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(The FASEB Journal. 2004;18:1209-1218.)
© 2004 FASEB

Inhibition of aldose reductase attenuates TNF-{alpha}-induced expression of adhesion molecules in endothelial cells

KOTA V. RAMANA*, ARUNI BHATNAGAR# and SATISH K. SRIVASTAVA*,1

* Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA; and
# Department of Medicine, University of Louisville, Louisville, Kentucky, USA

1Correspondence: Department of Human Biological Chemistry and Genetics, 6.644, Basic Science Building, University of Texas Medical Branch, Galveston, TX 77555, USA. E-mail: ssrivast{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased expression of adhesion molecules by the activated endothelium is a critical feature of vascular inflammation associated with several disease states such as atherosclerosis. However, mechanisms regulating the endothelial induction of adhesion molecules are not entirely clear. Herein we report that inhibition of the polyol pathway enzyme aldose reductase (AR) prevents the increase in ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs) and decreases monocyte adhesion to these cells. In TNF-{alpha}-stimulated HUVECs, treatment with AR inhibitors sorbinil and tolrestat diminished NF-{kappa}B activity, phosphorylation and degradation of I{kappa}-B{alpha}, and the nuclear translocation of NF-{kappa}B. Inhibition of AR abrogated TNF-{alpha}-induced activation and membrane translocation of PKC, and antisense ablation of AR prevented both TNF-{alpha}-induced PKC and NF-{kappa}B activation. However, inhibition of AR did not prevent phorbol ester-induced activation of PKC or NF-{kappa}B, indicating that inhibition of AR does prevents events upstream of PKC activation. These results identify a novel regulator of endothelial activation and suggest that AR is an obligatory mediator of TNF-{alpha} signaling leading to an increase in the expression of adhesion molecules and increased binding of monocytes to the endothelium.—Ramana, K. V., Bhatnagar, A., Srivastava, S. K. Inhibition of aldose reductase attenuates TNF-{alpha}-induced expression of adhesion molecules in endothelial cells.


Key Words: PKC activity • NF-{kappa}B • monocyte and adhesion molecules


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ENDOTHELIUM FORMS A nonthrombogenic interface between blood constituents and the vessel wall (1) . However, endothelial dysfunction due to vascular inflammation is associated with a diverse group of pathological processes including atherosclerosis (2 3 4) , diabetes (5 , 6) hypertension (4) and the pathogenesis of gram-negative bacteria-induced sepsis (7) . During atherogenesis, subendothelial accumulation of LDL causes activation of endothelial cells and increases the expression of adhesion molecules at the luminal surface of the endothelium. Increased adhesion of monocytes to the activated endothelium and their accumulation and transformation into macrophages generate foam cell-derived fatty streaks that develop into advanced atherosclerotic lesions (2 3 4) . Diabetes further exacerbates endothelial dysfunction and increases endothelial adhesion of leukocytes (5 , 6 , 8) . Remarkably similar endothelial changes have been reported for chronic infections by pathogens such as Chlamydia pneumoniae (9 , 10) .

Pathological endothelial changes are mediated in part by inflammatory cytokines such as TNF-{alpha}, which cause activation, increase adhesion, and, at high concentrations, induce apoptosis (11 , 12) . However, the mechanisms by which inflammatory cytokines cause endothelial dysfunction are not well understood. Previous studies have shown that cytokines such as TNF-{alpha} change the shape and motility of endothelial cells, which could contribute to vascular leakage at the site of inflammation (12) . TNF-{alpha} also stimulates an increase in the expression of cell adhesion molecules and genes involved in regulating the vessel tone and thrombosis (13 14 15 16) . It prevents endothelial recovery after injury (17) and activates signaling cascades that regulate the activation and translocation of redox-sensitive transcription factor NF-{kappa}B, an obligatory mediator of the inflammatory response that causes transcriptional activation of genes encoding adhesion molecules (13 , 17 18 19 20 21) . Multiple studies have shown that NF-{kappa}B activation is required for the up-regulation of adhesion molecules such as ICAM-1 and VCAM-1, which are responsible for monocyte adhesion and increased vascular inflammation (17 18 19 20 21) .

