Trichloroethylene Decreases Heat Shock Protein 90 Interactions with Endothelial Nitric Oxide Synthase: Implications for Endothelial Cell Proliferation

Jingsong Ou*,{dagger},1, Zhijun Ou*,{dagger}, D. Gail McCarver{ddagger}, Ronald N. Hines{ddagger}, Keith T. Oldham*,{dagger},§, Allan W. Ackerman* and Kirkwood A. Pritchard, Jr.*,{dagger},{ddagger},§

* Department of Surgery, Division of Pediatric Surgery, {dagger} Cardiovascular Center, {ddagger} Department of Pharmacology and Toxicology, § Free Radical Research Center, and the Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received November 18, 2002; accepted February 8, 2003

ABSTRACT

Trichloroetheylene (TRI) is an environmental pollutant that has been linked to congenital heart defects (CHD). Endothelial nitric oxide synthase (eNOS) generation of nitric oxide (NO) plays an important role in endothelial cell proliferation, which is considered essential for normal blood vessel growth and development. We hypothesized that TRI alters the balance of NO and superoxide anion (O2-) to impair endothelial cell proliferation. Proliferating endothelial cells were pretreated with TRI (5 µM) and then stimulated with the calcium ionophore, A23187 (5 µM), to determine changes in endothelial cell and eNOS function with respect to NO and O2- generation. Immunoblots of eNOS, phospho-eNOS at serine 1179 (S1179), and the levels of associated heat shock protein 90 (hsp90) were used to define the activation state of eNOS. The effects of TRI (0.05–100 µM) on vascular endothelial growth factor (VEGF, 0.58 nM) induced endothelial cell proliferation were determined from cell counts. TRI decreased A23187-stimulated nitrite + nitrate production from 1.99 ± 0.90 to 0.89 ± 0.51 pmol/mg protein (p < 0.05; n = 6). In controls, L{omega}-nitroargininemethylester (L-NAME) increased A23187-stimulated O2- production from 0.130 ± 0.089 to 0.214 ± 0.071 nmol/min/mg protein (p < 0.05; n = 5). In TRI-treated cultures, however, L-NAME decreased A23187-stimulated O2- production from 0.399 ± 0.121 to 0.199 ± 0.055 nmol/min/mg protein (p < 0.05; n = 5). TRI decreased hsp90 associated with eNOS by 46.7% and inhibited VEGF-stimulated endothelial cell proliferation by 12 to 35%. These data show that TRI alters hsp90 interactions with eNOS and induces eNOS to shift from NO to O2- generation. Our findings provide new insight into how TRI alters endothelial and eNOS function to impair VEGF-stimulated endothelial proliferation. Such changes in endothelial function may play an important role in the development of congenital heart defects.

Key Words: trichloroethylene (TRI); teratogens; nitric oxide (NO); superoxide anion (O2-); cardiac cells; septa.

Trichloroethylene (TRI) is an industrial solvent that is commonly used as a degreaser and cleaning agent. A growing body of evidence indicates that fetal exposure to TRI induces cardiac structural malformations. Case-controlled epidemiological studies in Arizona and California found strong associations between TRI-contaminated well water and increased incidence of congenital heart defects (Goldberg et al., 1990Go; Windham et al., 1991Go); animal studies in chicken, murine, and rodent models provide additional experimental evidence that TRI induces teratogenic cardiac defects (Johnson et al., 1998Go). Although these reports suggest that TRI can induce congenital heart defects (CHD) during development, other studies have found no effect of TRI on CHD in a Sprague-Dawley rodent model (Fisher et al., 2001Go). Adding to the confusion concerning teratogenic effects of TRI is the fact that reference values for exposure in the EPA’s IRIS were recently withdrawn. Such controversy suggests that more work is required to understand if and how TRI affects the developing heart.

