High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway.

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.

We have recently found that activation of Janus kinase 2 (JAK2) 1 was essential for the angiotensin II (Ang II)-induced proliferation of vascular smooth muscle cells (VSMC) and that high glucose (HG) augmented the Ang II induction of VSMC proliferation by increasing signal transduction through the activation of JAK2 (1,2). Current studies suggest that HG, via the polyol pathway, induces a rapid increase in intracellular reactive oxygen species (ROS) such as H 2 O 2 , which stimulates intracellular signal events similar to those activated by Ang II including stimulation of growth-promoting kinases such as JAK2 and extracellular signal-regulated kinase 1/2 (3)(4)(5). The polyol pathway generates ROS (H 2 O 2 and O 2 Ϫ ) (6,7), which can then act as signal mediators in the activation of mitogenic pathways, such as the JAK/STAT signaling cascade (8). For instance, in VSMC H 2 O 2 has been shown to play an important role in regulating cell growth (5). It has also recently been reported that Ang II induces a rapid increase in intracellular H 2 O 2 via NADPH oxidase, which subsequently activates growth-related responses plus the activation of JAK2 (5,9). Similar results have also been found for PDGF-induced cell proliferation, which was shown to be dependent on H 2 O 2 (8). Furthermore, PDGF uses H 2 O 2 as a second messenger to regulate the activation of JAK2 in rat fibroblasts (8).
The HG-induced activation of protein kinase C (PKC) has also been recently shown to increase the production of ROS and to enhance VSMC proliferation. In addition, the synthesis and characterization of a specific inhibitor for PKC-␤ isoforms has confirmed the role of PKC activation in mediating HG effects on VSMC, and it provides in vivo evidence that the activation of the PKC-␤ isoform could be responsible for the abnormal ROS production and vascular growth in diabetic animals (10). For example, a recent study has concluded that VSMC can produce ROS through NADPH oxidase via activation of PKC. The study found that exposure of cultured VSMC to HG significantly increased ROS production and that treatment of the cells with phorbol myristic acid, a PKC activator, also increased ROS production. Furthermore, it was also found that the HG-induced ROS production was completely inhibited by GF109203X, a PKC-specific inhibitor. These results suggest that HG stimulates ROS production through PKC-dependent activation of NADPH oxidase in VSMC (11). In addition, a very recent study has also shown that the PKC-␤2 isoform was essential for the activation of NADPH oxidase (12).
In the present study we have inhibited by either pharmacological or molecular methods the polyol pathway or PKC-␤ to examine their effects on the Ang II, HG, and Ang II plus HG-induced tyrosine phosphorylation of JAK2, ROS production, and VSMC proliferation. We hypothesize that HG augments the Ang II-induced activation of JAK2 and growth responses in VSMC through ROS generated via the polyol pathway activation of PKC-␤.

EXPERIMENTAL PROCEDURES
Materials-Molecular weight standards, acrylamide, SDS, N,NЈmethylenebisacrylamide, N,N,NЈ,NЈ-tetramethylenediamine, protein assay reagents, and nitrocellulose membranes were purchased from Bio-Rad. Bovine catalase was obtained from Roche Applied Science, and 2,7-dichlorofluorescin (DCFH) diacetate was from Molecular Probes. * This study was supported by National Institutes of Health Grants HL58139 and DK50268 and an American Heart Association Established Investigator Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Protein A/G-agarose was obtained from Santa Cruz Biotechnology, and Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, trypsin, and all medium additives were obtained from Mediatech Inc. Antibodies to phosphotyrosine (PY20), anti-SHP-1, anti-SHP-2, and the PKC-␤ isoforms were procured from Transduction Laboratories. Antiphosphotyrosine JAK2 and anti-JAK2 antibodies were obtained from BIOSOURCE International. The aldose reductase inhibitor zopolrestat and the PKC-␤ inhibitor LY379196 were gifts from Pfizer and Eli Lilly, respectively. The Supersignal substrate chemiluminescence detection kit was obtained from Pierce. Goat anti-mouse IgG and anti-rabbit IgG were acquired from Amersham Biosciences, and Tween 20 and all other chemicals were purchased from Sigma.
Aldose Reductase and PKC-␤ Antisense Oligonucleotide Treatment-Aldose reductase (15) and PKC-␤ (16) antisense oligonucleotides synthesis and treatments were carried out as previously described (17,18). After 12 h, medium was removed, calf serum (0.1%) in Dulbecco's modified Eagle's medium in NG (normal glucose) was added, and the cells were allowed to recover for 30 min. The VSMC were washed once with serum-free DMEM and growth-arrested in serum-free DMEM in NG for 24 h. Afterward, the VSMC were placed in either NG or HG media for 24 more hours.