We recently reported that aldose reductase (AR), a member of the aldo-keto reductase (AKR) superfamily that catalyzes the first and the rate-limiting step of the polyol pathway, plays an important role in vascular smooth muscle cell (VSMC) growth (22 , 23) . Inhibition of AR attenuates neointimal hyperplasia in balloon-injured carotid arteries and prevents activation of the transcription factor NF-{kappa}B in injured arteries as well as in cultured VSMC stimulated with growth factors or cytokines (22) . Our kinetic and structural studies on AR show that besides reducing glucose, the enzyme is an efficient catalyst for the reduction of a wide range of aliphatic and aromatic aldehydes and their glutathione conjugates (24 25 26) . Moreover, inhibition of AR increases the accumulation of lipid peroxidation products in smooth muscle cells (27) and ischemic hearts (28) , indicating that AR catalysis represents a novel paradigm of cardiovascular defense against oxidative stress (29) . However, it remains unclear whether inhibition of AR prevents the sequelae of inflammatory events leading to endothelial activation and increased adhesion. The study reported here was therefore designed to investigate the effect of AR inhibition on vascular adhesion-induced by TNF-{alpha}. Our results show that inhibition of AR prevents TNF-{alpha}-induced up-regulation of adhesion molecules in endothelial cells, identifying a novel target for preventing vascular inflammation.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and cell culture
Antibodies against I{kappa}B-{alpha} and p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-I{kappa}B-{alpha} (Ser32) antibodies were purchased from New England BioLabs (Beverly, MA, USA). Phosphorothioate AR antisense oligonucleotide was used to transfect HUVECs to prevent the translation of AR mRNA as described earlier (23) . Consensus oligonucleotides for NF-{kappa}B and AP-1 transcription factors were obtained from Promega Corp. Recombinant TNF-{alpha} protein and mouse anti-rabbit glyceraldehyde 3-phosphate dehydrogenase antibodies were obtained from Research Diagnostics (Flanders, NJ, USA) and anti-AR polyclonal antibodies against recombinant AR were raised in rabbits (22) . LipofectAMINE Plus and Opti-minimal essential medium were obtained from Life Technologies, Inc. (Grand Island, NY, USA). Sorbinil and tolrestat were a gift from Pfizer (Groton, CT, USA) and American Home Products (Madison, NJ, USA), respectively. All other reagents were of analytical grade and were obtained from Sigma (St. Louis, MO, USA). Human umbilical vascular endothelial cells (HUVECs) were obtained from ATCC (Manassas, VA, USA) and maintained and grown confluent in Ham’s F12K medium supplemented with 2 M L-glutamate, 0.1 mg/mL heparin, 0.05 mg/mL endothelial growth supplement, and 10% FBS at 37°C in a humidified atmosphere of 5% CO2.

Cell adhesion assay
These assays were performed as described previously (30) . Briefly, HUVECs were plated in 48-well plate, grown to 70% confluence, and treated with AR inhibitors in the absence and presence of TNF-{alpha} (2 nM) for 24 h. Cells were rinsed three times with serum-free medium and THP-1 cells were added to each well. After 12 h incubation, the nonadherent THP-1 cells were rinsed off and the adherent cells were collected by treatment with trypsin-EDTA for 1 min. The cells were counted under a light microscope. In some experiments, monoclonal antibodies against ICAM-1 and VCAM-1 (5 µg/mL) were added to prevent cell adhesion.

Measurement of the cell surface expressions of adhesion molecules by enzyme-linked immunosorbent assay (ELISA)
Cell surface expression of VCAM-1 and ICAM-1 by HUVECs was quantified using cell ELISA as described earlier (31) . Briefly, HUVECs were grown to 70% confluence in 96-well plates coated with gelatin. Indicated concentrations of AR inhibitors were added to each well in the absence and presence of TNF-{alpha} (2 nM). After incubation for 24 h, the cells were washed with phosphate-buffered saline pH 7.4 (PBS) and fixed with 4% paraformaldehyde for 30 min at 4°C. Non-fat dry milk (3.0% in PBS) was added to the monolayers to reduce nonspecific binding. After washing three times with PBS, cells were incubated with anti-VCAM-1 and anti-ICAM-1 monoclonal antibodies overnight at 4°C, washed with PBS, then incubated with peroxidase-conjugated goat anti-mouse secondary antibody. Cells were washed again with PBS and exposed to the peroxidase substrate. The increase in absorbance at 490 nm was measured using an automated ELISA microplate reader.

Western blot analysis
To examine changes in protein expression, Western blot analyses were carried out using antibodies raised against I{kappa}B-{alpha} and phospho-I{kappa}B, AR, ICAM-1, and VCAM-1. Equal amounts of cytoplasmic extracts were subjected to 10% SDS-PAGE. Proteins were transferred to nitrocellulose filters, probed with the indicated antibodies, and the antigen-antibody complex was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Electrophoretic mobility gel shift assay (EMSA)
Cells were pretreated with various concentrations of AR inhibitors for 24 h, then with TNF-{alpha} (0.1 nM) for 1 h at 37ºC. Cytosolic as well as nuclear extracts were prepared as described (32) . Consensus oligonucleotides for NF-{kappa}B and AP-1 transcription factors were 5'-end labeled using T4 polynucleotide kinase. EMSA was performed as described (32) . The specificity of the assay was examined by competition with an excess of unlabeled oligonucleotide and supershift assays with antibodies to p65.