The cellular mechanisms underlying TRI-induced cardiac teratogenicity are just beginning to be examined. In 2000, Boyer and colleagues used an in vitro chick-AV canal culture model to determine the effects of TRI on the development of cardiac valves and septa (Boyer et al., 2000Go). TRI (50–250 ppm) altered several elements of epithelial-mesenchymal cell transformation, including endothelial cell-to-cell separation. These separation processes may also be associated with endothelial activation (Boyer et al., 2000Go), such as they occur when endothelial cells are stimulated to proliferate with vascular endothelial growth factor (VEGF) (Battegay, 1995Go; Papapetropoulos et al., 1997bGo). Analysis of the markers mediating this cell transformation revealed that TRI inhibited expression of Mox-1 and fibrillin 2 but not {alpha}-smooth muscle actin, suggesting that endothelial transformation or proliferation may be a critical point at which TRI can alter cardiac development.

VEGF induces endothelial cells to proliferate by a nitric oxide (NO)-dependent mechanism (Keshet and Ben-Sasson 1999Go; Papapetropoulos et al., 1997bGo). Evidence for NO playing an important role in new blood vessel growth has come from studies using endothelial nitric oxide synthase (eNOS)-knockout mice or pharmacological and natural endogenous inhibitors of eNOS, in which responses for blood vessel growth were severely compromised (Jang et al., 2000Go; Matsunaga et al., 2000Go; Murohara et al., 1998Go; Papapetropoulos et al., 1997aGo). Recent studies find that the association of hsp90 with eNOS is critical for NO-dependent proliferation (Garcia-Cardena et al., 1998Go). Evidence for eNOS playing an important role in heart development comes from studies using eNOS knock-out mice, in that the eNOS-deficient mice had a greater incidence of developing congestive heart failure (Feng et al., 2002Go), congenital septal defects (Feng et al., 2002Go), impaired myocardial angiogenesis (Zhao et al., 2002Go) and formation of bicuspid aortic valves (Lee et al., 2000Go). With a growing number of investigations indicating that NO plays an important role in heart development and angiogenesis, we hypothesized that TRI might alter NO and superoxide anion (O2-) balance to impair endothelial proliferation.

Recent studies from this laboratory have shown that geldanamycin and radicicol, both anti-neoplastic agents, attenuate NO-dependent signaling by converting eNOS from an NO synthase into an NADPH oxygenase (Garcia-Cardena et al., 1998Go; Grenert et al., 1997Go; Ou et al., 2003Go; Pritchard et al., 2001Go). Angiostatin, a naturally occurring anti-neoplastic agent, increases endothelial O2- generation by an eNOS-dependent mechanism, which impairs vasodilation and proliferation (Koshida et al., 2003Go). As these pharmacological and naturally occurring antineoplastic agents impair vasodilation and endothelial cell proliferation, which may be important to cardiac developmental processes, we hypothesized that TRI perturbs hsp90 interactions with eNOS to impair endothelial cell proliferation. In this report, we examine the effects of TRI on endothelial cell generation of NO and O2- with the objective of determining whether TRI alters eNOS function and if changes in the balance of NO and O2- inhibit endothelial cell proliferation.

MATERIALS AND METHODS

Chemicals.
L{omega}-Nitroargininemethylester (L-NAME), NG-methyl-L-arginine • monoacetate (L-NMA), dimethyl sulfoxide (DMSO), antibiotics/mycotics, trypsin-EDTA, Hank’s balanced salts solution (HBSS), L-arginine, sodium nitrite, ferricytochrome c, superoxide dismutase (SOD), NaF, sodium deoxycholate, sodium dodecyl sulfate (SDS), 4-(2-aminoethyl benzene) sulfonyl fluoride hydrochoride, sodium orthovanadate, leupeptin, pepstatin A, aprotinin, and protein A-Sepharose were from Sigma Chemical Company (St. Louis, MO). RPMI 1640 was from Invitrogen Corporation (Grand Island, NY). Tris–HCl was from Baker (Phillipsburg, NJ). Triton X-100 was from Lab Chem (Pittsburg, PA). A23187, a calcium ionophore, was from Calbiochem (San Diego, CA). Laemmli buffer, polyacrylamide, nitrocellulose membranes were from BioRad (Hercules, CA). Fetal bovine serum (FBS) was from HyClone (Logan, UT). ECL reagents were from Amersham Pharmacia Biotech (Piscataway, NJ). X-OMAT film was from Kodak (Rochester, NY). H32 antibody was from BioMol (Plymouth Meeting, PA). Anti-eNOS was from Zymed Laboratories (San Francisco, CA) (Catalog# 33–4600, 9D10). Anti-hsp90 (H38220) was from Transduction Laboratories (Franklin, NJ). Anti-phospho-eNOS (Ser-1177) was from Cell Signaling Technology (Beverly, MA). Trichloroethylene and Vanadium (III) chloride were from Fluka-Aldrich Chemical Company (Milwaukee, WI).

