High Glucose Augments the Angiotensin II-induced Activation of JAK2 in Vascular Smooth Muscle Cells via the Polyol Pathway

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 was and was was and Dulbecco's modified Eagle's and to and the PKC-β isoforms JAK2 and antibodies The aldose reductase inhibitor and the PKC-β inhibitor and The was and and and of smooth muscle cells and in with mm and in a as M.B. Schieffer B. Schieffer Bernstein K.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.B. Duff J.L. B.C. Bernstein K.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and was and PKC-β reductase D. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and PKC-β S. T. Diabetes. PubMed Scopus Google antisense synthesis and as R.C. Venema V.J. Marrero M.B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Venema V.J. Wang X. Ju H. Venema R.C. Marrero M.B. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). was in Dulbecco's modified Eagle's in NG was and the cells to for The VSMC with and in in NG for the VSMC in either NG or HG for of the tyrosine phosphorylation of JAK2, VSMC in either NG or HG for and with Ang II or mm PDGF for to the of cells with with was then for with SDS, and The was as cell by for 25 of the was with the assay and as the by to a nitrocellulose and blocked by a in with plus The nitrocellulose was with JAK2 antibodies or the nitrocellulose membranes for with with and for with was on with a weight of the of and the and tyrosine phosphorylation, VSMC in HG for with Ang II for to the of cells with with was then for with SDS, and and the was as cell by for The cell was with either or antibodies for 2 and by the of of protein The then by and with and in of and of was by to a nitrocellulose and blocked by in with plus The nitrocellulose was with of and the antibodies a PKC activation was by the of M. M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). VSMC in either NG or HG for with Ang II for in and and to the of protein in and by and the of and PKC-β2 was by specific antibodies isoform of PKC-β. in cell and in for 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, M.B. Schieffer B. Schieffer Bernstein K.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B. Drexler H. Ling B.N. Marrero M.B. Am. J. Physiol. 1997; 272: PubMed Google Scholar), VSMC a Inc., that in and antibodies a of cells for an with DMEM, and in the of ROS production was by the of (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 with of cells stimulation with Ang II with modified Eagle's and in the for in mm a that it to the and 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 of a to the to a with a and and ROS was as a of the of The effect of was by the with a rapid and such as and for The cells then by of cells the in the for the was then for the and the was as the of the the in on a of cells for proliferation was the cell proliferation assay Inc., J. PubMed Scopus (290) Google Scholar). This assay on the of the 3,4-(5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium by found in in Dulbecco's was with the The of a assay with a to the of cells in the of proliferation the increase in cell was also with a The cells in a to and with cells and to for in with the cells in for and then with the the was to of of and then for an was then to the and the of was VSMC to either NG or HG for with or Ang II or to NG for by of VSMC then with with for with a modified and by for increased was for and of was of the on Both the and Ang II-induced of JAK2 in VSMC in of VSMC with an aldose reductase inhibitor J. X. T. T. C. J. Am. 1999; Google Scholar, A. K. PubMed Scopus Google Scholar), was found to the HG stimulation of the Ang II-induced JAK2 tyrosine phosphorylation However, had no effect on the Ang II-induced JAK2 tyrosine phosphorylation These results suggest that the Ang II-induced JAK2 activation dependent on the polyol pathway, but that Ang II and the polyol pathway JAK2 tyrosine phosphorylation via a PKC-β2 and the of HG and Ang II on Both the 2 2 and JAK2 in PKC-β2 has been shown to play an important in the hyperproliferation of smooth muscle a characteristic feature of diabetic macroangiopathy J. Y. Y. Naruse K. Y. K. 2001; PubMed Scopus Google Scholar). However, the precise remains unclear. in these studies we investigated the effects of a PKC-β-specific inhibitor (10Ishii H. Koya D. King G.L. J. Mol. Med. 2001; 76: 21-31Google on the Ang and HG-induced and HG of the Ang II-induced H2O2 production and tyrosine phosphorylation of JAK2. We found that with completely the increase in both the and Ang II-induced H2O2 production and JAK2 tyrosine phosphorylation These observations suggest that both the HG or Ang II production of H2O2, and JAK2 activation through either or we to the effects of HG and Ang II on the activation of the and PKC-β2 isoforms in The PKC isoforms by antibodies or PKC-β2 of PKC-β2 isoform protein in the of VSMC was significantly increased by Ang II in NG and HG when compared with those VSMC cultured in NG blocked the HG-induced activation of but it had no effect on the Ang II activation in both NG and HG significant in of the isoform between the and and Ang VSMC These studies evidence that both HG and Ang II induced the production of H2O2 or JAK2 activation