Determination of PKC activity
PKC activity was measured using the Promega-Sigma TECT PKC assay system (23) . Aliquots of the reaction (25 mM Tris-HCl pH 7.5, 1.6 mg/mL phosphatidylserine, 0.16 mg/mL diacylglycerol, and 50 mM MgCl2) were mixed with [{gamma}-32P] ATP (3,000 Ci/mmol, 10 µCi/µL) and incubated at 30°C for 10 min. To stop the reaction, 7.5 M guanidine hydrochloride was added and the phosphorylated peptide was separated on binding paper. The extent of phosphorylation was detected by measuring radioactivity retained on the paper.

Transfection with antisense oligonucleotides
The cells were incubated with 1 µM AR antisense or scrambled oligonucleotides using LipofectAMINE Plus (15 µg/mL) as the transfection reagent according to manufacturer’s instructions. After 12 h, the medium was replaced with freshF12K (containing 10% FBS) for another 24 h, followed by 24 h of incubation in serum-free F12K (0.1% FBS) before stimulation by TNF-{alpha}. Changes in the expression of AR were estimated by Western blot analysis using anti-AR antibodies and by measuring AR activity in the total cell lysates.

Statistical analysis
Data are presented as mean ± SE and P values were determined by unpaired Student’s ttest. P values of <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of AR inhibition on monocyte adhesion to activated endothelial cells
Increased adhesion of monocytes to the endothelium is a critical feature of vascular inflammation (4 5 6 7 8 9) . To determine whether AR regulates vascular adhesion, we examined whether inhibition of this enzyme would affect the adhesion of THP-1 cells to TNF-{alpha}-stimulated HUVECs. Unstimulated HUVECs displayed minimal adhesion to monocytes, but when these cells were stimulated with 2 nM TNF-{alpha}, a nearly sixfold increase in the adhesion of THP-1 cells to HUVECs was observed. However, TNF-{alpha}-induced stimulation of THP-1 cell adhesion to HUVECs was significantly attenuated by preincubating these cells with two structurally unrelated inhibitors of AR (i.e., sorbinil and tolrestat). The inhibition of THP-1 adhesion to activated HUVECs was prevented by AR inhibitors in a concentration-dependent manner (Fig. 1 A), but neither sorbinil nor tolrestat prevented monocyte adhesion to HUVECs in the absence of TNF-{alpha}, suggesting that AR inhibition selectively prevents endothelium-monocyte adhesion stimulated by TNF-{alpha}.



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  		Figure 1. Effect of AR inhibitors on THP-1 cell adhesion to HUVECs in the presence of TNF-{alpha}. A) Serum-starved cells were preincubated without or with the indicated concentrations of sorbinil/tolrestat for 24 h, then stimulated with 2 nM TNF-{alpha} for 24 h. After incubations, the cells were washed with PBS, THP-1 cells were added, and the incubation continued for another 12 h. Cell adhesion assays were performed as described in the text using a light microscope. B) Involvement of ICAM-1 and VCAM-1 in TNF-{alpha}-mediated THP-1 cell adhesion to HUVECs. Serum-starved cells treated without or with antibodies against ICAM-1 and VCAM-1 (5 µg/mL) were stimulated with TNF-{alpha} (2 nM) for 24 h. Cell adhesion assays were performed as described above. Data are presented as mean ± SE (n=5), *P <0.01 vs. TNF-{alpha}-treated cells.

To identify specific adhesion molecules that mediate TNF-{alpha}-induced endothelial adhesion to monocytes, we tested the involvement of VCAM-1 and ICAM-1, which has been reported to participate in the adhesion of monocytes to endothelial cells (4 , 5 , 13 , 14) . We examined the effects of monoclonal antibodies against VCAM-1 and ICAM-1 on the TNF-{alpha}-induced adhesion of THP-1 cell to endothelial cells. Treatment with anti-VCAM-1 or anti-ICAM-1 antibodies resulted in a 30–35% inhibition of TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. However, combined incubation with both antibodies blocked TNF-{alpha}-induced THP-1 cell adhesion to HUVECs by >55% (Fig. 1B ). Based on these results we conclude that, under the experimental conditions tested, ICAM-1 and VCAM-1 together play a significant role in mediating the adhesion of THP-1 cells to HUVECs and that inhibition of AR may be preventing monocyte-endothelial interactions by preventing up-regulation of ICAM-1 and VCAM-1.

AR inhibition attenuates TNF-{alpha}-induced cell surface expressions of VCAM-1 and ICAM-1 in HUVECs
We next determined whether inhibition of cell adhesion by AR inhibitors is related to changes in the expression and activity of ICAM-1 and VCAM-1. As before, we tested the effects of two different AR inhibitors sorbinil and tolrestat. For these experiments, HUVECs were pretreated with AR inhibitors and changes in the expression of ICAM-1 and VCAM-1 after stimulation with TNF-{alpha} were measured using ELISA. As shown in Fig. 2 A, stimulation with TNF-{alpha} (2 nM) increased ICAM-1 expression by ~fivefold compared with unstimulated HUVECs, and this increase was significantly attenuated by sorbinil and tolrestat in a dose-dependent manner. At the maximal concentration of the drugs tested only basal levels of expression of ICAM-1 were observed. Similarly, cell surface expression of VCAM-1 was significantly (~8-fold) increased by TNF-{alpha} and markedly inhibited in cells pretreated with either sorbinil or tolrestat (Fig. 2B ).