Endothelial cell culture.
Bovine coronary endothelial cells (BCEC) were provided by William B. Campbell, Medical College of Wisconsin (Milwaukee, WI). BCEC were cultured in RPMI 1640 media containing 10% fetal bovine serum, penicillin, streptomycin and amphotericin B as before (Pritchard et al., 2001Go). BCEC were passaged with trypsin-EDTA and used for experiments between passages 6–8. To obtain proliferating endothelial cells, BCEC were passaged on to test plates at 16,700/cm2 for all culture conditions and used the second day after passage as described (Arnal et al., 1994Go).

Preparation of TRI working stock solutions.
Stock solutions of TRI were made by adding TRI (neat) directly to a varying volume of ethanol (95%) so as to be able to add constant volume of the working-stock TRI solutions to the culture media (final concentration of ethanol, 15 mM) to achieve the desired final concentration (0–100 µM). An equal volume of the ethanol vehicle was added to all paired controls.

Determination of nitrite + nitrate.
To assess changes in nitrite + nitrate production in the presence and absence of TRI, TRI (5 µM, final concentration) or ethanol vehicle was added to proliferating BCEC in 6-well dishes overnight. The next day, L-NMA (1 mM, final concentration) was added in parallel at time 0. At 30 min, the cultures were washed three times with HBSS. After the third wash, the test-groups were incubated in 1 ml HBSS containing A23187 (5 µM, final concentration) and L-arginine (25 µM, final concentration) for 30 min. The HBSS solutions were removed and nitrite + nitrate was quantified using a Sievers NOA analyzer as described (Braman and Hendrix, 1989Go).

Determination of O2-.
TRI (5 µM, final concentration) was added to proliferating BCEC in 6-well plates as described above. The next day, L-NAME (1 mM, final concentration) was added to proliferating BCEC cultures at time 0. At 30 min, the test groups were washed three times with HBSS. After the final HBSS wash, the four test groups in 6-well dishes (Control, L-NAME, TRI, and TRI + L-NAME) were incubated in 1 ml of HBSS containing ferricytochrome c (50 µM, final concentration) and A23187 (5 µM, final concentration) with and without L-NAME (1 mM, final concentration) for 30 min. Superoxide anion production was calculated from the absorbance of ferricytochrome c at 550 nm and the molar extinction coefficient ({varepsilon} = 21,000 M-1cm-1) as described (Pritchard et al., 2001Go). SOD-inhibitable O2- data were calculated from the release of O2- from independent wells incubated with HBSS containing ferricytochrome c, with and without SOD (1000 units/ml, final concentration).

Western blots for eNOS, hsp90, and phosphorylation of eNOS (S1179).
Western blot analysis was used to determine if TRI altered endothelial cell expression of eNOS or hsp90 or altered the phosphorylation of eNOS at S1179 in control and TRI-treated cultures. Briefly, proliferating BCEC cultures in 60-mm dishes were pretreated with TRI (5 µM, final concentration) overnight. The cultures were washed three times with HBSS and then incubated with 10 µM L-arginine supplemented HBSS with and without A23187 (5 µM, final concentration). After incubation for 10 min at 37°C, the HBSS solutions were removed by aspiration, and cell proteins were harvested in 500 µl of modified RIPA buffer, as described (Pritchard et al., 2001Go). Aliquots (50 µl) were combined with an equal volume of Laemmeli buffer and heated (95°C, 5 min) and stored on ice until fractionated by sodium dodecyl sulfate-polyacrylamide (7%) electrophoresis gel (SDS–PAGE) (15 µg protein/lane). The proteins were transferred to nitrocellulose membranes and blotted with anti-phospho-eNOS (Ser-1177), anti-eNOS (9D10) and anti-hsp90 overnight at 4°C. Bands were visualized using horseradish peroxidase (HRP)-linked secondary antibodies and ECL reagents (Pritchard et al., 2001Go).