Western Blotting of JAK2-To ascertain the tyrosine phosphorylation of JAK2, growth-arrested VSMC were placed in either NG or HG for 24 h and stimulated with 0.1 M Ang II or 0.33 mM PDGF for various times ranging from 0 to 10 min. At the end of stimulation, cells were washed twice with ice-cold phosphate-buffered saline with 1 mmol/liter Na 3 VO 4 . Each dish was then treated for 60 min with ice-cold lysis buffer (20 mmol/liter Tris-HCl, pH 7.4, 2.5 mmol/liter EDTA, 1% Triton X-100, 10% glycerol, 1% deoxycholate, 0.1% SDS, 10 mmol/liter Na 4 P 2 O 7 , 50 mmol/liter NaF, 1 mmol/liter Na 3 VO 4 , and 1 mmol/liter phenylmethylsulfonyl fluoride). The supernatant fraction was obtained as cell lysate by centrifugation at 58,000 ϫ g for 25 min at 4°C. Protein concentration of the lysate was measured with the Bio-Rad detergent-compatible assay kit and bovine serum albumin as the standard.
Subsequently, samples were resolved by 10% SDS-PAGE gel electrophoresis, transferred to a nitrocellulose membrane, and blocked by a 60-min incubation at room temperature (22°C) in Tris-buffered saline with 0.05% Tween 20, pH 7.4, plus 5% skimmed milk powder. The nitrocellulose membrane was incubated overnight at 4°C with affinitypurified anti-phospho-specific JAK2 antibodies or non-phospho anti-JAK2 antibodies. Subsequently, the nitrocellulose membranes were washed twice for 10 min each with Tris-buffered saline with 0.05% Tween 20, pH 7.4, and incubated for various times with goat anti-rabbit IgG horseradish peroxidase conjugate. After extensive washing, bound antibody was visualized on Kodak Biomax film with a Pierce Supersignal substrate chemiluminescence detection kit. Molecular weight markers assessed specificity of the bands.
Immunoprecipitation Studies of SHP-1 and SHP-2-To determine the protein-tyrosine phosphatase SHP-1 and SHP-2 tyrosine phosphorylation, serum-starved VSMC grown in HG for 24 h were stimulated with 0.1 M Ang II for various times ranging from 0 to 10 min. At the end of stimulation, cells were washed twice with ice-cold phosphatebuffered saline with 1 mmol/liter Na 3 VO 4 . Each dish was then treated for 60 min with ice-cold lysis buffer (20 mmol/liter Tris-HCl, pH 7.4, 2.5 mmol/liter EDTA, 1% Triton X-100, 10% glycerol, 1% deoxycholate, 0.1% SDS, 10 mmol/liter Na 4 P 2 O 7 , 50 mmol/liter NaF, 1 mmol/liter Na 3 VO 4 , and 1 mmol/liter phenylmethylsulfonyl fluoride), and the supernatant fraction was obtained as cell lysate by centrifugation at 58,000 ϫ g for 20 min at 4°C. The cell lysate was incubated with 10 g/ml either anti-SHP-1 or anti-SHP-2 monoclonal antibodies at 4°C for 2 h and precipitated by the addition of 50 l of protein A/G-agarose at 4°C overnight. The immunoprecipitates were then recovered by centrifugation and washed 3 times with ice-cold wash buffer (Trisbuffered saline, 0.1% Triton X-100, 1 mmol/liter phenylmethylsulfonyl fluoride, and 1 mmol/liter Na 3 VO 4 ). Immunoprecipitated proteins were dissolved in 100 l of Laemmli sample buffer, and 80 l of each sample was resolved by SDS-PAGE gel electrophoresis. Samples were transferred to a nitrocellulose membrane and blocked by 60-min incubation at 22°C in Tris-buffered saline with 0.05% Tween 20, pH 7.4, plus 5% skimmed milk powder. The nitrocellulose membrane was incubated overnight at 4°C with 10 g/ml of affinity-purified anti-phosphoty-rosine antibodies, and the bound antibodies were visualized using a Pierce Supersignal chemiluminescence detection kit.
PKC Assay-PKC activation was determine by the method of Tardif et al. (19). Briefly, serum-starved VSMC grown in either NG or HG for 24 h were treated with Ang II (0.1 M) for 1 min. Cells were washed, resuspended in buffer, and sonicated, and homogenates were ultracentrifuged to isolate the plasma membrane fractions. Equal amounts of plasma membrane protein levels in each sample were loaded and separated by SDS-PAGE under reducing conditions, and the plasma membrane distribution of PKC-␤1 and PKC-␤2 was visualized by Western blot using specific monoclonal antibodies against each isoform of PKC-␤.
