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• W2013171495 abstract "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-β, 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-β antisense oligonucleotide, preincubation with a PKC-β-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-β-ROS activation of JAK2. 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-β, 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-β antisense oligonucleotide, preincubation with a PKC-β-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-β-ROS activation of JAK2. We have recently found that activation of Janus kinase 2 (JAK2) 1The abbreviations used are: JAK2, Janus kinase 2; Ang II, angiotensin II; VSMC, vascular smooth muscle cells; HG, high glucose; NG, normal glucose; ROS, reactive oxygen species; STAT, signal transducers and activators of transcription; PDGF, platelet-derived growth factor; PKC, protein kinase C; DCFH, 2,7-dichlorofluorescin; DMEM, Dulbecco's modified Eagle's medium; MTS, 3,4-(5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt. 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 (1Amiri F. Venema V.J. Wang X. Ju H. Venema R.C. Marrero M.B. J. Biol. Chem. 1999; 274: 32382-32386Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 2Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Current studies suggest that HG, via the polyol pathway, induces a rapid increase in intracellular reactive oxygen species (ROS) such as H2O2, 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 (3Berk B.C. Corson M.A. Circ. Res. 1997; 80: 607-616Crossref PubMed Scopus (283) Google Scholar, 4Berk B.C. Duff J.L. Marrero M.B. Bernstein K.E. Sowers J. Contemporary Endocrinology of the Vasculature. Humana Press Inc., Totowa, NJ1996: 187-204Google Scholar, 5Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). The polyol pathway generates ROS (H2O2 and O2- ) (6Chappey O. Dosquet C. Wautier M.P. Wautier J.L. Eur. J. Clin. Invest. 1997; 27: 97-108Crossref PubMed Scopus (211) Google Scholar, 7Ha H. Lee H.B. Kidney Int. 2000; 58: 19-25Abstract Full Text Full Text PDF Scopus (290) Google Scholar), which can then act as signal mediators in the activation of mitogenic pathways, such as the JAK/STAT signaling cascade (8Simon A.R. Rai U. Fanburg B.L. Cochran B.H. Am. J. Physiol. 1998; 275: C1640-C1652Crossref PubMed Google Scholar). For instance, in VSMC H2O2 has been shown to play an important role in regulating cell growth (5Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). It has also recently been reported that Ang II induces a rapid increase in intracellular H2O2 via NADPH oxidase, which subsequently activates growth-related responses plus the activation of JAK2 (5Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 9Schieffer B. Luchtefeld M. Braun S. Hilfiker A. Hilfiker-Kleiner D. Drexler H. Circ. Res. 2001; 87: 1195-1201Crossref Scopus (240) Google Scholar). Similar results have also been found for PDGF-induced cell proliferation, which was shown to be dependent on H2O2 (8Simon A.R. Rai U. Fanburg B.L. Cochran B.H. Am. J. Physiol. 1998; 275: C1640-C1652Crossref PubMed Google Scholar). Furthermore, PDGF uses H2O2 as a second messenger to regulate the activation of JAK2 in rat fibroblasts (8Simon A.R. Rai U. Fanburg B.L. Cochran B.H. Am. J. Physiol. 1998; 275: C1640-C1652Crossref PubMed Google Scholar). 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-β 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-β isoform could be responsible for the abnormal ROS production and vascular growth in diabetic animals (10Ishii H. Koya D. King G.L. J. Mol. Med. 2001; 76: 21-31Google Scholar). 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 (11Inoguchi T. Li P. Umeda F. Yu H.Y. Kakimoto M. Imamura M. Aoki T. Etoh T. Hashimoto T. Naruse M. Sano H. Utsumi H. Nawata H. Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1272) Google Scholar). In addition, a very recent study has also shown that the PKC-β2 isoform was essential for the activation of NADPH oxidase (12Korchack H.M. Kilpatrick L.E. J. Biol. Chem. 2001; 276: 8910-8917Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the present study we have inhibited by either pharmacological or molecular methods the polyol pathway or PKC-β 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-β. Materials—Molecular weight standards, acrylamide, SDS, N,N′-methylenebisacrylamide, N,N,N′,N′-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. 