Diabetes & Metabolism Journal

Original Articles

Effects of Anti-Vascular Endothelial Growth Factor (VEGF) on Pancreatic Islets in Mouse Model of Type 2 Diabetes Mellitus.: Ji Won Kim, Dong Sik Ham, Heon Seok Park, Yu Bai Ahn, Ki Ho Song, Kun Ho Yoon, Ki Dong Yoo, Myung Jun Kim, In Kyung Jeong, Seung Hyun Ko; Korean Diabetes J. 2009;33(3):185-197. Published online June 1, 2009; DOI: https://doi.org/10.4093/kdj.2009.33.3.185

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Vascular endothelial growth factor (VEGF) is associated with the development of diabetic complications. However, it is unknown whether systemic VEGF treatment has any effects on the pancreatic islets in an animal model of type 2 diabetes mellitus. METHODS: Anti-VEGF peptide (synthetic ATWLPPR, VEGF receptor type 2 antagonist) was injected into db/db mice for 12 weeks. We analyzed pancreatic islet morphology and quantified beta-cell mass. Endothelial cell proliferation and the severity of islet fibrosis were also measured. VEGF expression in isolated islets was determined using Western blot analysis. RESULTS: When anti-VEGF was administered, db/db mice exhibited more severe hyperglycemia and associated delayed weight gain than non-treated db/db mice. Pancreas weight and pancreatic beta-cell mass were also significantly decreased in the anti-VEGF-treated group. VEGF and VEGF receptor proteins (types 1 and 2) were expressed in the pancreatic islets, and their expression was significantly increased in the db/db group compared with the db/dm group. However, the elevated VEGF expression was significantly reduced by anti-VEGF treatment compared with the db/db group. The anti-VEGF-treated group had more prominent islet fibrosis and islet destruction than db/db mice. Intra-islet endothelial cell proliferation was also remarkably reduced by the anti-VEGF peptide. CONCLUSION: Inhibition of VEGF action by the VEGF receptor 2 antagonist not only suppressed the proliferation of intra-islet endothelial cells but also accelerated pancreatic islet destruction and aggravated hyperglycemia in a type 2 diabetes mouse model. Therefore, the potential effects of anti-VEGF treatment on pancreatic beta cell damage should be considered.

Vascular Endothelial Growth Factor (VEGF) and Advanced Glycation End Products (AGEs) Overexpression in the Retina and Serum and Lens Opacities of Streptozotocin-induced Diabetic Rats.: Young Sook Kim, Eun Jin Sohn, Chan Sik Kim, Yun Mi Lee, Dong Ho Jung, Nan Hee Kim, Hyun Young Lee, Jung Yeon Kim, Jin Sook Kim; Korean Diabetes J. 2008;32(1):44-52. Published online February 1, 2008; DOI: https://doi.org/10.4093/kdj.2008.32.1.44

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Vascular Endothelial Growth Factor (VEGF) and Advanced Glycation End products (AGEs) have been implicated in the development of diabetic retinopathy. In this study, we examined the expression of VEGF and AGEs in the retina and serum, apoptosis in the retina, and lens opacities in streptozotocin (STZ)-induced diabetic rats. METHODS: The localization of VEGF and AGEs in the retina of STZ-induced diabetic rats was determined by immunohistochemical analysis, and apoptotic cell death was assessed using the TUNEL assay. In the serum, STZ-induced diabetic rats were assayed for VEGF and AGEs by ELISA. Lenses were also isolated to detect the opacity. RESULTS: Expression of VEGF and accumulation of AGEs were significantly increased in the retinal ganglion cell layers (GCL) and nuclear cell layers (NCL) of STZ-induced diabetic rats compared to normal control rats. In addition to cellular expression, serum VEGF and AGEs levels were also increased significantly in STZ-diabetic rats compared to normal rats (both P < 0.001) and there was a significant correlation between the serum VEGF and AGEs levels (r = 0.504). The lens opaque density of STZ-induced diabetic rats were significantly higher than in normal rats (P < 0.001). CONCLUSIONS: AGEs could be involved in the development of diabetic retinopathy through the induction of VEGF. One could possibly correlate this lens opaque formation with elevation of AGE induced VEGF level. Thus, this study should be considered as a basic research for studying pathology of the retina and lens in diabetic experimental models.