To determine if TRI altered protein interactions between hsp90 and eNOS, the experiment described above was repeated on proliferating BCEC cultures in 100-mm dishes to increase the amount of protein in cell lysates for immunoprecipitation of eNOS. After incubation with TRI overnight, the four test groups (control, TRI, control + A23187, and TRI + A23187) were washed three times with 6 ml of HBSS and then incubated at 37°C in a tissue culture incubator in 6 ml of HBSS containing L-arginine (10 µM) with and without A23187 (5 µM). After 10 min, the HBSS solutions were removed and the cells were lysed in modified RIPA buffer and samples processed as previously described (Pritchard et al., 2001Go). An aliquot was removed (300 µg) and the volume adjusted to 100 µg cell protein/100 µl. Aliquots of the cell lysates were precleared with protein A-Sepharose beads for 1–2 h to minimize nonspecific binding of cell proteins to the beads used to isolate the eNOS immunocomplex. The precleared supernatant fractions were transferred to another microfuge tube and incubated 24 h with the anti-eNOS antibody, H32 from BioMol (1 µg/100 µl of cell lysate) to immunoprecipitate eNOS. The H32-eNOS immunocomplex was isolated by incubation (2 h) with 50 µl of a 50% slurry of protein A-Sepharose beads. The beads were washed 3 times with 750 µl of Tris-HCL-buffer saline (TBS); mixed with Laemmeli buffer (60 µl); heated (95°C, 5 min); mixed, and stored on ice until fractionated by 7% SDS–PAGE (20 µl/lane). The separated proteins in the gel were electrotransferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in TBS-Tween 20 (0.1%) and immunoblotted for eNOS using 9D10 the antibody (1:1000) from Zymed Laboratories, Inc., for hsp90 using H38220 (1:1000), and phospho-eNOS (S1177, human) using anti-phospho-eNOS (S1177). Autoradiograms were obtained using horseradish peroxidase (HRP)-linked secondary antibodies and chemiluminescence. Images of the bands of interest in the autoradiograms were obtained with UMAX Magicscan, ver. 4.4 and Adobe PhotoShop, ver. 5.5 software (UMAX Data Systems, Inc., Taipei, Taiwan). Densities of the bands were quantified from the scanned images using NIH Image 1.62.

Relative changes in eNOS phosphorylation in cell lysates and eNOS phosphorylation and hsp90 association with eNOS in eNOS immunoprecipitates were determined as follows. The band densities for p-eNOS and hsp90 were divided by the band densities for the corresponding eNOS and hsp90, respectively. The resulting ratios for p-eNOS/eNOS and hsp90/hsp90 for the controls was used to calculate relative ratios for the four test groups, thereby making C = 1.0. To obtain relative changes for eNOS and hsp90 across treatments in cell lysates and eNOS across treatments in eNOS immunoprecipitates, the band densities for all treatment groups (C, TRI, C + A23187 and TRI + A23187) were divided by the band densities of eNOS and hsp90 for the control, making the eNOS/eNOS and hsp90/hsp90 ratio for C =1.0. Relative band densities, normalized to control, are shown as mean ± SD, n = 5.

Endothelial cell proliferation.
BCEC were passaged onto 6-well cluster plates at 16,700 cells/cm2. On the 2nd day after passage, the endothelial cells were serum-starved in RPMI 1640 media containing 0.5% fetal bovine serum, for 48 h. The medium was removed from serum-starved endothelial-cell cultures and changed to RPMI 1640 media containing 2% fetal bovine serum. VEGF (0.58 nM, final concentration) and increasing concentrations of TRI (0.05–100 µM, final concentrations) were added, and the endothelial cell cultures incubated for 48 h. At the end of this incubation period, the cells in each well were counted using a Coulter Counter Model ZM (Coulter Electronics LTD, Luton, BEDS, England).