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-β isoforms were procured from Transduction Laboratories. Anti-phosphotyrosine JAK2 and anti-JAK2 antibodies were obtained from BIOSOURCE International. The aldose reductase inhibitor zopolrestat and the PKC-β 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. Preparation of Rat Aorta VSMC—Rat aorta smooth muscle cells were harvested and maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 5.5 mm glucose, 10 mg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 5% CO2 enriched, humidified atmosphere as previously described (13Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 14Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar). Cells from passages 5–6 were routinely subcultured 1:5 at 7-day intervals, and medium was changed every 24 h. Aldose Reductase and PKC-β Antisense Oligonucleotide Treatment— Aldose reductase (15Ramana K.V. Chandra D. Srivastava S. Bhatnagar A. Aggarwal B.B. Srivastava S.K. J. Biol. Chem. 2002; 277: 32063-32070Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and PKC-β (16Venugopal S.K. Devaraj S. Yang T. Jialal I. Diabetes. 2002; 51: 3049-3054Crossref PubMed Scopus (165) Google Scholar) antisense oligonucleotides synthesis and treatments were carried out as previously described (17Venema R.C. Venema V.J. Eaton D.C. Marrero M.B. J. Biol. Chem. 1998; 273: 30795-30800Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 18Liang H. Venema V.J. Wang X. Ju H. Venema R.C. Marrero M.B. J. Biol. Chem. 1999; 274: 19846-19851Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 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 μm 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 Na3VO4. 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 Na4P2O7, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, and 1 mmol/liter phenylmethylsulfonyl fluoride). The supernatant fraction was obtained as cell lysate by centrifugation at 58,000 × 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 affinity-purified 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 μm Ang II 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 Na3VO4. 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 Na4P2O7, 50 mmol/liter NaF, 1 mmol/liter Na3VO4, and 1 mmol/liter phenylmethylsulfonyl fluoride), and the supernatant fraction was obtained as cell lysate by centrifugation at 58,000 × g for 20 min at 4 °C. The cell lysate was incubated with 10 μg/ml either anti-SHP-1 or anti-SHP-2 monoclonal antibodies at 4 °C for 2 h and precipitated by the addition of 50 μl 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 (Tris-buffered saline, 0.1% Triton X-100, 1 mmol/liter phenylmethylsulfonyl fluoride, and 1 mmol/liter Na3VO4). Immunoprecipitated proteins were dissolved in 100 μl of Laemmli sample buffer, and 80 μl 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 μg/ml of affinity-purified anti-phosphotyrosine 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. (19Tardif M. Savard M. Flamand L. Gosselin J. J. Biol. Chem. 2002; 277: 24148-24154Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Briefly, serum-starved VSMC grown in either NG or HG for 24 h were treated with Ang II (0.1 μm) 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-β1 and PKC-β2 was visualized by Western blot using specific monoclonal antibodies against each isoform of PKC-β. 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 (2Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Schieffer B. Drexler H. Ling B.N. Marrero M.B. Am. J. Physiol. 1997; 272: C2019-C2030Crossref PubMed Google Scholar), VSMC were electroporated using a Multi-Coaxial electrode (Model P/N 747, BTX Inc., San Diego, CA) that performed in Ca2+- and Mg2+-free Hanks' balanced salt solution containing anti-p47phox antibodies at a final concentration of 10 μg/ml. After electroporation, cells were incubated for an additional 30 min at 37 °C, washed once with serum-free DMEM, and left in serum-free DMEM before the experiments. Assay of Intracellular ROS—Intracellular ROS production was measured by the method of Ushio-Fukai et al. (5Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) 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 (5Ushio-Fukai M. Alexander R.W. Akers M. Yin Q. Fujio Y. Walsh K. Griendling K.K. J. Biol. Chem. 1999; 274: 22699-22704Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). 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 ×20 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™ AQueous nonradioactive cell proliferation assay (Promega, Inc., Madison, WI) (21Buttke T.M. McCubrey J.A. Owen T.C. J. Immunol. Methods. 1993; 157: 233-240Crossref PubMed Scopus (290) Google Scholar). This assay is based on the cellular conversion of the colorimetric reagent 3,4-(5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-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-mm2 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 μl/100 μl 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 3× 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 < 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 (22Kapor-Drezgic J. Zhou X. Babazono T. Dlugosz J.A. Hohman T. Whiteside C. J. Am. Soc. Nephrol. 1999; 10: 1193-1203PubMed Google Scholar, 23Beyer-Mears A. Mistry K. Diecke F.P. Cruz E. Pharmacology. 