Electroporation Procedure-Cells were plated in 100-mm cell plates and growth-arrested in serum-deprived DMEM for 24 h before experiments. As previously described (2,13,20), VSMC were electroporated using a Multi-Coaxial electrode (Model P/N 747, BTX Inc., San Diego, CA) that performed in Ca 2ϩ -and Mg 2ϩ -free Hanks' balanced salt solution containing anti-p47phox antibodies at a final concentration of 10 g/ml. After electroporation, cells were incubated for an additional 30 min at 37°C, washed once with serum-free DMEM, and left in serumfree DMEM before the experiments.
Assay of Intracellular ROS-Intracellular ROS production was measured by the method of Ushio-Fukai et al. (5) with some modifications. Briefly, dishes of confluent cells after stimulation with Ang II were washed with modified Eagle's medium without phenol red and incubated in the dark for 5 min in Krebs-Ringer solution containing 5 mM DCFH diacetate. DCFH diacetate is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the non-fluorescent polar derivative DCFH and thereby trapped within the cells (5). In the presence of a proper oxidant, DCFH is oxidized to the highly fluorescent 2,7-dichlorofluorescein. Culture dishes were transferred to a Zeiss inverted microscope equipped with a ϫ20 Neofluor objective and Zeiss LSM 410 confocal attachment, and ROS generation was detected as a result of the oxidation of DCFH (excitation, 488 nm; emission, 515-540 nm). The effect of DCFH photo-oxidation was minimized by collecting the fluorescent image with a single rapid scan (line average, 4; total scan time, 4.33 s) and identical parameters, such as contrast and brightness, for all samples. The cells were then imaged by differential interference contrast microscopy. Five groups of 20 -30 cells each were randomly selected from the image in the digital interference contrast channel for each sample, the fluorescence intensity was then measured for each group from the fluorescence image, and the relative fluorescence intensity was taken as the average of the five values. Therefore, the relative fluorescence intensity (given in arbitrary units) reflects measurements performed on a minimum of 100 cells for each sample. All experiments were repeated at least six times.
Cell Proliferation Assay-VSMC proliferation was measured using the Cell Titer 96™ AQ ueous nonradioactive cell proliferation assay (Promega, Inc., Madison, WI) (21). This assay is based on the cellular conversion of the colorimetric reagent 3,4-(5-demethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) into soluble formazan by dehydrogenase enzymes found only in metabolically active, proliferating cells. MTS in Dulbecco's phosphate-buffered saline, pH 6.0, was mixed with the electron-coupling reagent phenazine methyl sulfate. The absorbance of formazan, measured at 490 nm using a 96-well enzyme-linked immunosorbent assay plate reader interfaced with a personal computer is directly proportional to the number of living cells in culture. To confirm the accuracy of our MTS proliferation assay, the actual increase in cell number was also directly assessed with a Coulter counter (Model ZM, Coulter Corp., Hialeah, FL). The cells were grown in a 75-mm 2 flask to confluence and detached with trypsin-EDTA (0.05% trypsin, 0.53 mol/liter EDTA). 20,000 cells were plated into 96-well plates and allowed to settle for 4 h in DMEM supplemented with 10% fetal bovine serum. Before the experiments, cells were growth-arrested in serum-deprived DMEM for 24 h and then stimulated with the various ligands. After timed ligand exposure, the phenazine methyl sulfate/MTS mix was added to each well (final volume of 20 l/100 l of medium) and then incubated for an additional 60 min. A 10% SDS solution was then added to stop the reaction, and the absorbance of formazan was measured at 490 nm.
Apoptosis Assessment-To assess apoptosis VSMC are exposed to either NG or HG for 48 h with or without Ang II or to NG for 48 h followed by 15 min of ultraviolet light. VSMC are then washed with 3ϫ phosphate-buffered saline at room temperature, fixed with 100% methanol for 5-7 min, stained with a modified Wright-Giemsa stain, and analyzed by light microscopy for increased nuclear particulate staining (representing apoptosis). Average nuclear pixel intensity was measured using NIH Image analysis software (NIH, Bethesda, MD).
Data Analysis-All statistical comparisons were made using Student's t test for paired data and analysis of variance. p Ͻ 0.05 was considered significant.

Effects of the Aldose Reductase Inhibitor, Zopolrestat, on Both the Basal and Ang II-induced Tyrosine Phosphorylation of JAK2 in VSMC Preincubated in HG-Preincubation of VSMC
with zopolrestat, an aldose reductase inhibitor (22,23), was found to inhibit the HG stimulation of the Ang II-induced JAK2 tyrosine phosphorylation (Fig. 1). However, zopolrestat had no effect on the Ang II-induced JAK2 tyrosine phosphorylation (Fig. 1). These results suggest that the Ang II-induced JAK2 activation is not dependent on the polyol pathway, but rather, that Ang II and the polyol pathway induce JAK2 tyrosine phosphorylation separately, perhaps via a common system.