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  		Figure 2. Effect of AR inhibitors on the surface expression of ICAM-1 and VCAM-1 in TNF-{alpha}-stimulated cells. Serum-starved cells were preincubated without or with the indicated concentrations of sorbinil/tolrestat for 24 h, then stimulated with 2 nM TNF-{alpha} for 24 h. After incubations, the cells were fixed in 4% formaldehyde and the expression of ICAM-1 and VCAM-1 was measured by ELISA using anti-VCAM-1 and anti-ICAM-1 antibodies, respectively. Data are presented as mean ± SE (n=5), *P <0.001 vs. TNF-{alpha}-treated cells.

To confirm the results obtained with ELISA, we measured the protein levels of ICAM-1 and VCAM-1 by Western blot analysis. As shown in Fig. 3 , TNF-{alpha} significantly increased VCAM-1 and ICAM-1 protein levels in HUVECs compared with unstimulated HUVECs. No significant difference in ICAM-1 and VCAM-1 levels was observed when HUVECs were either left untreated or were pretreated with sorbinil or tolrestat in the absence of TNF-{alpha}. However, pretreatment with sorbinil and tolrestat significantly (80 to 100%) prevented the TNF-{alpha}-induced up-regulation of ICAM-1 and VCAM-1 proteins (Fig. 3) . These observations indicate that inhibition of AR by itself does not affect the expression of adhesion molecules, but it specifically prevents TNF-{alpha}-induced endothelial activation.



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  		Figure 3. Inhibition of AR prevents TNF-{alpha}-induced up-regulation of adhesion molecules. Serum-starved cells treated without or with 10 µM sorbinil or tolrestat in the absence and presence TNF-{alpha} (2 nM) for 24 h. The pooled cell extracts from 3 independent experimental sets were subjected to SDS-PAGE and Western blots were developed using antibodies anti-GAPDH, anti-VCAM-1, and anti-ICAM-1 antibodies. Data shown are representative of 3 independent experiments.

Attenuation of TNF-{alpha}-induced NF-{kappa}B activation by AR inhibitors
Up-regulation of the endothelial expression of adhesion molecules by TNF-{alpha} is mediated by a complex array of signaling cascades in which NF-{kappa}B activation plays a pivotal role (17 18 19 20 21) . Hence, we systematically examined how inhibition of AR affects TNF-{alpha}-induced NF-{kappa}B signaling in endothelial cells. For these experiments, endothelial cells were grown to confluency and preincubated for 24 h with sorbinil (0 to 100 µM), then stimulated with 0.1 nM TNF-{alpha} for 60 min at 37°C. The NF-{kappa}B activity was measured by EMSA. As shown in Fig. 4A, preincubation of HUVECs with sorbinil caused a concentration-dependent inhibition of NF-{kappa}B activation. Treatment with sorbinil (10 µM) caused >70% inhibition of NF-{kappa}B binding to its cognate DNA sequence. However, sorbinil alone did not affect the NF-{kappa}B activity.

To examine the time course of inhibition, quiescent HUVECs were preincubated with 0–48 h with 10 µM sorbinil before a 60 min exposure to TNF-{alpha}, and NF-{kappa}B binding was determined by EMSA. As shown in Fig. 4B , the inhibitory effects of sorbinil were evident after 12–24 h of preincubation; maximal inhibition was observed in cells preincubated with sorbinil for 24 h. No additional inhibition was observed when the preincubation time was extended to 48 h. To determine whether sorbinil chemically interacts with TNF-{alpha} and attenuates TNF-{alpha}-initiated signaling, the HUVECs were incubated with TNF-{alpha} and sorbinil or tolrestat for 60 min. Under these conditions, treatment with sorbinil or tolrestat did not inhibit NF-{kappa}B activity (Fig. 4C , lanes 3, 4), indicating that preincubation with AR inhibitors is essential for inhibiting NF-{kappa}B and that these inhibitors do not directly interfere with NF-{kappa}B activation once the signaling cascade is initiated. To ascertain that the gel-retarded band visualized in EMSA in TNF-{alpha}-stimulated cells was indeed due to NF-{kappa}B, we incubated the nuclear extract from TNF-{alpha}-activated cells with antibodies to p65 subunit prior to EMSA. Inclusion of anti-p65 antibodies supershifted the band to a higher molecular weight, whereas, preimmune serum did not affect NF-{kappa}B binding (Fig. 4 , lane 6). Moreover, competition with a 50-fold excess of unlabeled NF-{kappa}B oligonucleotide eliminated these bands, indicating that the bands seen were specifically due to NF-{kappa}B (Fig. 4 , lane 8).



