposure, using fluorescein isothiocyanate labeled bovine serum albumin (FITC–BSA) as a permeable tracer that will pass across endothelial monolayers, by the method described by Maruo et al.  with minor modifications. Endothelial monolayers were grown on the surface of 8-μm pore membrane filters (Becton Dickinson). These cell monolayers were washed twice with Hank's balanced salt solution (HBSS), and then placed on 24-well plates (Becton Dickinson) with 1 ml HBSS in the lower chamber. Five-hundred μl HBSS containing 10 μg/ml FITC–BSA were put into the luminal chamber, and then the apparatus was placed in a CO2 incubator at 37°C. After incubation for 30 min, a 100 μl sample was taken from the lower chamber and the absorbance of FITC–BSA was determined at 492 nm with a spectrophotometer (Titertek MCC 340). The data were expressed as follows:EQUATION
Our preliminary data indicate that as the trans- endothelial electrical resistance decreases, the endothelial permeability index increases (data not shown). Permeability measurements were made under control conditions, following exposure to Tat and following pretreatment with the protein kinase inhibitors which were used at their IC50 concentration (the concentration giving 50% inhibition of enzyme activity), genistein (20 μM), PD98059 (20 μM), KT5823 (0.5 μM) (Calbiochem). After cells were pretreated with these inhibitors for 1 h, Tat was added to the culture, and permeability was measured at the time points described.
Western analysis of cell lysates
Equal quantities of protein (75 μg) from each sample were separated by electrophoresis on 7.5% acrylamide gels containing sodium dodecyl sulphate. Proteins were transferred to nitrocellulose membranes (Sigma) and blocked with 5% milk powder in phosphate buffered saline at 4°C overnight. The membranes were washed twice for 10 min in wash buffer (0.1% milk powder in phosphate buffered saline). Primary mouse anti-phosphotyrosine monoclonal (Santa Cruz Biotechnology Inc. Santa Cruz, California, USA) or rabbit anti-active MAP kinase polyclonal antibody (Promega Corporation, Madison, Michigan, USA) was added at a concentration of 0.05 μg/ml and incubated at room temperature for 1 h. The membrane was washed twice with wash buffer. Secondary goat anti-mouse horseradish peroxidase conjugated secondary antibody (Sigma) or goat anti-rabbit alkaline phosphatase conjugate antibody (Sigma) was added at a 1 : 5000 dilution for 1 h. Finally, membranes were washed three times and developed using the enhanced chemiluminescence detection system (Amersham, La Jolla, California, USA) or nitro blue tetrazolium/bromo chloro indolyl phospate (Sigma).
Immunofluorescence staining of junctional proteins
HUVEC were grown to confluency on coverslips and exposed to Tat, with and without genistein, PD98059, or appropriate Tat buffer controls. Samples were fixed with ethanol and acetone  and stained for phosphotyrosine. Mouse anti-phosphotyrosine monoclonal antibody was used at a concentration of 4 μg/ml. Cy3 goat anti-mouse secondary antibody (Jackson Immunoresearch Laboratories Inc. Westgrove, Pasadena, USA) was used at a 1 : 250 dilution. Photomicrographs were taken at 1000 × magnification using 12 s exposure times with T-Max 400 film.
All values are expressed as mean ± SE. Data were analyzed using one-way ANOVA with Bonferroni's correction for multiple comparisons. Significance was accepted at P < 0.05.
Effect of Tat on HUVEC monolayer resistance
The electrical resistance of confluent HUVEC monolayers was 16.29 ± 0.72 Ω/cm2. Incubation of HUVEC monolayers with Tat (50, 150 ng/ml) for 24 and 48 h elicited dose-dependent decreases in the electrical resistance with a maximum by 24 h (fig. 1). Tat (150 ng/ml) significantly decreased electrical resistance to 72.00 ± 3.45%, by 24 h, whereas Tat (50 ng/ml) did not have a significant effect. The effects of 150 ng/ml Tat were not significant before 24 h. Tat (100 and 500 ng/ml) also decreased electrical resistance as 150 ng/ml Tat (data not shown). Subsequent removal of Tat resulted in recovery of monolayer resistance within 48 h (data not shown).