via activation of the PKC-β2 of the PKC-β inhibitor on Ang and high tyrosine phosphorylation of JAK2. VSMC with or of in either NG (5.5 mm) or HG mm) and with Ang II for and and with either or with either the JAK2 or JAK2 (JAK2) of on the high activation of and PKC isoforms by antibodies or PKC-β2 The of PKC-β2 isoform protein in the of VSMC cultured in HG or to Ang II significant when compared with those VSMC cultured in blocked the HG-induced activation of but it had no effect on the Ang II 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 However, very and that antibodies or we the antisense to the of both aldose reductase and PKC-β in the HG of the Ang II-induced production of H2O2 and activation of JAK2. We have and in and antisense to in VSMC the of JAK2, and the kinases and in the of and tyrosine phosphorylation and activation in VSMC R.C. Venema V.J. Marrero M.B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Venema V.J. Wang X. Ju H. Venema R.C. Marrero M.B. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google In addition, recent studies that both aldose reductase D. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and PKC-β S. T. Diabetes. PubMed Scopus Google antisense the synthesis of these VSMC for with either the antisense or the oligonucleotide to aldose reductase and PKC-β, and the of of these by shown in both aldose reductase and the PKC-β antisense completely their of In the had no then in VSMC, which with either the antisense or oligonucleotide for stimulation of the cells with HG or Ang II in either NG or HG. We found that the VSMC with the aldose reductase antisense oligonucleotide the JAK2 significantly inhibited the HG stimulation of the Ang II-induced JAK2 tyrosine phosphorylation However, with the aldose reductase antisense oligonucleotide had no effect on the Ang II-induced JAK2 tyrosine phosphorylation These results which showed that the Ang II-induced JAK2 activation dependent on the polyol pathway that Ang II and the polyol pathway JAK2 tyrosine phosphorylation via a of aldose reductase antisense on the high glucose on the angiotensin II-induced JAK2 tyrosine VSMC with either aldose reductase or antisense for in in the VSMC for an in either NG (5.5 mm) or HG mm) and with Ang II for and and with either or with either the JAK2 or JAK2 (JAK2) results with the PKC-β antisense oligonucleotide to studies with PKC-β specific inhibitor For example, we found that with the PKC-β antisense oligonucleotide completely the increase in both the and Ang II-induced H2O2 production and JAK2 tyrosine phosphorylation These observations which that both the or Ang II-induced production of H2O2 and JAK2 activation through the PKC-β2 of PKC-β antisense on the high glucose on the angiotensin II-induced JAK2 tyrosine VSMC with either PKC-β or antisense for in in the VSMC for an in either NG (5.5 mm) or HG mm) and with Ang II for and and with either or with either the JAK2 or JAK2 (JAK2) of and of the NADPH on Both the and Ang II-induced of JAK2 in VSMC in of recent studies have that of the H2O2 by Ang II stimulation in cells the oxidase (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). has been shown to be a specific inhibitor of the oxidase (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 these we found that of VSMC with apocynin completely the increase in ROS production in VSMC with Ang II both NG and HG on the blocked the HG-induced production of In addition, apocynin also completely blocked the JAK2 tyrosine phosphorylation in VSMC with Ang II both NG and HG These results that oxidase be the responsible for of the H2O2 with Ang II in VSMC cultured in either NG or HG and that the intracellular H2O2 via NADPH oxidase subsequently activates JAK2, as shown by (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 of the NADPH oxidase inhibitor apocynin on the high glucose on the angiotensin II-induced JAK2 tyrosine VSMC in for to apocynin in either normal glucose (5.5 mm) or high glucose mm) and with Ang II for and and with either or with either the JAK2 or JAK2 (JAK2) very we also used to the of the NADPH oxidase on both the and the Ang II-induced JAK2 The has been shown to the NADPH oxidase in VSMC B. Luchtefeld M. Braun S. Hilfiker A. Hilfiker-Kleiner D. Drexler H. Circ. Res. 2001; 87: 1195-1201Crossref Scopus (240) Google Scholar). In these we found that electroporation of VSMC with antibodies completely the JAK2 tyrosine phosphorylation in electroporation of a had no effect These results which suggest that NADPH oxidase be the responsible for of the H2O2 with Ang II in VSMC cultured in either NG or HG. of Ang II and HG on and the of the and shown in VSMC to Ang II for NG in a significant increased in cell proliferation when compared with of cells to HG also in a significant increase in cell proliferation when compared with In addition, the Ang II-induced cell proliferation was also significantly in cells in HG when compared with cells in we also as we had found for the ROS production and JAK2 tyrosine phosphorylation, that the VSMC with the apocynin significantly inhibited the the Ang and Ang II plus HG-induced cell proliferation inhibited the HG-induced effects In a similar the of cells the of was also to the in cell proliferation, Ang II and HG cell and a significant increase in cell Ang the VSMC with apocynin significantly inhibited the the Ang and Ang II plus HG-induced increased in cell inhibited the HG-induced effects of Ang II and HG on cell was The no significant in the of between and cells Ang II of and or with Ang II of NG Ang II, HG Ang II, the VSMC to for in NG significant of on Both the and Ang II-induced of and in VSMC in have shown that the phosphorylation of JAK2 by the and M.B. Venema V.J. Ju H. Venema R.C. Am. J. Physiol. 