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  		Figure 4. Attenuation of TNF-{alpha}-induced NF-{kappa}B activation by AR inhibitors. A) Concentration-dependent inhibition of NF-{kappa}B activation by sorbinil. Serum-starved cells were preincubated without or with the indicated concentrations of sorbinil for 24 h, then stimulated with 0.1 nM TNF-{alpha} for 1 h. B) Time course of inhibition. The cells were incubated with sorbinil (10 µM), then stimulated with 0.1 nM TNF-{alpha} for 1 h. C) Supershift and competition assays. Cells were serum-starved and left untreated or stimulated under the indicated conditions. Nuclear extracts were prepared and EMSA assay was performed as described in the text. Nuclear extracts of untreated cells (lane 1), after stimulation with 0.1 nM TNF-{alpha} for 1 h (2) , after combined treatment with 0.1 nM TNF-{alpha} and 10 µM sorbinil for 1 h (3) or with 0.1 nM TNF-{alpha} and 10 µM tolrestat for 1 h (4) . Lanes 5, 6: supershift with p65 antibody or the preimmune sera. Lanes 7, 8: competition with 20x and 50x unlabeled oligonucleotide probe.

Inhibition of AR prevents nuclear translocation of the p50/p65 dimer
To examine signaling events upstream to NF-{kappa}B activation, we measured nuclear translocation of NF-{kappa}B and the phosphorylation and degradation of I{kappa}B-{alpha}. As shown in Fig. 5 A, most of the inactive form of NF-{kappa}B was present in the cytosol of the unstimulated cells, as evident by the diffused staining obtained with anti-p65 antibodies identified using Alexa Fluor 488 fluorescence secondary antibodies. Incubation with TNF-{alpha} led to sharp nuclear localization of fluorescence that corresponded with the intracellular staining of the Hoechst nuclear dye (Fig. 5B ) and change to cyno color when superimposed (Fig. 5C ), indicating that TNF-{alpha} induces the nuclear localization of p65/p50 dimer. Incubation of these cells with tolrestat alone did not affect the nuclear localization of p65, as evident from the diffused staining, comparable to that observed in the unstimulated cells. However, when tolrestat pretreated cells were stimulated with TNF-{alpha}, no nuclear staining was observed, and these cells showed diffused perinuclear staining. This was further confirmed by Western blot analysis using p65 antibodies in cytosolic and nuclear extracts 60 min after stimulation with TNF-{alpha} (Fig. 5D ). Exposure of HUVECs to TNF-{alpha} for 20 min resulted in a gradual translocation of NF-{kappa}B from cytosol to the nucleus, which was maximal in 60 min. In the sorbinil pretreated cells, however, only a partial translocation of NF-{kappa}B was observed 60 min after exposure to TNF-{alpha} (Fig. 5D ). Based on these results, we conclude that inhibition of AR prevents TNF-{alpha}-induced nuclear translocation of p65.



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  		Figure 5. Inhibition of AR prevents nuclear translocation of NF-{kappa}B and phosphorylation and degradation of I{kappa}B-{alpha}. Serum-starved cells were either untreated (I), treated with 0.1 nM TNF-{alpha} for 1 h (II), pretreated with tolrestat for 24 h (III), or preincubated with 10 µM tolrestat for 24 h, followed by stimulation with 0.1 nM TNF-{alpha} for 1 h (IV). Photomicrographs (60x): A) NF-{kappa}B localization by immunostaining using antibodies against p65; B) HUVECs nuclei stained with Hoechst-33342; and C) composite images generated by superimposing photomicrographs shown in panels A, B. D) Time course analysis of NF-{kappa}B translocation. Western blots were developed using anti-p65 antibodies of cytosolic (CE) or nuclear (NE) extracts prepared from serum-starved cells pretreated without (upper panel) or with (lower panel) 10 µM sorbitol for 24 h, followed by stimulation with 0.1 nmol/L TNF-{alpha}. Cytosolic extracts were used for developing Western blots using anti-phospho-I{kappa}-B{alpha} (E) or anti-I{kappa}-B{alpha} antibodies (F).

Inhibition of AR attenuates phosphorylation and degradation of I{kappa}B-{alpha}
The nuclear translocation of NF-{kappa}B is preceded by phosphorylation and proteolytic degradation of I{kappa}B-{alpha} (18 , 19) . Hence, we tested whether inhibition of AR prevents phosphorylation and degradation of I{kappa}B-{alpha} on Western blots developed with antibodies specific to unphosphorylated and phosphorylated I{kappa}B-{alpha}. In untreated HUVECs, partial I{kappa}B-{alpha} phosphorylation was observed within 10 min of stimulation with TNF-{alpha}; maximal phosphorylation was evident at 20 min, after which a progressive decrease in the immunoreactive band was observed (Fig. 5E ). In the presence of sorbinil, the increase in phosphorylation appeared to be less than in its absence. Moreover, the decrease in the I{kappa}-B{alpha} protein after 20 min of TNF-{alpha} exposure was blunted in sorbinil pretreated cells. These observations indicate that stimulation with TNF-{alpha} leads to rapid phosphorylation and degradation of I{kappa}B-{alpha}, followed by complete resynthesis in 60 min. This sequence of events was dramatically affected by inhibiting AR. In the sorbinil-treated cells, little phosphorylation of I{kappa}B-{alpha} was observed upon stimulation with TNF-{alpha}, and there was no change in the cellular abundance of the I{kappa}B-{alpha} protein (Fig. 5F ). Together, these results show that inhibition of AR prevents TNF-{alpha}-induced phosphorylation and resultant proteolytic degradation of I{kappa}B-{alpha}, thereby inhibiting activation and translocation of NF-{kappa}B.