PKC-␤2 and the Effects of HG and Ang II on Both the H 2 O 2 Production and JAK2 Activation in VSMC-
The PKC-␤2 isozyme has been shown to play an important part in the hyperproliferation of smooth muscle cells, a characteristic feature of diabetic macroangiopathy (24). However, the precise mechanism remains unclear. Therefore, in these studies we investigated the effects of a PKC-␤-specific inhibitor LY379196 (10) on the Ang II-and HG-induced and HG augmentation of the Ang II-induced H 2 O 2 production and tyrosine phosphorylation of JAK2. We found that incubation with 0.2 g/ml LY379196 completely suppressed the increase in both the HGand Ang II-induced H 2 O 2 production (Fig. 2) and JAK2 tyrosine phosphorylation (Fig. 3). These observations suggest that both the HG or Ang II induce production of H 2 O 2 , and JAK2 activation occurs through either PKC-␤1 or PKC-␤2. Therefore, we next tried to determine the effects of HG and Ang II on the activation of the PKC-␤1 and PKC-␤2 isoforms in VSMC. The PKC isoforms were characterized by using monospecific polyclonal antibodies against PKC-␤1 or PKC-␤2 isoforms. Expression of PKC-␤2 isoform protein in the plasma membrane fraction of VSMC was significantly (#, p Ͻ 0.01) increased by Ang II in NG and HG (*, p Ͻ 0.01) alone when compared with those VSMC cultured in NG (Fig. 4). Zopolrestat blocked the HGinduced activation of PKC-2, but it had no effect on the Ang II activation in both NG and HG (Fig. 4). No significant differences in expression of the PKC-␤1 isoform were detected between the HG-and NG-and Ang II-treated VSMC plasma membrane fractions (data not shown). These studies provide evidence that both HG and Ang II induced the production of H 2 O 2 or JAK2 activation via activation of the PKC-␤2 isoform.
Antisense Studies-Our studies with the aldose reductase inhibitor (zopolrestat) and the PKC-␤ isoform inhibitor (LY379196) suggest that HG augments the Ang II-induced production of H 2 O 2 and activation of JAK2 via the polyol pathway. However, most inhibitors are usually not very specific, and acknowledging that not all antibodies block or neutralize biologic activities, we consider the antisense approach to further confirm the roles of both aldose reductase and PKC-␤ in the HG augmentation of the Ang II-induced production of H 2 O 2 and activation of JAK2. We have experience and success in designing and using antisense oligonucleotides to inhibit gene expression in VSMC cultures (e.g. blocking the expression of , an H 2 O 2 -sensitive dye that is incorporated into the cell. We found that both HG (ϩ, p Ͻ 0.01) or Ang II (*, p Ͻ 0.01) in NG caused a significant increase in H 2 O 2 production when compared with the NG control cells. In VSMC exposed to HG plus Ang II, a significant difference (#, p Ͻ 0.01) in H 2 O 2 production was observed when compared with cells treated with HG alone. LY379196 blocked the HG-induced H 2 O 2 production significantly (ϩϩ, p Ͻ 0.01) and also the Ang II-induced H 2 O 2 production in both NG (**, p Ͻ 0.01) and HG (##, p Ͻ 0.01). Data represent the mean Ϯ S.E. of six independent cultures. MKP-1, JAK2, and the Src kinases Src and Fyn in studying the regulation of STAT1 and STAT3 tyrosine phosphorylation and activation in VSMC (17,18)). In addition, two recent studies show that both aldose reductase (15) and PKC-␤ (16) antisense oligonucleotides inhibit the synthesis of these two proteins. VSMC were treated for various times with either the antisense or the sense oligonucleotide to aldose reductase and PKC-␤, and the levels of expression of these two proteins were demonstrated by immunoblotting. As shown in Fig. 5, both aldose reductase and the PKC-␤ antisense completely suppressed their expression after 12 h of treatment. In contrast, the sense oligonucleotides had no effect. Experiments were then carried out in VSMC, which were treated with either the antisense or sense oligonucleotide for 12 h before stimulation of the cells with HG alone or Ang II in either NG or HG. We found that preincubating the VSMC with the aldose reductase antisense oligonucleotide (but not the JAK2 sense oligonucleotide) significantly inhibited the HG stimulation of the Ang II-induced JAK2 tyrosine phosphorylation (Fig. 6). However, just like with zopolrestat, the aldose reductase antisense oligonucleotide had no effect on the Ang II-induced JAK2 tyrosine phosphorylation (Fig. 6). These results further support our previous findings, which showed that the Ang II-induced JAK2 activation is not dependent on the polyol pathway but, rather, that Ang II and

FIG. 4. Effects of zopolrestat on the high glucose-induced activation of PKC-␤1 and PKC-␤2
. PKC isoforms were characterized by using monospecific antibodies against PKC-␤1 or PKC-␤2 isoforms. The expression of PKC-␤2 isoform protein in the plasma membrane fraction of VSMC cultured in HG or exposed to Ang II were significant (p Ͻ 0.01) when compared with those VSMC cultured in NG. Zopolrestat blocked the HG-induced activation of PKC-␤2, but it had no effect on the Ang II activation.