Statistical analysis.
All data are presented as the mean ± SD. Nitrite + nitrate, superoxide anion and cell count data were examined by paired sample ANOVA. The Newman-Kuel’s post hoc test was employed to determine the level of significance between means. The minimum level of significance was set at p < 0.05.

RESULTS

TRI inhibits NO and increases O2- generation by eNOS.
TRI inhibited A23187-stimulated nitrite + nitrate production by proliferating BCEC from 1.99 ± 0.90 to 0.89 ± 0.51 pmol/mg protein (Fig. 1AGo p, < 0.05, n = 6). In contrast to the effects on nitrite + nitrate production, TRI increased the release of O2- by A23187-stimulated proliferating BCEC cultures (Fig. 1BGo, p < 0.05, n = 5). L-NAME, a substrate analogue inhibitor of NOS, increased the release of O2- from A23187-stimulated control cultures from 0.130 ± 0.089 to 0.214 ± 0.071 nmol/min/mg protein (Fig. 1BGo, p < 0.05, n=5), confirming that, under normal conditions, eNOS in control cells generates NO that scavenges intracellular O2- before it has a chance to escape from the endothelial cell and react with ferricytochrome c (Pritchard et al., 1995Go). In A23187-stimulated cultures exposed to TRI, however, L-NAME decreased the release of O2- 0.399 ± 0.121 to 0.199 ± 0.055 nmol/min/mg protein (Fig. 1BGo p, < 0.05, n = 5) indicating that TRI induced proliferating BCEC cultures to generate O2- by an eNOS-dependent mechanism. Calculation of the relative change in O2- generation due to L-NAME inhibition reveals the extent to which TRI induces proliferating BCEC to generate O2- by an eNOS-dependent mechanism (Fig. 1CGo; p < 0.01, n = 5).



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FIG. 1. Effects of TRI on A23187-stimulated eNOS-dependent NO production and O2- generation. Data are presented as mean ± SD (A) Nitrite + nitrate production by stimulated proliferating BCEC cultures pretreated with (closed bar) and without (open bar) 5 µM TRI (**p < 0.01; n = 6). Nitrite + nitrate data were calculated from the difference in production between A23187-stimulated cultures with and without L-NMA. Experimental conditions were performed in triplicate and samples analyzed in duplicate or triplicate. Cell protein assays were determined in duplicate. (B) Superoxide anion generation by A23187-stimulated proliferating BCEC cultures pretreated with (closed bars) and without (open bars) 5 µM TRI, plus or minus L-NAME (1 mM) (*p < 0.05; **p < 0.01, n = 5). The assays were performed in triplicate and cell proteins analyzed in duplicate. (C) The relative difference in A23187-stimulated O2- generation when eNOS activity in control (open bar) and TRI-treated cultures (closed bar) was inhibited with L-NAME (1 mM) (**p < 0.01, n = 5).

 
TRI inhibits endothelial cell proliferation.
As TRI seems to target epithelial-mesenchymal cells and block endothelial cell–cell separation processes, we reasoned that TRI might also alter mechanisms involved in endothelial proliferation. Accordingly, we examined the effects of TRI on the balance of endothelial NO and O2- generation, which is considered essential for proliferation (Papapetropoulos et al., 1997bGo). Figure 2Go shows that TRI inhibited proliferation of BCEC in a threshold-dependent manner. Incubation of proliferating BCEC with TRI (0.05–100 µM) for two days significantly inhibited VEGF-stimulated endothelial cell proliferation by 12 to 35% compared to controls with an apparent threshold of 0.1 µM (p < 0.05, n = 7).