Comparison of Minicircle with Conventional Plasmid for the Non-viral Vascular Endothelial Growth Factor (VEGF) Gene Therapy.: Minjeong Kwon, Soonhee Lee, Heysook Chung, Changshin Yoon, Mikyung Kim, Jeonghyun Park; Korean Diabetes J. 2007;31(6):465-471. Published online November 1, 2007; DOI: https://doi.org/10.4093/jkda.2007.31.6.465

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Abstract PDF: BACKGROUND
Delayed wound healings in diabetic patients are related with the impairment of the expressions of various growth factors. Treatments using growth factors have been attempted on diabetic foot ulcer. VEGF (vascular endothelial growth factor) accelerates neo-angiogenesis on the early phase of the wound healing and exerts chemo-attractive effect for the other growth factors and cytokines. Non-viral gene transfer strategies are attractive tool for the gene therapy due to the safety and the versatility, but the low efficiency has been the serious problem. METHODS: We performed the VEGF gene therapy using reconstructed minicircle MINI-pbetaVEGF DNA with a polymeric carrier, polyethylenimine (PEI, 25 kDa) in HEK293, CHO, and NIH3T3 cell lines, and compared its efficiency with the conventional VEGF plasmid pbetaVEGF. RESULTS: The levels of expressed VEGF were higher in the groups using BPEI (branched PEI) as a gene carrier than naked plasmid transfer in all cell lines (P < 0.05). The minicircle MINI-pbetaVEGF DNA showed much higher VEGF expression than conventional plasmid pbetaVEGF (P < 0.05). CONCLUSION: Minicircle DNA MINI-pbetaVEGF showed much higher transfection efficiency than conventional plasmid pbetaVEGF. It might be used in actual human clinical trial due to its higher efficiency and possible safety for the treatment of diabetic foot ulcer.

Effects of Troglitazone on the Expression of VEGF and TGF-beta in Cultured Rat Mesangial Cells.: Dong Lim Kim, Nan Hee Kim, Dong Seop Choi; Korean Diabetes J. 2007;31(3):220-229. Published online May 1, 2007; DOI: https://doi.org/10.4093/jkda.2007.31.3.220

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Clinical study reported that troglitazone ameliorated microalbuminuria in diabetic nephropathy. However, the mechanism of action is not fully understood. Vascular endothelial growth factor (VEGF) is known as vascular permeability factor and it is considered the most likely cause of glomerular hyperfiltration and proteinuria in diabetic nephropathy. Transforming growth factor-beta (TGF-beta) is a potent inducer of extracellular matrix production and fibrosis in renal cells and one of the important cytokine in the pathogenesis of diabetic nephropathy. To determine whether troglitazone affects VEGF and TGF-beta production in diabetic nephropathy, we examined the effects of troglitazone on the VEGF and TGF-beta expression in cultured rat mesangial cells exposed to high glucose concentration. METHODS: Rat mesangial cells were cultured in media with D-glucose 5.5 mM (NG) or D-glucose 30 mM (HG), or D-glucose 30 mM/troglitazone 20 micrometer(HTz) and for 6, 24, or 72 hours, respectively. VEGF and TGF-beta expression were assessed by semiquantitative RT-PCR and western blot analysis. RESULTS: Troglitazone decreased the VEGF164 and VEGF120 mRNA expressions in cultured rat mesangial cells exposed to high glucose concentration with incubation for 24 and 72 hours, respectively. VEGF protein was also decreased in experimental group treated with troglitazone (HTz) than in those with HG for 24 and 72 hours. However troglitazone had no effect on the expression of TGF-beta mRNA in mesangial cells. CONCLUSION: This study suggested that troglitazone may modulate the development and progression of diabetic nephropathy by reducing the expression of VEGF in mesangial cells

Effective Glycemic Control Achieved by the Transplantation of VEGF-Transfected Islets in STZ-induced Diabetic Mice.: Byung Wan Lee, Hee Young Chae, You Ran Ahn, Seung Hoon Oh, Ji Youn Kim, Yun Jae Chung, Sang Young Kim, Kyun Yung Cho, Jae Hoon Chung, Yong Ki Min, Myung Shik Lee, Moon Kyu Lee, Kwang Won Kim; Korean Diabetes J. 2005;29(4):282-294. Published online July 1, 2005