FIG. 5. Effect of aldose reductase and PKC-␤ antisense oligonucleotides on aldose reductase and PKC-␤2 expression in vascular smooth muscle cells.
VSMC were treated with aldose reductase and PKC-␤ sense and antisense oligonucleotides for the times indicated, the cells were lysed, and aldose reductase and PKC-␤ were immunoprecipitated from the lysates with the specific anti-aldose reductase or anti-PKC-␤ antibody. Precipitated proteins were then immunoblotted with the specific anti-aldose reductase or anti-PKC-␤ antibody.
FIG. 6. Effects of aldose reductase antisense on the high glucose augmentation on the angiotensin II-induced JAK2 tyrosine phosphorylation. Quiescent VSMC were transfected with either aldose reductase (AR) sense or antisense without for 12 h in serum-free medium in NG. Afterward the VSMC were further incubated for an additional 24 h in serum-free medium containing either NG (5.5 mM) or HG (25 mM) and treated with Ang II (0.1 M) for 5 and 10 min. Cells were lysed, and lysates were immunoblotted with either phosphotyrosine-specific or nonphospho-specific anti-JAK2 antibodies. Representative immunoblots (of three experiments) probed with either the JAK2 phosphotyrosine-specific antibody (pJAK2) or JAK2 antibody (JAK2) are shown. the polyol pathway induce JAK2 tyrosine phosphorylation separately perhaps via a common system.
Last, our results with the PKC-␤ antisense oligonucleotide were almost identical to our studies with PKC-␤ specific inhibitor LY379196. For example, we found that incubation with the PKC-␤ antisense oligonucleotide completely suppressed the increase in both the HG-and Ang II-induced H 2 O 2 production (Fig. 7) and JAK2 tyrosine phosphorylation (Fig. 8). These observations further support our hypothesis, which suggests that both the HG-or Ang II-induced production of H 2 O 2 and JAK2 activation occurs through the PKC-␤2 isoform.
Effects of Apocynin and Electroporation of the NADPH Oxidase-neutralizing Antibody, anti-p47phox, on Both the Basal and Ang II-induced Tyrosine Phosphorylation of JAK2 in VSMC Preincubated in HG-A number of recent studies have suggested that most of the H 2 O 2 produced by Ang II stimula-tion in cells comes from the NAD(P)H oxidase system (5,9). Apocynin has been shown to be a specific inhibitor of the NAD(P)H oxidase system (5). In these studies, we found that incubation of VSMC with apocynin completely suppressed the increase in ROS production ( Fig. 9) in VSMC treated with Ang II under both NG and HG conditions. Zopolrestat, on the other hand, only blocked the HG-induced production of H 2 O 2 . In addition, apocynin also completely blocked the JAK2 tyrosine , an H 2 O 2 -sensitive dye that is incorporated into the cell. We found that both HG (ϩ, p Ͻ 0.01) or Ang II (*, p Ͻ 0.01) in NG caused a significant increase in H 2 O 2 production when compared with the NG control cells. In VSMC exposed to HG plus Ang II a significant difference (#, p Ͻ 0.01) in H 2 O 2 production was observed when compared with cells treated with HG alone. Apocynin blocked both the HG-induced H 2 O 2 production significantly (ϩϩ, p Ͻ 0.01) and also the Ang II-induced H 2 O 2 production in both NG (**, p Ͻ 0.01) and HG (##, p Ͻ 0.01). Zopolrestat, on the other hand, only blocked the HG-induced H 2 O 2 production significantly (:, p Ͻ 0.01). Data represent the mean Ϯ S.E. of six independent cultures. phosphorylation in VSMC treated with Ang II under both NG and HG conditions (Fig. 10). These results suggests that NAD(P)H oxidase might be the ROS-generating system responsible for most of the H 2 O 2 produced after experimental treatments with Ang II in VSMC cultured in either NG or HG and that the intracellular H 2 O 2 produced via NADPH oxidase subsequently activates JAK2, as shown by other investigators (5,9).