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FIG. 2. Effects of TRI on BCEC VEGF-stimulated proliferation. TRI inhibited VEGF-induced proliferation of BCEC as a function of TRI concentration (0.05–100 µM). Data shown are mean ± SD. (*p < 0.05, n = 7)

 
Effects of TRI on expression of eNOS and hsp90 and on phosphorylation of eNOS at S1179.
Immunoblots of control and TRI-treated cells revealed that TRI had no effect on the expression of eNOS or hsp90 in proliferating BCEC cultures or on the phosphorylation of eNOS (S1179) in unstimulated or A23187-stimulated cultures relative to controls (Fig. 3AGo). The effects of TRI on the stability of eNOS and hsp90 expression can be seen in the bar chart showing the mean of relative band densities from 5 independent experiments (Fig. 3BGo).



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FIG. 3. Effects of TRI on p-eNOS, eNOS and hsp90 expression. (A) Immunoblots examining the effect of overnight treatment TRI (5 µM) on the phosphorylation of eNOS at S1179 (upper), eNOS concentration (middle panel) or hsp90 concentration (lower) in proliferating BCEC cultures (15 µg protein) that were unstimulated or stimulated with A23187 (5 µM). (B) The bar graphs represent mean ± SD of changes in band densities of autoradiograms relative to band densities of the control (lane 1) set at 1.0; n = 5.

 
Effects of TRI on association of Hsp90 with eNOS and phosphorylation of eNOS at S1179.
To determine the effects of TRI on eNOS protein interactions, eNOS was immunoprecipitated from unstimulated and A23187-stimulated control and TRI-treated BCEC cultures. Immunoblots for phospho-eNOS, eNOS, and hsp90 on the immunoprecipitated eNOS complex revealed that A23187 stimulation increased phospho-eNOS (S1179) levels compared to the levels detected in unstimulated control cultures (Fig. 4AGo, top). Pretreatment with TRI alone had little effect on the levels of phospho-eNOS (S1179) in unstimulated or A23187-stimulated BCEC cultures compared to their corresponding controls (Fig. 4AGo). TRI increased the association of hsp90 with eNOS over controls without increasing phospho-eNOS levels in unstimulated cultures. In contrast to the effects of TRI on the association of hsp90 with eNOS in unstimulated cultures, TRI decreased the association of hsp90 with eNOS compared to control levels in A23187-stimulated BCEC cultures (Fig. 4AGo). The effects of TRI on association of hsp90 with eNOS relative to controls (C) can be seen in the bar chart showing the mean of relative band densities from 5 independent experiments (Fig. 4BGo).



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FIG. 4. (A) eNOS immunoprecipitation from 300 µg protein of total cell lysates from control, TRI, and/or A23187-treated BCEC cultures were fractionated (see Materials and Methods). After transfer to nitrocellulose membrane, Western-blot analysis with antibodies specific for phosphorylated eNOS (s1179)(p-eNOS), eNOS or hsp90 was performed as described in Methods. (B) Relative densities of phosphorylated eNOS (S1179) and hsp90 to total eNOS are shown in the bar graphs. Data are presented as mean ± SD of changes in relative band densities compared to controls (lane 1), which is set at 1.0. (*p < 0.05, **p < 0.01, n = 5).

 
DISCUSSION

These data indicate that TRI shifts the balance of NO and O2- production in proliferating endothelial cells from NO to O2-. Incubation of control and TRI-treated cultures with L-NAME revealed that in proliferating BCEC, eNOS generates NO, and that exposure to TRI alters eNOS function such that, upon endothelial stimulation, eNOS generates O2-. Exposure of endothelial cell cultures to TRI also impairs proliferative responses to VEGF, a potent mitogen that is well recognized for its involvement in proliferation and angiogenesis (Morbidelli et al., 1996Go; Ziche et al., 1997Go). Analysis of the activation state of eNOS reveals that TRI decreases the association of hsp90 with eNOS in stimulated cultures without significantly inhibiting the phosphorylation of eNOS at S1179, a change in protein interaction that allows eNOS to generate O2- (Pritchard et al., 2001Go, 2002Go). Recently, we found radicicol, a selective inhibitor of hsp90 that does not redox cycle, can shift the balance of NO and O2- production in proliferating endothelial cells from NO to O2-(Ou et al., 2003Go). Findings here are consistent with these previous reports and suggest that TRI disrupts protein interactions with eNOS to induce endothelial and eNOS dysfunction rather than altering expression of hsp90 or eNOS. Our observations provide new insight into the cellular mechanisms by which TRI converts eNOS from an NO synthase to a O2-, generating NADPH oxygenase. As eNOS generation of NO is intimately involved in numerous critical aspects of vascular physiology and endothelial cell biology, such changes in radical species generation may play a role in endothelial dysfunction to impair cardiac development.