Effect of Tat on HUVEC monolayer permeability
We next determined whether solute-flux measurements of FITC–BSA would give similar results to the change of the electrical resistance. Incubation of HUVEC monolayer with Tat (10, 100, 500 ng/ml) for 24 h elicited a dose-dependent increase in FITC–BSA permeability (P < 0.05) (fig. 2a). For further experiments a dose of 100 ng/ml Tat that showed a measurable disruption of monolayer barrier integrity was chosen for subsequent studies. Incubation of HUVEC monolayer with Tat (100 ng/ml) for 12, 24, and 48 h elicited a time-dependent increase in FITC–BSA permeability with a maximum increase by 24 h and a continued decrease up to 48 h (fig. 2b).
Effects of protein kinase inhibitors on Tat-induced endothelial permeability
To examine the effect of kinase inhibitors, HUVEC were pretreated with genistein, PD98059, or KT5823. Pre-incubation of monolayers with 20 μM genistein prevented Tat-dependent increases in permeability (fig. 2c). The effects of Tat on permeability were also blocked by 20 μM PD98059, a specific inhibitor of MAP kinase kinase-1, but not by 0.5 μM KT5823, protein kinase G inhibitor (fig. 2c). Inhibitors alone had no effect on permeability (data not shown).
Effect of Tat on tyrosine phosphorylation
The effect of extracellular Tat on tyrosine phosphorylation was next investigated. When analyzed using phosphotyrosine immnoblotting, Tat altered the tyrosine phosphorylation pattern in a time-dependent manner (fig. 3). The tyrosine phosphorylation of several proteins (68, 75, 85, 98, 125, 142, 170, 180, 190, and 205 kDa molecular weight) was increased by Tat.
Immunofluorescent staining of phosphotyrosine in vitro
Phosphotyrosine expression on HUVEC was assessed by indirect immunofluorescence. The cells treated with control medium exhibited weak junctional and filamentous staining (fig. 4a). Tat (100 ng/ml) disorganized the junction associated staining, and induced a marked increase in the immunofluorescent staining of slash-like filaments, with an increase in the number of these immunostaining structures (fig. 4b). Pretreatment with the tyrosine kinase inhibitor, genistein, or the MAP kinase inhibitor, PD98059 attenuated the increase in the immunofluorescent staining of these structures induced by Tat (100 ng/ml) (Figs 4c and 4d).
Effects of protein kinase inhibitors on Tat-induced tyrosine phosphorylation
To examine the effects of protein kinase inhibitors, HUVEC were pretreated with genistein, or PD98059. Pre-incubation of monolayers with 20 μM genistein prevented Tat-dependent increases in protein phosphorylation (fig. 5a). The effects of Tat on protein phosphorylation were also blocked by 20 μM PD98059.
The effect of both classes of kinase inhibitors (genistein, PD98059) on Tat-induced MAP kinase phosphorylation was next investigated. Pre-incubation of monolayers with 20 μM genistein prevented Tat-induced MAP kinase phosphorylation. The effect of Tat on MAP kinase phosphorylation was also blocked by 20 μM PD98059 (fig. 5b).
In the present report, we examined the effect of Tat protein on endothelial permeability. We found that endothelial permeability was increased by Tat in a time- and dose-dependent manner (Figs 1 and 2). We chose HUVEC as a model system because endothelial cells appear to be significantly activated by Tat protein . One important index of activation, increased expression of adhesion molecules on endothelial cells, can be induced by Tat protein exposure [16,20]. These observations suggest that Tat alone can activate human vascular endothelium, indicating that this increased vascular permeability may facilitate permeation of HIV into the tissue surrounding infected cells and may explain some of the vascular leakage phenomena related to HIV infection. In fact, it has been proposed that Tat vaccines could be beneficial in that they might bind Tat, prevent permeability, and slow the exchange of HIV and HIV infected cells , as after HIV gains entry into extravascular tissue spaces, infection may be transferred to endogenous tissue macrophages either by direct infection or by cell fusion.