As stated before, because most inhibitors are usually not very specific, we also used another additional approach to determine the influence of the NADPH oxidase system on both the HG-and the Ang II-induced JAK2 activation. The anti-p47phox antibody has been shown to neutralize the NADPH oxidase system in VSMC (9). In these studies, we found that electroporation of VSMC with anti-p47phox antibodies completely suppressed the JAK2 tyrosine phosphorylation in all groups tested, whereas electroporation of a control rabbit IgG had no effect (Fig. 11). These results again strongly support our hypothesis, which suggest that NADPH oxidase might be the ROS-generating system responsible for most of the H 2 O 2 pro-duced after experimental treatments with Ang II in VSMC cultured in either NG or HG.
Effect of Ang II and HG on Vascular Smooth Muscle Cell Growth and the Role of the Polyol Pathway and Reactive Oxygen Species-As shown in Fig. 12a, VSMC exposed to Ang II for FIG. 10. Effects of the NADPH oxidase inhibitor apocynin on the high glucose augmentation on the angiotensin II-induced JAK2 tyrosine phosphorylation. Quiescent VSMC in serum-free medium were exposed for 24 h to 10 M apocynin in either normal glucose (5.5 mM) or high glucose (25 mM) and treated with Ang II (0.1 M) for 5 and 10 min. Cells were lysed, and lysates were immunoblotted with either phosphotyrosine-specific or nonphospho-specific anti-JAK2 antibodies. Representative immunoblots (of three experiments) probed with either the JAK2 phosphotyrosine-specific antibody (pJAK2) or JAK2 antibody (JAK2) are shown.
FIG. 11. Effects of electroporating anti-IgG or anti-p47phox antibodies on the high glucose augmentation on the angiotensin II-induced JAK2 tyrosine phosphorylation. VSMC were electroporated as described under "Experimental Procedures." Quiescent VSMC in serum-free medium were then exposed for 24 h to normal glucose (5.5 mM) or high glucose (25 mM) and treated with Ang II (0.1 M) for 5 and 10 min. Cells were lysed, and lysates were immunoblotted with either phosphotyrosine-specific or nonphospho-specific anti-JAK2 antibodies. Representative immunoblots (of three experiments) probed with either the JAK2 phosphotyrosine-specific antibody (pJAK2) or JAK2 antibody (JAK2) are shown. FIG. 12. a, effects of zopolrestat and apocynin on the angiotensin II-induced cell proliferation in VSMC grown in normal or high glucose. VSMC were incubated for with or without 100 M zopolrestat or 10 M apocynin in normal glucose (5.5 mM) or high glucose (25 mM) and then treated with Ang II at 100 nM for 48 h. Cell proliferation was determined as described under "Experimental Procedures" and is expressed as absorbance (A) of formazan at 490 nm. We found that both HG (ϩ, p Ͻ 0.01) or Ang II (*, p Ͻ 0.01) in NG caused a significant increase in VSMC proliferation when compared with the NG control cells. In VSMC exposed to HG plus Ang II a significant difference (#, p Ͻ 0.01) in VSMC proliferation was also observed when compared with cells treated with HG alone. Apocynin blocked the HG-induced VSMC proliferation significantly (ϩϩ, p Ͻ 0.01) and also the Ang II-induced VSMC proliferation in both NG (**, p Ͻ 0.01) and HG (##, p Ͻ 0.01). Zopolrestat, on the other hand, only blocked the HG-induced VSMC proliferation significantly (:, p Ͻ 0.01). Data represent the mean Ϯ S.E. of six independent cultures. b, effects of zopolrestat and apocynin on the angiotensin II-induced increase in cell number in VSMC grown in normal or high glucose. VSMC were incubated with or without 100 M zopolrestat or 10 M apocynin in normal glucose (5.5 mM) or high glucose (25 mM) and then treated with Ang II at 100 nM for 48 h. Both HG (ϩ, p Ͻ 0.01) or Ang II (*, p Ͻ 0.01) in NG caused a significant increase in VSMC cell number when compared with the NG control cells. In VSMC exposed to HG plus Ang II a significant increase in increasing cell number (#, p Ͻ 0.01) was also observed when compared with cells treated with HG alone. Apocynin blocked the HG-induced VSMC increase in cell number significantly (ϩϩ, p Ͻ 0.01) and also the Ang II-induced VSMC. VSMC increased in cell number in both NG (**, p Ͻ 0.01) and HG (##, p Ͻ 0.01). Zopolrestat, on the other hand, only blocked the HG-induced VSMC. VSMC increased in cell number significantly (:, p Ͻ 0.01). Data represent the mean Ϯ S.E. of six independent cultures. 48 h under NG conditions resulted in a significant increased in cell proliferation when compared with control cells. Exposure of cells to HG alone also resulted in a significant increase in cell proliferation when compared with NG. In addition, the Ang II-induced cell proliferation was also significantly enhanced in cells incubated in HG when compared with cells incubated in NG. Finally, we also found, as we had previously found for the ROS production and JAK2 tyrosine phosphorylation, that preincubating the VSMC with the apocynin significantly inhibited all the conditions tested (i.e. the Ang II-, HG-, and Ang II plus HG-induced cell proliferation (Fig. 12a), whereas zopolrestat only inhibited the HG-induced effects (Fig. 12a). In a similar experimental condition, the number of cells at the end of respective incubations was also counted. Parallel to the alterations in cell proliferation, Ang II and HG individually stimulated cell number and produced a significant increase in cell replication under Ang II/HG conditions (Fig. 12b). Again pre-incubating the VSMC with apocynin significantly inhibited all the conditions tested (i.e. the Ang II-, HG-, and Ang II plus HG-induced increased in cell number (Fig. 12b), whereas zopolrestat only inhibited the HG-induced effects (Fig. 12b).