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Hypoxic damage is one of the major causes of early islet graft failure, and VEGF is known to play a crucial role in revascularization. We tried to evaluate whether the VEGF transgene in an islet graft can increase islet revascularization and; therefore, increase the survival rate of transplanted islets in order to achieve effective glycemic control in diabetic mice models using a non-viral cationic lipid reagent for gene delivery into non- dividing islet cells. METHODS: Human VEGF165 cDNA was transfected into Balb/c mice islets using Effectene, and the vascular neogenesis and glucose levels examined in the recipient syngeneic Balb/c mice. A minimal number of VEGF-transfected islets(100 IEQ/animal) were transplanted into STZ-induced diabetic mice. The recipient mice were classified into three groups: islet transplantation(IT) without intervention(IT-alone group, n=8), IT with an islets transduced rhoJDK-control vector(IT-rhoJDK group, n=8), and IT with an islets transduced rhoJDK-VEGF vector(IT-rhoJDK-VEGF group, n=8). RESULTS: The transfection efficiency was highest with 4microgram/microliter cDNA and 25microliter Effectene(1: 6 weight ratio), with satisfactory cell viability under these conditions. The overproductions of VEGF mRNA and proteins from the conditioned cells were confirmed. A minimal number of the VEGF-transfected islets(100 IEQ/animal) were transplanted into STZ-induced diabetic mice. The control of hyperglycemia in the IT-alone(0/8) and IT-rhoJDK groups(0/8) failed. However, complete abrogation of hyperglycemia and viable islets, and an increased vascularization of the VEGF-transfected grafts were identified in the renal capsules of the IT-rhoJDK-VEGF group(8/8). CONCLUSION: These studies support the utility of VEGF-transfected islet delivery using a cationic lipid reagent to achieve euglycemia with minimal islets via neovascularization.

VEGF-Angiopoietin-Tie2 System in Diabetic Retinopathy.: Nan Hee Kim, Hee Young Kim, Ji A Seo, Kye Won Lee, Sin Gon Kim, Kyung Mook Choi, Sei Hyun Baik, Yoon Shin Park, Inho Jo, Dong Seop Choi; Korean Diabetes J. 2005;29(2):122-132. Published online March 1, 2005

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Ischemia-induced neovascularization can cause the loss of vision in retinal disorders such as diabetic retinopathy. Recent studies have shown that the angiopoietin-Tie2 system is a major regulator of vascular integrity and it is involved in pathologic angiogenesis. However, its role in the pathophysiology of diabetic retinopathy is not yet known. We examined the regulation of the VEGF-angiopoietin-Tie2 system in both in vitro and in vivo studies to discover their possible role in diabetic retinopathy. METHODS: We investigated the effects of a well-known angiogenic stimulus, hypoxia(2% O2 concentration) and vascular endothelial growth factor(VEGF, 10 ng/mL) on the expression of the angiopoietin-Tie2 mRNA in bovine retinal pericytes(BRP) and bovine aortic endothelial cells(BAEC). We also examined the expressions of VEGF-angiopoietin-Tie2 mRNA in retinas of type 2 diabetic OLETF(Otsuka-Long-Evans-Tokushima-Fatty) rats at 30 and 50 weeks. We also investigated the effect of angiotensin II receptor type 1(AT1) antagonist on the VEGFangiopoietin-Tie2 expression. RESULTS: Hypoxia and VEGF treatment significantly increased angiopoietin-1(Ang1) mRNA expression in the BRPs. In contrast, the angiopoietin-2(Ang2) mRNA expression was unaltered in the BRPs treated with hypoxia and VEGF. Significant up-regulation of Tie2 mRNA expression was found and this lasted up to 12 h. However, using BAECs, we found that only the Ang2 expression responded to these two angiogenic stimuli. In OLETF rats, the Ang-Tie2 expression patterns were similar with those of the BAECs. Ang2 and VEGF mRNA were increased at 30 and 50 weeks for the OLETF rats, whereas the Ang1 expression was not changed. The up-regulation of Ang2 and VEGF was decreased with the losartan treatment, an AT1 receptor antagonist. Tie2 mRNA expression was increased only at 50 weeks and it did not show any decrement by the losartan treatment. CONCLUSION: Our data suggest that hypoxia and VEGF treatment differentially regulate the angiopoietin-Tie2 system in the two vascular cells. Ang2 and VEGF expressions were predominantly increased in type 2 diabetic rats, and the unopposed action of Ang2 with VEGF might be involved in the development of diabetic retinopathy. The renin-angiotensin system may be a potential mechanism for the up-regulated VEGF-Ang2 system

Effect of Peroxisome Proliferator Activated Receptor-gamma Agonist, Angiotensin II Receptor Blocker and alpha-lipoic Acid on Renal VEGF Expression in Diabetic Nephropathy.: Jang Hyun Koh, Yeon Lee, Mi Jin Kim, Young Goo Shin, Eun Young Lee, Choon Hee Chung; Korean Diabetes J. 2004;28(5):367-376. Published online October 1, 2004