Inhibition of AR prevents PKC activation
To determine whether PKC is a critical target of AR inhibitors in endothelial cells, we measured PKC activity in TNF-{alpha}- and PMA-stimulated cells. As shown in Fig. 6 A, sorbinil and tolrestat by themselves did not alter the PKC activity in these cells. Stimulation with TNF-{alpha} led to a significant increase in membrane-bound PKC activity, as has been reported (23) . PKC activity was dramatically increased in these cells upon stimulation with PMA. Pretreatment of the cells with sorbinil or tolrestat prevented TNF-{alpha}-induced PKC activity. However, the AR inhibitors did not prevent PMA-induced activation of PKC. Collectively, these results suggest that inhibition of AR does not directly affect PKC activity, but prevents PKC activation by interrupting upstream signaling events, and that the pathways downstream to PKC are insensitive to AR.



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  		Figure 6. Inhibition of AR attenuates of PKC activation. A) Serum-starved cells were preincubated without (control) or with 10 µM sorbinil or tolrestat for 24 h, stimulated with TNF-{alpha} (0.1 nM) or PMA (10 nM) for 4 h, and the membrane-bound PKC activity was measured. B) The HUVECs were either left untreated, treated with lipofectAMINE, or transiently transfected with AR antisense or scrambled oligonucleotides. The cells were stimulated with TNF-{alpha} (0.1 nM) or PMA (10 nM) for 4 h and membrane-bound PKC activity was measured. Bars represent mean ± SE (n=4). **P<0.001 compared with the activity without the inhibitor (A) or in cells transfected with the scrambled control oligonucleotides (B). C, left: PMA induced NF-{kappa}B is not inhibited by AR inhibitors. Growth arrested HUVECs preincubated without or with AR inhibitors, followed by stimulation with TNF-{alpha} (0.1 nM) for 1 h or PMA (10 nM) for 2 h and activation of NF-{kappa}B was measured by EMSA. C, right: Antisense ablation of AR attenuates TNF-{alpha}-induced NF-{kappa}B activation. The HUVECs were either untreated, treated with lipofectAMINE, or transiently transfected with AR antisense or scrambled oligonucleotides. Cells were stimulated with TNF-{alpha} (0.1 nM) for 1 h and NF-{kappa}B activation was measured by EMSA.

To further confirm the specificity of the AR inhibitors, we transfected the HUVECs with AR antisense oligonucleotides, which abolished >90% of the AR protein (Fig. 6B , inset). The ablation of AR expression was accompanied by a decrease in the membrane-bound PKC activity and the activation of NF-{kappa}B in TNF-{alpha}-treated cells (Fig. 6B, C , right panel).

To determine whether inhibition of AR prevents only PKC activation or also directly affects the NF-{kappa}B pathway, we examined whether inhibition of AR would prevent the activation of NF-{kappa}B induced by phorbol ester phorbol 12-myristate 13-acetate (PMA), which bypasses the upstream signaling and directly stimulates PKC. For this series of experiments, growth-arrested VSMC were preincubated without and with AR inhibitors, followed by incubation with PMA. Stimulation with PMA resulted in marked NF-{kappa}B activation, which was not prevented by either sorbinil or tolrestat (Fig. 6C , left). These observations indicate that the locus of AR inhibition is upstream of PKC and, when PKC is directly activated, AR inhibition does not abolish downstream signaling. These observations suggest that the AR inhibitors do not directly interfere with any of the signaling events downstream of PKC.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that inhibition of AR prevents the TNF-{alpha}-induced up-regulation of adhesion molecules in endothelial cells by inhibiting the PKC-I{kappa}B-{alpha}-NF-{kappa}B signaling pathway. This identifies a novel mechanism that regulates endothelial signaling and leads to activation of the major axis of vascular inflammation. The results obtained from this study may be of significance in understanding and regulating endothelial dysfunction associated with a wide range of pathological states.