Effect of Ang II and HG on Vascular Smooth Muscle Cell
Apoptosis-VSMC cell apoptosis was examined using Wright-Giemsa staining (which detects damaged nuclear DNA). The staining revealed no significant difference in the intensity of nuclear staining between NG-and HG-exposed cells without Ang II (arbitrary units of pixel intensity per nucleus, 41 Ϯ 5 and 40 Ϯ 3, respectively) or with Ang II (arbitrary units of pixel intensity per nucleus; NG ϩ Ang II, 43 Ϯ 5; HG ϩ Ang II, 40 Ϯ 3). On the other hand, VSMC exposed to ultraviolet light for 15 min in NG demonstrated significant (p Ͻ 0.05) nuclear staining (pixel intensity per nucleus, 121 Ϯ 9), indicating apoptosis.
Effects of Aldose Reductase Antisense Oligonucleotide on Both the Basal and Ang II-induced Tyrosine Phosphorylation of SHP-1 and SHP-2 in VSMC Preincubated in HG-We have previously shown that the phosphorylation state of JAK2 is tightly regulated by the two cytoplasmic protein-tyrosine phosphatases SHP-1 and SHP-2 (25). Furthermore, we have also shown that HG alters the tyrosine phosphorylation and activation of these protein-tyrosine phosphatases (1). Therefore, we investigated the effects of blocking the polyol pathway on the activation of these two cytosolic protein-tyrosine phosphatases under HG conditions in VSMC by examining their tyrosine phosphorylation states. We found that transfection of the VSMC with the aldose reductase antisense oligonucleotide was able to prevent the HG-induced abrogation of SHP-1 tyrosine phosphorylation (Fig. 13). On the other hand, the HG augmentation of the Ang II-induced tyrosine phosphorylation of SHP-2 was blocked by pretreatment with the aldose reductase antisense oligonucleotide (Fig. 13). These results suggest that the JAK2-sustained tyrosine phosphorylation, which occurs under HG concentration in VSMC, might be due to changes on SHP-1 and SHP-2 activation influenced by the polyol pathway.
Effects of the Aldose Reductase Antisense on Both the Basal and PDGF-induced Tyrosine Phosphorylation of JAK2 in VSMC Preincubated in HG-Recently it has been shown that the growth factor PDGF, which is another growth factor just like Ang II, employs H 2 O 2 as a second messenger to regulate the activation of JAK2 in rat fibroblasts (8). Therefore, we were interested in examining if HG also augments the PDGF-induced activation of JAK2 via the polyol pathway. We found that transfection of VSMC with the aldose reductase antisense oligonucleotide inhibited the HG stimulation of the PDGF-induced JAK2 tyrosine phosphorylation (Fig. 14). However, just FIG. 14. Effects of aldose reductase antisense oligonucleotides on the high glucose augmentation of the PDGF-induced JAK2 tyrosine phosphorylation. Quiescent VSMC were transfected with either aldose reductase sense or antisense for 12 h in serum-free medium in NG. Afterward the VSMC were further incubated for an additional 24 h in serum-free medium containing either NG (5.5 mM) or HG (25 mM) and treated with PDGF (0.33 mM) for 5 and 10 min. Cells were lysed, and lysates were immunoblotted with either phosphotyrosinespecific or nonphospho-specific anti-JAK2 antibodies. Representative immunoblots (of three experiments) probed with either the JAK2 phosphotyrosine-specific antibody (pJAK2) or JAK2 antibody (JAK2) are shown. as we previously showed with Ang II, the aldose reductase antisense had no effect on the PDGF-induced JAK2 tyrosine phosphorylation (Fig. 14). These results suggest that the PDGF-induced JAK2 activation, just as we hypothesized with Ang II, is not dependent on the polyol pathway but, rather, that both PDGF and the polyol pathway induce JAK2 tyrosine phosphorylation independently, perhaps via complementary pathways.