1998; 275: PubMed Google Scholar). Furthermore, we have also shown that HG the tyrosine phosphorylation and activation of these (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). we investigated the effects of the polyol pathway on the activation of these HG in VSMC by their tyrosine phosphorylation We found that transfection of the VSMC with the aldose reductase antisense oligonucleotide was to the HG-induced of tyrosine phosphorylation the the HG of the Ang II-induced tyrosine phosphorylation of was blocked by with the aldose reductase antisense oligonucleotide These results suggest that the tyrosine phosphorylation, which HG in VSMC, be to on and activation by the polyol of the on Both the and PDGF-induced of JAK2 in VSMC in it has been shown that the growth PDGF, which growth Ang II, 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). we in HG also augments the PDGF-induced activation of JAK2 via the polyol We found that transfection of VSMC with the aldose reductase antisense oligonucleotide inhibited the HG stimulation of the PDGF-induced JAK2 tyrosine phosphorylation However, as we showed with Ang II, the aldose reductase antisense had no effect on the PDGF-induced JAK2 tyrosine phosphorylation These results suggest that the PDGF-induced JAK2 as we with Ang II, dependent on the polyol pathway that both PDGF and the polyol pathway JAK2 tyrosine phosphorylation via with an that both and of JAK2. of JAK2 and for the that the with the antibodies to in the of JAK2 in both the Ang increases in H2O2 and Ang II-induced JAK2 tyrosine phosphorylation in the was inhibited when cells with a specific J. Med. PubMed Google Scholar), that induction by Ang II was The specific activated in VSMC have been completely and (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 found that increased both the and Ang II-induced VSMC proliferation, tyrosine phosphorylation, and of JAK2 with the Ang II and the of the tyrosine and phosphorylation of and also found that Ang II-induced tyrosine phosphorylation and the activities of and (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). These results suggest that increased activation of tyrosine kinase (JAK2), tyrosine and and such as and be for the increased Ang II-induced VSMC growth in to the molecular responsible for the of Ang II-induced activation of JAK2 in VSMC by be to the activation of JAK2 by For example, it has recently been shown that ROS the of JAK2 in both fibroblasts and cells (8Simon A.R. Rai U. Fanburg B.L. Cochran B.H. Am. J. Physiol. 1998; 275: C1640-C1652Crossref PubMed Google Scholar). Furthermore, Schieffer and B. Luchtefeld M. Braun S. Hilfiker A. Hilfiker-Kleiner D. Drexler H. Circ. Res. 2001; 87: 1195-1201Crossref Scopus (240) Google that activation of JAK2 and by Ang II in VSMC was significantly inhibited by the NADPH oxidase inhibitor and electroporation of the NADPH oxidase These the that generated by NADPH oxidase ROS to activation of JAK2 and in to Ang these suggest that the pathway to intracellular ROS and that the Ang II uses ROS as a second messenger to regulate JAK2 glucose has also been shown to the activation of PKC, and PKC has also been shown to increase the production of ROS, extracellular and and to enhance and vascular cell proliferation. the synthesis and characterization of specific to the PKC-β isoforms have confirmed the role of PKC activation in mediating effects on vascular cells and in vivo evidence that PKC-β activation could be responsible for abnormal ROS production and vascular growth in diabetic animals (10Ishii H. Koya D. King G.L. J. Mol. Med. 2001; 76: 21-31Google Scholar). recent studies also suggest that high via the polyol pathway, induces a rapid increase in intracellular ROS such as H2O2 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 (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 activates PKC, which in activates NADPH oxidase, which then 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). These ROS can then act as signal transducers via in the activation of mitogenic such as JAK2 (8Simon A.R. Rai U. Fanburg B.L. Cochran B.H. Am. J. Physiol. 1998; 275: C1640-C1652Crossref PubMed Google Scholar). recent studies that ROS the of the and T. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). In study we have of JAK2 HG augments the Ang II-induced ROS production, VSMC proliferation, and tyrosine phosphorylation of JAK2 via the polyol pathway activation of which in activates NADPH oxidase to produce ROS regulate the of the and and and in regulate the activation of JAK2. In the results study which that the polyol pathway activation of PKC-β2 an important by which HG augments the Ang II-induced ROS production and the activation of JAK2, which to VSMC proliferation that with