Tat might activate cells at either the plasma membrane, or within the cytoplasm or nucleus. Thus, Tat could be taken up by the target cells and affect genes, or might act like a hormone, binding to cell surface receptors, to generate intracellular signals. Extracellularly applied exogenous Tat rapidly enters into cells and accumulates in the nucleus [21,26]. However, it has also been reported recently that Tat can activate endothelial cells by binding to the Flk-1/KDR receptor for vascular endothelial growth factor (VEGF), promoting tyrosine autophosphorylation of the receptor and its associated downstream effects which might include increased permeability [17,18]. Our results also show that HIV Tat protein, when applied extracellularly, induces the activation of protein kinases in endothelial cells (Figs 3 and 4). Tat produced a striking increase in tyrosine phosphorylation of several endothelial proteins. The Tat-induced tyrosine phosphorylation is both rapid and concentration-dependent (data not shown).
There are several reports that show Tat activates paxillin, p125FAK, p130CAS and MAP kinase [27–30]. Our results also show that Tat activates MAP kinase in endothelial cells (fig. 5b) and suggests that Tat contributes to the tyrosine phosphorylation of these proteins in endothelial cells. Although protein phosphorylation is observed within 1 h of exposure to Tat, permeability responses were not observed until at least 24 h later (Figs 1 and 2). The Tat-dependent increases in permeability were blocked by tyrosine and MAP kinase blockers (fig. 2c). Therefore, despite the difference in time, these early tyrosine phosphorylation changes appear to be critical in the 24 h permeability response. Co-incubation of monolayers with Tat plus genistein or PD98059, attenuated Tat-dependent immunofluorescent staining changes (fig. 4), Tat-dependent protein phosphorylation and MAP kinase phosphorylation (fig. 5). By 24 h, we no longer observed a significant difference in the phosphotyrosine pattern within HUVEC (data not shown).
We have previously shown that VEGF-mediated permeability occurs through disorganization of endothelial junctions, e.g., occludin, and that this effect is blocked by PD98059 . In another report, VEGF was shown to stimulate focal adhesion kinase and paxillin tyrosine phosphorylation . Although we failed to demonstrate a change in junctional proteins (e.g., occludin, VE-cadherin), the change of phosphotyrosine immunofluorescent staining to a focal adhesion `slashes' may represent the phosphorylation of focal adhesion molecules such as paxillin, FAK and p130CAS. To define precisely the events which modulate adhesive plaque reorganization following Tat exposure clearly requires further investigation.
We previously demonstrated that VEGF-dependent endothelial permeability was attenuated significantly by tyrosine and MAP kinase inhibitors . Therefore the data presented here appear superficially to be consistent with permeability changes occurring as a result of FLK-1 receptor binding. However, in our previous study, we reported that FLK-1 receptor binding altered endothelial permeability in a very rapid (< 1 h) manner, which reversed on a similar time scale. Our results in this current report show that unlike FLK-1 receptor binding, Tat-mediated permeability changes do not occur until at least 24 h after Tat exposure, and this effect did not reverse until 48 h after Tat washout. Therefore, while Tat-mediated permeability shares the tyrosine and MAP kinase signal dependence seen with FLK-1, the timing of these events are inconsistent with permeability responses as a result of FLK-1 receptor binding.
The targets responsible for Tat-mediated permeability have still not been identified. Besides its ability to bind to FLK-1 receptors and enter the cytoplasm, Tat also binds to several proteins including a 90 kDa binding protein and RGD-motif containing matrix proteins including fibronectin, laminin and fibrinogen . Tat is also thought to play a significant role in tissue remodeling through its ability to promote matrix metalloproteinase (MMP) formation leading to vessel injury. Interactions of monocytes with human endothelial cells stimulate monocyte MMP production and monocytes appear to be the dominant source of MMP in this type of injury [33–35]. However, it has been reported that cytokines such as tumor necrosis factor-α may contribute to endothelial permeability through the synthesis and activation of endothelial MMP ; therefore we cannot exclude the possibility that Tat may increase endothelial solute permeability by the enhanced endothelial expression of MMP in this model.
In summary, we have provided evidence that the HIV transcriptional regulator, Tat, increases endothelial monolayer solute permeability, and that Tat-induced permeability increase is apparently mediated through the tyrosine kinase and MAP kinase signal transduction cascades. The release of Tat protein in to the vascular microenvironment by infected cells may contribute to the dissemination of HIV-infected cells into tissue and therefore, may enhance disease progression. Therefore, it is possible that MAP kinase and tyrosine kinase antagonists might be beneficial in limiting some of the effects of Tat following HIV infection.
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