Lysates were all immunoblotted with an anti-JAK2 antibody that recognizes both phosphorylated and nonphosphorylated forms of JAK2. Equal amounts of JAK2 (Figs. 1, 3 , 6, 8, 10, 11, 13, and 14) were detected for all the conditions tested, indicating that the differences detected with the phosphotyrosinespecific antibodies (pJAK2) were not due to differences in the amount of total JAK2 loaded in each lane. Finally, both the Ang II-stimulated increases in H 2 O 2 and Ang II-induced JAK2 tyrosine phosphorylation in all the experiments tested was inhibited when cells were preincubated with candesartan, a specific AT 1 receptor blocker (26), indicating that this induction by Ang II was AT 1 receptor-specific (data not shown).

DISCUSSION
The specific cellular signals activated in VSMC under hyperglycemic conditions have not been completely elucidated. Recently Amiri and co-workers (1) found that hyperglycemia increased both the basal and Ang II-induced VSMC proliferation, tyrosine phosphorylation, and complex formation of JAK2 with the Ang II AT 1 receptor and the extent of the tyrosine and serine phosphorylation of STAT1 and STAT3. They also found that hyperglycemia altered Ang II-induced tyrosine phosphorylation and the activities of SHP-1 and SHP-2 (1). These results suggest that increased and/or altered activation of tyrosine kinase (JAK2), tyrosine phosphatases (SHP-1 and SHP-2), and downstream transcription factors, such as STAT1 and STAT3, may be key mechanisms for the increased Ang IIinduced VSMC growth potential in response to hyperglycemic conditions.
But what are the molecular mechanisms responsible for the augmentation of Ang II-induced activation of JAK2 in VSMC by HG? A candidate mechanism might be related to the activation of JAK2 by ROS. For example, it has recently been shown that ROS stimulate the activity of JAK2 in both fibroblasts and A-431 cells (8). Furthermore, Schieffer and coworkers (9) demonstrate that activation of JAK2 and STAT proteins by Ang II in VSMC was significantly inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and electroporation of the NADPH oxidase neutralizing antibody anti-p47phox IgG. These data support the hypothesis that oxygen-free radicals generated by NADPH oxidase ROS may contribute to activation of JAK2 and STATs in response to Ang II. Therefore, these findings suggest that the JAK-STAT pathway responds to intracellular ROS and that the vasoactive peptide Ang II uses ROS as a second messenger to regulate JAK2 activation.
High glucose has also been shown to induce the activation of PKC, and PKC has also been shown to increase the production of ROS, extracellular matrix, and cytokines and to enhance contractility, permeability, and vascular cell proliferation. Recently, the synthesis and characterization of specific inhibitors to the PKC-␤ isoforms have confirmed the role of PKC activation in mediating hyperglycemic effects on vascular cells and provide in vivo evidence that PKC-␤ activation could be responsible for abnormal ROS production and vascular growth in diabetic animals (10). Finally, recent studies also suggest that high glucose, via the polyol pathway, induces a rapid increase in intracellular ROS such as H 2 O 2 via activation of PKC, which stimulates intracellular signal events similar to those activated by Ang II, including stimulation of growth-promoting kinases such as JAK2 and extracellular signal-regulated kinase 1/2 (3)(4)(5). Therefore, the polyol pathway activates PKC, which in turn activates NADPH oxidase, which then generates ROS (H 2 O 2 and O 2 Ϫ ) (6, 7). These ROS can then act as signal transducers via protein-tyrosine phosphatases in the activation of mitogenic enzymes such as JAK2 (8). Indeed, recent studies show that ROS regulates the activity of the protein-tyrosine phosphatases SHP-1 and SHP-2 (27,28). In this study we have demonstrated this mechanism of JAK2 activation. That is, HG augments the Ang II-induced ROS production, VSMC proliferation, and tyrosine phosphorylation of JAK2 via the polyol pathway activation of PKC-␤2, which in turn activates NADPH oxidase to produce ROS. ROS regulate the activity of the protein-tyrosine phosphatases SHP-1 and SHP-2, and SHP-1 and SHP-2 in turn regulate the activation of JAK2.
In conclusion, the results from this study support our hypothesis, which states that the polyol pathway activation of PKC-␤2 is an important mechanism by which HG augments the Ang II-induced ROS production and the activation of JAK2, which leads to VSMC proliferation that is associated with diabetes mellitus (Fig. 15).