Increased expression of cell adhesion molecules is an important aspect of inflammatory changes associated with atherosclerosis and contributes to the activation and recruitment of lymphocyte from adventitial vessels and the arterial lumen to the vessel wall (2 3 4) . Adhesion molecules are induced in endothelial cells by inflammatory cytokines, and increased endothelial adhesion of leukocytes has been linked to another diverse group of physiological and pathological processes, including B cell development (33 , 34) , tumor metastasis (35 , 36) , and angiogenesis (37 , 38) . Up-regulation of adhesion molecules and activation of the endothelium are the major causes of cardiovascular events during bacterial infections (9 10 11) . Although monocyte adhesion to the endothelium is a complex process, earlier studies have shown that ICAM-1 and VCAM-1 are the main adhesion molecules induced in the activated endothelium (11 12 13 14 , 19) . In agreement with these reports, we found that stimulation with TNF-{alpha} led to a robust increase in the expression of these molecules. The functional significance of these changes was demonstrated by the ability of anti-ICAM-1 and anti-VCAM antibodies-1 to significantly prevent TNF-{alpha}-induced adhesion of the endothelial cells to monocytes. However, even in the presence of both anti-ICAM-1 and anti-VACM-1 antibodies, residual monocyte-endothelial cell adhesion was observed, indicating that other adhesion molecules participate in these interactions. This residual binding may be due to other adhesion molecules such as E-selectin or ELAM-1, also expressed by the activated endothelium (19 , 21 , 39 , 40) . Nevertheless, our demonstration that inhibition of AR decreases the cellular abundance and surface expression of ICAM-1 and VCAM-1 and, at the same time, prevents the binding of THP-1 cells to HUVECs indicates that AR-dependent endothelial processes are essential for ICAM-1- and VCAM-1-mediated adhesion to monocytes. Furthermore, because treatment with tolrestat or sorbinil abrogated the induction of these molecules by TNF-{alpha}, it appears that inhibition of AR interferes with the transcriptional regulation of these proteins.

The transcription of adhesion molecules ICAM-1 and VCAM-1 is regulated by the activity of NF-{kappa}B (18 19 20 21) . In atherosclerotic lesions, activated NF-{kappa}B localizes to sites expressing high levels of adhesion molecules (4 , 5) . Although other transcriptional mechanisms relating to SP-1, GATA, and ORF activation have been identified in regulating the expression of adhesion molecules, increased expression of these molecules is associated with increased NF-{kappa}B activity, and inhibition of NF-{kappa}B has been shown to prevent the increase in ICAM-1 and VCAM-1 (18 , 20 , 21) . Consistent with these observations, we found that activation of ICAM-1 and VCAM-1 in TNF-{alpha}-stimulated endothelial cells are preceded by the activation of NF-{kappa}B. As described before (19 , 21) , NF-{kappa}B activation was associated with the phosphorylation and degradation of I{kappa}B-{alpha} and the nuclear translocation of p65. However, this entire series of events was prevented by AR inhibitors, suggesting that AR catalysis is permissive of signaling events that lead to I{kappa}B-{alpha} phosphorylation and that inhibition of AR does not prevent the binding of NF-{kappa}B to its cognate DNA sequences.

The view that AR inhibitors do not directly interact with the NF-{kappa}B activation pathway is further supported by the observation that activation of NF-{kappa}B by PMA was not inhibited by sorbinil or tolrestat. Based on these observations, we propose that inhibition of AR extinguishes signaling events upstream of NF-{kappa}B activation. The NF-{kappa}B-dependent mechanism of inhibition of the induction of adhesion molecules by AR inhibition is similar to mechanism of inhibition due to NO (41 , 42) , glucocorticoids (43 , 44) , or aspirin (45) but distinct from the NF-{kappa}B-independent mechanisms by which selenium (46) , metal chelators (47) , or sheer stress (48) prevent the induction of adhesion molecules in cytokine-treated endothelial cells. However, inhibition of AR prevents NF-{kappa}B activation by mechanisms different from those of glucocorticoids or NO. Glucocorticoids increase the transcription of I{kappa}-B{alpha} gene (49 , 50) or inhibit NF-{kappa}B transactivation (44 , 51) , whereas NO inhibits the expression and nuclear translocation of I{kappa}-B{alpha} (42) . In contrast, inhibition of AR does not prevent NF-{kappa}B-dependent gene transcription (vide supra) or increase the abundance of I{kappa}-B{alpha} protein or its nuclear translocation, but prevents phosphorylation and degradation of I{kappa}-B{alpha}, suggesting that inhibition of AR does not interfere with NF-{kappa}B signaling pathway per se, but affects events upstream to I{kappa}-B{alpha} phosphorylation.

