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1 Vol. 30, pp , mg/dl 3 MTS Assay VEGF EIA 600 mg/dl VEGF VEGF VEGF VEGF VEGF vascular neogenesis vascular formation

2 4 VEGF: vascular endothelial growth factor VEGF VEGF 1 VEGF II Fig. 1 Time schedule of fibroblast cell culture. Fig. 2 Fibroblast cell culture with normal and high glucose medium mm mm FALCON 10 10% JRH BIOSCIENCES DME Dulbecco's Modified EagleGIBCO BRL 3 80% 0.05% trypsin DIFCO LABORRATORIES 0.02% EDTA PBS 2500 cell/cm mg/dl 200, 400, 600 mg/dl DME 3 Fig FALCON cell/well 226

3 VEGF 1 MTS assay Cell Titer 96 AQueous: PromegaFig mg/dl 600 mg/dl mg/dl600 mg/dl 96 1, 4 VEGF 1, VEGF165 Human VEGF Immunoassay: Quantikine EIA Enzyme Immuno Assay 4 VEGF Fig. 3 FGF 100 mg/dl 600 mg/dl Heparin 10 ng/ml bfgf : basic fibroblast growth factorsigma 0.05, 0.5, 5.0 ng/ml 48 VEGF SIGMA0.5, 5.0, 50.0 ng/ml 48 VEGF Fig. 3 ± SEEM ANOVA Dunnet's 5% 100 mg/dl 200, 400, 600 mg/dl Fig. 3 VEGF assay in the medium maintained with normal and high glucose level. Fig. 4 Influence of glucose level change on fibroblast. *p<0.05, **p< mg/dl 600 mg/dl Fig mg/dl 600 mg/ dl 100 mg/dl Fig. 5 VEGF VEGF 1 VEGF Fig

4 Fig. 5 Growth rate of fibroblasts following change in glucose concentration. *p<0.001, **p<0.05. Fig. 7 Production of VEGF following change in glucose concentration. *p<0.05, **p<0.05. Fig. 6 Changes in production of VEGF by fibroblasts. *p<0.05, **p<0.05. Fig. 8 Changes in production of VEGF caused by addition of FGF. VEGF VEGF Fig. 7 FGF bfgf Heparin bfgf VEGF 0.5, 5.0 ng/ml bfgf, VEGF, Fig. 8 Fig. 9 Effects of adding different concentrations of insulin on production of VEGF. VEGF VEGF Fig

5 VEGF 100 mg/dl VEGF VEGF 400 mg/dl 600 mg/dl VEGF VEGF VEGF VPF: vascular permeability factor 6~8 PDGF: platelet-derived growth factor 9 10 bfgf VEGF 12 VEGF 13~ VEGF VEGF 22 bfgf VEGF bfgf VEGF VEGF VEGF AGE: advanced glycation endproducts VEGF 23 SMC: smooth muscle cell PDGF 4 24 HGF: hepatocyte growth factor 229

6 VEGF VEGF Molecular Medicine 1998; 35; 10; ; 22; 3: Rubanyi GM. The role of endothelium in cardiovascular homeostasis and disease. J Cardiovasc Pharmacol 1993; 22: S1-S PDGF. 1997; 45; 11: VEGF. Molecular Medicine 1998; 35; 10: Dvorak HE and Brown IF. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995; 146: Ferrara N and Heinsohn H. The regulation of bloodvessel growth factor. Ann NY Acad Sci 1995; 752: Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem 1996; 271: VEGF. 1999; 39 (3): Roberts WG and Palade GE. Increased microvascular permeability and endthelial fenestration induced by vascular endothelial growth factor. J Cell Sci 1995; 108: ; 200; 3: I. 2000; 34; 4: Mohle R, Green D and Moore MA. Constitutive production and thrombin indused release of vascular endthelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA 1997; 94: Banks RE and Forbes MA. Release of the angiogenic cytokine vascular endthelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer 1998; 77: Weltermann A, Wolzt M and Petersmann K. Large amounts of vascular endthelial growth factor at the site of homostatic plug formation in vivo. Arterioscler Thromb Vasc Biol 1999; 19: Banks RE, Forbs MA and Stanley A. Presense of vascular endthelial growth factor (VEGF) in megakaryocyte and platelets (Abstract). Br J Haematol 1996; 93: A ; 57; 6: ; II: Miriam AM Loots. Cultured fibroblasts from chronic diabetic wounds the lower extremity (non-insulin dependent diabetes mellitus) show disturbed proliferation. Arch Dermaol Res 1999; 291: , ; 194; 10: Aygit AC and Ayhan MS. Vascular effects of epinephrine, lisinopril, and chlorpromazin in diabetic and non-diabetic rats. J Reconstract Microsurg 1999; 15 (6): ; 2; 5: ,,.. Molecular Medicine 1998; 35; 10: Kawano M. Diabetes mellitus induces accelerated growth of aortic smooth muscle cells; association with overexpression of PDGFβ-receptors. Eur J Clin Invest 1993; 23:

7 VEGF Abstract Effects of Hyperglycemic Conditions on Growth of Fibroblasts and Secretion of Growth Factors Noriko Kondo, Hajime Inoue, Kyoichi Matsuzaki, Masaki Aihara, and Norio Kumagai Vascular proliferation and granulation on wound surface are essential for wound healing, but in diabetes patients, this process is often delayed due to poor granulation. It has recently been reported that hyperglycemia alters the function of vascular endothelial cell receptors, which disrupts endothelial cell function and ultimately impairs vascular proliferation. Since several studies have also confirmed that growth of fibroblasts is suppressed on the surface of diabetic ulcers, the relationship between blood sugar and cellular function is being closely analyzed. In the present study, we conducted a basic investigation in order to determine the changes in function of normal fibroblasts that were cultured for an extended period of time under hyperglycemic conditions. Fibroblasts were collected from healthy skin and then subcultured for at least three passages using media containing different concentrations of glucose ( mg/dl). Growth of these cells was measured using MTS assay. In addition, release of vascular endothelial growth factor (VEGF) was measured by EIA. Although fibroblast growth decreased in a dose-dependent manner with regard to glucose, when fibroblasts grown in high-glucose (600 mg/dl) medium were subcultured in normal-glucose (100 mg/dl) medium their growth rate normalized. Both VEGF levels and VEGF production per cell were significantly lower in the high-glucose medium. However, when fibroblasts grown in high-glucose (600 mg/dl) medium were subcultured in normal-glucose medium, the levels of VEGF did not normalize. Taken together, these results indicate that high concentrations of glucose not only decrease growth of fibroblasts, but also affect production of growth factors such as VEGF. (St. Marianna Med. J., 30: , 2002) Department of Plastic and Reconstructive Surgery St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki , Japan 231

STUDIES ON THE RELATION BETWEEN LATE DUMPING SYNDROME AND GLUCAGON RESPONCES TO GLUCOSE Taisuke MATSUI The First Department of Surgery, Nara Medical U

STUDIES ON THE RELATION BETWEEN LATE DUMPING SYNDROME AND GLUCAGON RESPONCES TO GLUCOSE Taisuke MATSUI The First Department of Surgery, Nara Medical University, Kashihara (Director: Prof. H. Nakano) This