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Abstract-We have previously reported that high glucose stimulates osteopontin (OPN) expression through protein kinase C-dependent pathways as well as hexosamine pathways in cultured rat aortic smooth muscle cells. The finding prompted us to study in vivo expression of OPN in diabetes mellitus. In the present study, we found by immunohistochemistry that medial layers of the carotid arteries of streptozotocin-induced diabetic rats and the forearm arteries of diabetic patients stained positively for OPN antibodies, whereas the staining from arteries of control rats and nondiabetic patients was negative. We also found that OPN stimulated the migration and enhanced platelet-derived growth factor (PDGF)-mediated DNA synthesis of cultured rat aortic smooth muscle cells. OPN and PDGF synergistically activated focal adhesion kinase as well as extracellular signal-regulated kinase; this finding seems to explain the OPN-induced enhancement of PDGF-mediated DNA synthesis. Taken Key Words: diabetic macroangiopathy Ⅲ osteopontin Ⅲ platelet-derived growth factor Ⅲ vascular smooth muscle cells I t is generally accepted that diabetic patients often suffer from atherosclerotic vascular diseases, such as ischemic heart disease and arteriosclerosis obliterans. 1,2 It is also known that diabetic vascular lesions tend to undergo restenosis after angioplasty and that diffuse calcification of the affected arteries is a characteristic feature of diabetic vascular diseases. [3][4][5] However, the reason for the accelerated atherogenesis in diabetes mellitus has not been fully elucidated.Osteopontin (OPN) is a multifunctional phosphoprotein secreted by many cell types, such as osteoclasts, lymphocytes, macrophages, epithelial cells, and vascular smooth muscle cells (SMCs). 6 Overexpression of OPN has been found in several physiological as well as pathological conditions, including immunologic disorders, 7 neoplastic transformation, 8 progression of metastases, 8 formation of urinary stones, 9 and wound healing. 10 It has been reported that OPN protein and mRNA are expressed in the neointima as well as in calcified atheromatous plaque. 11 A neutralizing antibody against OPN has been found to inhibit rat carotid neointimal formation after endothelial denudation. 12 It has also been reported that OPN inhibits the calcification of vascular SMCs in culture. 13 These reports have suggested that OPN contributes not only to the tissue calcification process but also to the development of atherosclerosis, especially in the process of intimal thickening.We have recently reported that high glucose levels stimulate OPN expression through protein kinase C-dependent pathways as well as hexosamine pathways in cultured rat aortic SMCs. 14 The present study was undertaken to gain more insight into the mechanism of diabetic vascular complications. We first demonstrate that OPN protein is highly expressed in the medial layers of the arteries of diabetic rats and patients. Furthermore, OPN stimulates migration and enhances platelet-derived growth factor (PDG...

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100 -26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC 50 ‫؍‬ 1-3 nM) and did not require Mg 2؉ or Ca 2؉ , suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M r ‫؍‬ 150,000) and consisted of structural (A, 65 kDa), catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B␣ isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase a, and pyruvate kinase, and the K m values were 0.8 M, 3.7 M, and 2.2 M, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K a value for Xu 5-P was 20 M. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined.These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P 2 level in liver after high glucose administration.Many cellular processes and signaling transductions are controlled by reversible phosphorylation of proteins. Fru 2,6-P 2 1 is the most potent activator of phosphofructokinase and plays an important role in regulation of glycolysis, especially in liver (reviewed in Ref. 1). Synthesis and degradation of Fru 2,6-P 2 are catalyzed by a bifunctional enzyme, Fru-6-P,2-kinase:Fru-2,6-bisphosphatase. Liver Fru-6-P,2-kinase:Fru-2,6-Pase is phosphorylated by cAMP-dependent protein kinase (2-4). When blood glucose level falls, glucagon level increases which raises the cAMP level in hepatic cells. The elevated cAMP activates cAMP-dependent protein kinase, which phosphorylates Fru-6-P,2-kinase:Fru-2,6-Pase, leading to the inhibition of Fru-6-P,2-kinase and the activation of Fru-2,6-Pase. This results in a rapid decrease in Fru 2,6-P 2 , inhibition of phosphofructokinase and glycolysis, and activation of gluconeogenesis. Mechanism for regulation of the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase remains unclear. Pelech et al. (5) sought to identify the nature of protein phosphatases in liver involved in dephosphorylation of some of the known ...

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These results demonstrate that leukocyte-depleted reperfusion is potentially beneficial as an adjunct to terminal cardioplegia during cardiac surgery to attenuate reperfusion injury in patients with left ventricular hypertrophy.