Previous studies demonstrated that infants born with CHD in TRI contaminated regions often suffer from valve and septal defects (Goldberg et al., 1990Go). Recent reports showed that valve and septal defects was much higher in eNOS knockout mice (Feng et al., 2002Go; Lee et al., 2000Go). As the valves and septa are lined with endothelium (Nakajima et al., 1997Go; Wunsch et al., 1994Go), our findings suggest that not only eNOS deficiency, but also changes in endothelial cell and eNOS function play an important role in altering the development of these structures. The current studies were performed with fully differentiated endothelial cells that allowed us to obtain mechanistic data demonstrating a proof of concept that may be relevant to mechanisms by which TRI impairs development in vivo. For example, TRI has been shown to alter the transformation of epithelial-mesenchymal cells, the immediate precursors to differentiated endothelial cells (Boyer et al., 2000Go). Analysis of three markers of epithelial-mesenchymal cell transformation revealed that TRI dramatically inhibited the expression of Mox-1 and fibrillin 2 (Boyer et al., 2000Go). Interestingly, TRI had no effect on expression of {alpha}-smooth muscle actin. It is important to note that changes in Mox-1 and fibrillin 2 are seen in the developing AV canal where separation processes are required for development (Boyer et al., 1999Go, 2000Go). In angiogenesis, endothelial cell proliferation is governed by vascular remodeling of the subendothelial matrix (Gasparini 1999Go), which is similar to the separation processes seen in development. Interestingly, Zhao reported that deficiency in eNOS also impaired myocardial angiogenesis (Zhao et al., 2002Go). This is an important observation in that, during proliferation, endothelial cells exist in what appears to be an activated state with respect to NO and O2- generation (Arnal et al., 1994Go; Papapetropoulos et al., 1997bGo). Although cultured endothelial cells are not the same as epithelial-mesenchymal cells, our findings reveal a novel aspect of eNOS function that mimics eNOS deficiency when endothelial cell are exposed to the environmental pollutant, TRI. When TRI uncouples eNOS activity in proliferating endothelial cells, not only is less NO made but also greater amounts of O2- are generated. Such changes in enzyme function, for all practical purposes, results in a loss of NO activity that may also contribute to congenital heart defects.

Immunoprecipitation of eNOS, followed by immunoblotting for eNOS, phospho-eNOS, (S1179, bovine) and hsp90, revealed that exposure to TRI altered hsp90 interactions with eNOS. When such changes in the cellular participants are interpreted in light of the radical products eNOS generates upon activation, we find that a decrease in the association of hsp90 with eNOS correlates with an increase in eNOS-dependent O2- generation. Our findings that TRI induces eNOS O2- generation and decreases the association of hsp90 with eNOS in proliferating BCEC cultures are consistent with the effects of atherogenic lipoproteins such as native low-density lipoprotein (LDL) and minimally modified LDL on hsp90 interactions with eNOS and eNOS O2- generation (Pritchard et al., 2002Go; Stepp et al., 2002Go) and with reports that antineoplastic agents uncouple eNOS activity by altering hsp90 interactions with eNOS (Koshida et al., 2003Go; Ou et al., 2003Go; Pritchard et al., 2001Go).

This investigation shows that VEGF-induced endothelial cell proliferation is sensitive to TRI in a threshold-dependent manner. As VEGF may be an essential component in the development of valves and septa (Dor et al., 2001Go), our findings suggest that TRI alters VEGF-induced signaling in proliferating endothelial cells. The inhibitory effects of TRI on VEGF-induced proliferation are consistent with the fact that TRI decreases mesenchymal cell numbers within the endothelial layer (Boyer et al., 2000Go).