In endothelial cells, TNF-{alpha}-induced phosphorylation of I{kappa}B-{alpha} and its proteolytic degradation is mediated by PKC, and inhibition of PKC has been shown to prevent TNF-{alpha} induced NF-{kappa}B activation (52 53 54) . Our results support the possibility that TNF-{alpha}-induced NF-{kappa}B activation is mediated by PKC since inhibition of NF-{kappa}B by AR inhibitors was associated with inhibition of PKC activation and membrane translocation. Because ablation of AR by antisense oligonucleotides also resulted in the inhibition of PKC activity, it appears that inhibition of the PKC-NF-{kappa}B pathway by AR inhibitors in endothelial cells is not due to nonspecific effects of the drugs used to inhibit AR, but are a result of decreased AR catalysis. Nonetheless, inhibition of AR did not prevent PMA-stimulated NF-{kappa}B. We suggest this indicates that inhibition of AR prevents signaling events upstream of DAG formation. Consequently, direct stimulation of PKC by PMA, which bypasses these events, is insensitive to AR. However, these results do not rule out the alternate possibility that PMA-activated PKC isoforms are insensitive to AR inhibition, which only prevents activation of atypical PKCs such has PKC{zeta}, which has been shown to be a critical mediator of TNF-{alpha} induced NF-{kappa}B activation and ICAM-1 induction in endothelial cells (55) .

Preventing NF-{kappa}B activation by inhibiting AR represents a new approach for decreasing vascular inflammation. A number of AR inhibitors have been synthesized for treating diabetic complications (56 , 57) and could be used for their anti-inflammatory effects. Moreover, the observation that inhibition of AR prevents endothelial activation may provide new insight into the mechanisms by which AR inhibitors prevent or delay diabetic complications. Our previous studies have shown that inhibition of AR prevents smooth muscle cell growth and decreases neointimal hyperplasia in balloon-injured arteries, suggesting that inhibition of AR may be beneficial in injured arteries denuded of the endothelium (23) . However, smooth muscle cells in noninjured arteries do not express AR, although the enzyme is basally expressed in the endothelium (22) . Therefore, the endothelial rather than smooth muscle cell-specific effects of AR are likely to be more general and relevant to a larger set of vascular pathologies. We expect that chronic vascular dysfunction in the presence of intact endothelium, such as that associated with diabetes, bacterial infections, or hypertension, is likely to be regulated by AR-dependent pathways and consequently responsive to AR inhibition.

AR was first identified as an enzyme catalyzing the reduction of glucose to sorbitol (56 , 57) . The view that AR participates in glucose metabolism is consistent with extensive data showing increased AR-dependent sorbitol formation in the presence of high glucose. In keeping with this view, inhibition of AR has been shown to prevent or delay renal and neurological complications of long-term diabetes (56 , 57) . However, structural and kinetic studies of the enzyme suggest otherwise. In vitro, glucose is a poor substrate of the enzyme, which is more efficient in catalyzing the reduction of medium-chain hydrophobic aldehydes such as those generated as by-products of lipid peroxidation (24) . Moreover, the active site of the enzyme lacks hydrophilic groups characteristic of carbohydrate binding proteins but offers a large hydrophobic surface capable of binding to hydrophobic aldehydes, steroids, and glutathione conjugates (24 , 56) . Thus, glucose seems to be an incidental substrate of the enzyme that appears to participate in the detoxification of lipid peroxidation products and related electrophiles. How these properties of AR relate to the regulation of TNF-{alpha}-NF-{kappa}B signaling remains unclear, but we speculate that AR may be regulating PKC and NF-{kappa}B activation by containing the oxidative stress and redox changes that accompany TNF-{alpha} stimulation (55) , and that inhibition of the enzyme prevents PKC/NF-{kappa}B activation due to excessive accumulation of oxidants.

Alternatively, AR may be essential for providing cofactor(s)/substrate(s) essential for PKC activation. Our observation that prolonged pretreatment with AR inhibitors was necessary for preventing the up-regulation of adhesion molecules by TNF-{alpha} suggests an indirect role of the enzyme as a regulator of metabolic and genetic events permissive of cytokine signaling. Although extensive investigations are required to identify AR-dependent products required for signaling and growth, the observation that inhibition of AR does not prevent PMA-induced PKC activation suggests that inhibition of AR interferes with the availability of diacylglycerol (DAG). Previous investigations have shown that inhibition of AR prevents high glucose-induced DAG formation (58) , which may relate to the ability of the enzyme to alter the redox state of pyridine nucleotides (56 , 57) . Whether AR regulates cytokine signaling by similar mechanisms remains unknown. Clearly, additional experiments are required to identify the role of AR in redox signaling and its relationship to the PKC-NF-{kappa}B axis.

In summary, the results of the present study show that inhibition of AR prevents the induction of adhesion molecules in activated endothelial cells. Inhibition of AR prevents TNF-{alpha}-induced activation of NF-{kappa}B and PKC, indicating that continuous activity of AR is essential for proinflammatory signaling and that AR is a critical element in the development of inflammation. These results identify a novel component regulating endothelial activation and offer a new target for anti-inflammatory interventions.


   ACKNOWLEDGMENTS
 
This work was supported in part by National Institutes of Health grants DK36118 and EY01677 (to S.K.S.) and HL55477 (to A.B.). Technical assistance by Mr. Brian Friedrich is gratefully acknowledged.

Received for publication March 23, 2004. Accepted for publication April 23, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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