1996; 52: 292-302Crossref PubMed Scopus (30) Google Scholar), 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-β2 and the Effects of HG and Ang II on Both the H 2 O 2 Production and JAK2 Activation in VSMC—The PKC-β2 isozyme has been shown to play an important part in the hyperproliferation of smooth muscle cells, a characteristic feature of diabetic macroangiopathy (24Nakamura J. Kasuya Y. Hamada Y. Nakashima E. Naruse K. Yasuda Y. Kato K. Hotta N. Diabetologia. 2001; 44: 480-487Crossref PubMed Scopus (67) Google Scholar). However, the precise mechanism remains unclear. Therefore, in these studies we investigated the effects of a PKC-β-specific inhibitor LY379196 (10Ishii H. Koya D. King G.L. J. Mol. Med. 2001; 76: 21-31Google Scholar) on the Ang II- and HG-induced and HG augmentation of the Ang II-induced H2O2 production and tyrosine phosphorylation of JAK2. We found that incubation with 0.2 μg/ml LY379196 completely suppressed the increase in both the HG- and Ang II-induced H2O2 production (Fig. 2) and JAK2 tyrosine phosphorylation (Fig. 3). These observations suggest that both the HG or Ang II induce production of H2O2, and JAK2 activation occurs through either PKC-β1 or PKC-β2. Therefore, we next tried to determine the effects of HG and Ang II on the activation of the PKC-β1 and PKC-β2 isoforms in VSMC. The PKC isoforms were characterized by using monospecific polyclonal antibodies against PKC-β1 or PKC-β2 isoforms. Expression of PKC-β2 isoform protein in the plasma membrane fraction of VSMC was significantly (#, p < 0.01) increased by Ang II in NG and HG (*, p < 0.01) alone when compared with those VSMC cultured in NG (Fig. 4). Zopolrestat blocked the HG-induced 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-β1 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 H2O2 or JAK2 activation via activation of the PKC-β2 isoform.Fig. 3Effects of the PKC-β inhibitor LY379196 on Ang II- and high glucose-induced tyrosine phosphorylation of JAK2. Quiescent VSMC were incubated with or without 0.2 μg/ml of LY379196 in serum-free medium containing either NG (5.5 mm) or HG (25 mm) and treated with Ang II (0.1 μm) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Effects of zopolrestat on the high glucose-induced activation of PKC-β1 and PKC-β2. PKC isoforms were characterized by using monospecific antibodies against PKC-β1 or PKC-β2 isoforms. The expression of PKC-β2 isoform protein in the plasma membrane fraction of VSMC cultured in HG or exposed to Ang II were significant (p < 0.01) when compared with those VSMC cultured in NG. Zopolrestat blocked the HG-induced activation of PKC-β2, but it had no effect on the Ang II activation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Antisense Studies—Our studies with the aldose reductase inhibitor (zopolrestat) and the PKC-β isoform inhibitor (LY379196) suggest that HG augments the Ang II-induced production of H2O2 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-β in the HG augmentation of the Ang II-induced production of H2O2 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 MKP-1, JAK2, and the Src kinases Src and Fyn in studying the regulation of STAT1 and STAT3 tyrosine phosphorylation and activation in VSMC (17Venema R.C. Venema V.J. Eaton D.C. Marrero M.B. J. Biol. Chem. 1998; 273: 30795-30800Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 18Liang H. Venema V.J. Wang X. Ju H. Venema R.C. Marrero M.B. J. Biol. Chem. 1999; 274: 19846-19851Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar)). In addition, two recent studies show that both aldose reductase (15Ramana K.V. Chandra D. Srivastava S. Bhatnagar A. Aggarwal B.B. Srivastava S.K. J. Biol. Chem. 2002; 277: 32063-32070Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and PKC-β (16Venugopal S.K. Devaraj S. Yang T. Jialal I. Diabetes. 2002; 51: 3049-3054Crossref PubMed Scopus (165) Google Scholar) 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-β, and the levels of expression of these two proteins were demonstrated by immunoblotting. As shown in Fig. 5, both aldose reductase and the PKC-β 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 the polyol pathway induce JAK2 tyrosine phosphorylation separately perhaps via a common system.Fig. 6Effects 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 μm) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Last, our results with the PKC-β antisense oligonucleotide were almost identical to our studies with PKC-β specific inhibitor LY379196. For exampl" @default.
• W2013171495 created "2016-06-24" @default.
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• W2013171495 creator A5030589572 @default.
• W2013171495 creator A5033393374 @default.
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• W2013171495 date "2003-08-01" @default.
• W2013171495 modified "2023-10-16" @default.
• W2013171495 title "High Glucose Augments the Angiotensin II-induced Activation of JAK2 in Vascular Smooth Muscle Cells via the Polyol Pathway" @default.
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