To determine how TRI altered signaling in proliferating endothelial cells, we considered the possibility that TRI may alter eNOS generation of NO and O2- similar to atherogenic LDLs and antineoplastic agents mentioned above. A23187 was chosen as the agonist because of its ability to activate eNOS by a receptor-independent and calcium-dependent mechanism, which allowed for direct enzyme activation. Taking this approach, we observed that TRI inhibited nitrite + nitrate production while increasing O2- generation by an L-NAME-inhibitable mechanism. Such reciprocal changes in O2- production due to inhibition of eNOS with L-NAME is strong evidence that TRI altered eNOS function in proliferating BCEC cultures such that, upon activation, eNOS switched from a NO synthase into a NADPH oxygenase. The importance of such a change in eNOS function cannot be understated. Based on the stoichiometry of the NOS reaction alone, for every one NO made during coupled activity, 2 O2- can be made during uncoupled activity. This represents a major change in function that, to date has been shown to adversely impact vascular function (Pritchard et al., 2002Go; Stepp et al., 2002Go; Vergnani et al., 2000Go). Indeed, uncoupled eNOS activity has been observed in a wide variety of animal and tissue culture models of diabetes, atherosclerosis, and hypertension (Hink et al., 2001Go; Kerr et al., 1999Go; Pritchard et al., 2002Go; Stepp et al., 2002Go). Angiostatin, a proteolytic product of plasminogen and matrix metalloproteinases (MMP) (Patterson and Sang, 1997Go) that antagonizes the trophic effects of VEGF (Keshet and Ben-Sasson, 1999Go), has recently been shown to inhibit endothelial proliferation and impair vasodilation by uncoupling eNOS activity (Koshida et al., 2003Go). Taken together, these reports and our data suggest that the balance of endothelial NO and O2- generation plays an important role in endothelial cell proliferation, which if altered or disturbed by an environmental pollutant such as TRI, may induce cardiovascular defects during development. As NO has been shown to promote endothelial cell proliferation (Fulton et al., 1999Go; Garcia-Cardena et al., 1998Go), the finding that TRI shifts the balance of NO and O2- at the level of eNOS provides new insight into the mechanisms by which endothelial and eNOS dysfunction may play a role in development of cardiac defects after exposure to a known teratogen.

Boyer and colleagues reported TRI could regulate the expression of more than 40 cellular proteins (Boyer et al., 2000Go). Our findings reveal that TRI has no effects on the expression of eNOS or hsp90 but rather alters fundamental protein interactions to induce a change in endothelial cell phenotype. Such changes in protein interactions underscores the importance of hsp90 in promoting coupled eNOS activity and maintaining proliferation. Others have shown that this abundant, constitutive chaperone binds to eNOS and acts as a scaffolding protein for aligning Akt with it target site of phosphorylation on eNOS at S1177 (Fontana et al., 2002Go). Although our data clearly indicate that TRI decreases hsp90 interactions with eNOS in proliferating endothelial cells, it remains unclear how a decrease in hsp90 association with eNOS promotes uncoupled enzyme activity.

In conclusion, TRI shifts the balance of NO and O2- in proliferating endothelial cells, and decreases the association of hsp90 with eNOS without impairing the ability of endothelial cells to phosphorylate eNOS at S1179. When this occurs, electron flow through eNOS occurs under less than ideal conditions, which can result in uncoupled eNOS activity. Measurements of NO and O2- confirm that TRI induces eNOS to generate O2- rather than NO, upon stimulation. If such changes in eNOS and endothelial function transpire at crucial time points during development, such changes might contribute to heart malformations.

ACKNOWLEDGMENTS

This work was supported in part by the Marie Z. Uihlein endowed chair award (to K.T.O.) from Children’s Hospital Foundation (Milwaukee, WI), and by NIH grants HL61417 and HL71214 (to K.A.P.) and ES011789 (to D.G.M.).

NOTES

1 To whom correspondence should be addressed at the Medical College of Wisconsin, Department of Surgery, Division of Pediatric Surgery, Cardiovascular Center–M4060, 8701 Watertown Plank Road, Milwaukee, WI 53226. Fax: (414) 456-6473. E-mail: JOU{at}mcw.edu. Back

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