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Diabetic nephropathy is one of the most serious complications in diabetes mellitus, and it is the leading cause of end stage renal disease. It has been reported that angiotensin converting enzyme inhibitor (ACEi) reduces the vascular endothelial growth factor (VEGF) expression, and so it plays an important role in reducing the renal damage. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonist is known to reduce insulin resistance in type 2 diabetic patients. In the previous study, PPAR-gamma agonist was shown to lower VEGF expression in the retina, but it increased the plasma VEGF level. Alpha-lipoic acid (alpha-LA), which is an antioxidant, lowers the increased level of VEGF in retina as well. The precise role of PPAR-gamma agonist and alpha-LA on renal VEGF expression in diabetic nephropathy is still uncertain. We studied the effect of PPAR-gamma agonist, angiotensin II receptor blocker (ATIIRB) and alpha-LA on the renal VEGF expression in diabetic rats. METHODS: We used 60 Sprague-Dawley male rats, those were 8 weeks old and weighted about 300 g each as the study subjects. Among them, 48 rats were chosen and injected with streptozotocin (70 mg/kg) into peritoneal cavity to induce diabetes mellitus. The rast were than divided into 5 groups. Group I was a normal control group (n=12), group II was diabetic control group (n=12), group III was diabetic group that was given with PPAR-gamma agonist (n=12), group IV was the diabetic group that was given ATIIRB (n=12), and group V was the diabetic rats that were given alpha-LA (n=12). We measured their body weight, blood glucose levels, 24 hour urine protein and albumin levels at the baseline, the 8th and the 16th weeks of the experiment. On the 16th weeks of our experiment we extracted the kidneys to measure the glomerular volume, the optical density of the VEGF staining and VEGF mRNA expression. RESULTS: At the beginning of the study, the 5 groups all showed similar 24 hour urine albumin levels. At the 8th week, group II showed an increased urine albumin level of 143.4 +/- 117.2 mg/day; this was greater than that of group IV (60.7+/-30.6 mg/day) (p<0.05). The glomerular volume and optical densities of VEGF expression were significantly reduced in group III, IV and V compared to group II. For group IV and V, the renal VEGF mRNA expression was significantly lower than that of group II, but group III showed no significant difference. from group II. CONCLUSION: Angiotensin II receptor blocker delayed the progression of diabetic nephropathy. PPAR-gamma agonist and alpha-lipoic acid did not have any protective effect against the progression of diabetic nephropathy in spite of the decreased VEGF expression noted in this study.

Plasma and urinary Vascular Endothelial Growth Factor and Diabetic Nephropathy in Type 2 Diabetes Mellitus.: Jeong Heon Oh, Hye Jin Yoo, Soo Yeon Park, Ohk Hyun Ryu, Sang Soo Park, Soon Beom Kwon, Hee Young Kim, Ji A Seo, Kye Won Lee, Sin Gon Kim, Nan Hee Kim, Kyung Mook Choi, Dae Ryong Cha, Sei Hyun Baik, Dong Seop Choi; Korean Diabetes J. 2004;28(2):111-121. Published online April 1, 2004

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Abstract PDF: BACKGROUND
VEGF(vascular endothelial growth factor) has been implicated in the pathogenesis of neovascularization and endothelial dysfunction in diabetes mellitus. However, its precise role in diabetic nephropathy is still unknown. Our aims were to determine whether alterations of plasma and urinary VEGF levels were related to diabetic microvascular complications, especially nephropathy in type 2 diabetic patients. METHODS: 107 type 2 diabetic patients, without non-diabetic kidney diseases, and 47 healthy control subjects were studied. The urinary albumin excretion was defined as the albumin-to-creatinine ratio(ACR) in 24 hour urine samples. The study subjects were divided into four groups: a nondiabetic healthy control group(n=47), a normoalbuminuric diabetic group(ACR <30mug/mg, n=37), a microalbuminuric diabetic group(ACR 30~299mug/mg, n=37) and an overt proteinuric diabetic group(ACR=300mug/mg, n=33). The plasma and urinary VEGF levels were measured in these subjects by enzyme-linked immunosorbent assays. RESULTS: 1) The urinary VEGF concentrations were significantly higher in the diabetic groups than in the controls, even in the normoalbuminuric stage(log VEGF/Cr, normoalbuminuria; 4.33+/-1.06 vs. control; 3.53+/-0.79, p=0.009). The levels of urinary VEGF excretions increased with advancing diabetic nephropathy stage. 2) The plasma and urinary VEGF levels were higher in the hypertensive diabetic than the normotensive diabetic patients. 3) In the diabetic patients, the level of plasma VEGF was positively correlated with the BUN(r=0.398, p=0.039) and urinary ACR (r=0.251, p=0.044). The level of urinary VEGF was positively correlated with the urinary ACR(r=0.645, p<0.001), and creatinine(r=0.336, p=0.009), but negatively correlated with the level of serum albumin(r=-0.557, p<0.001). Both the levels of urinary VEGF and serum creatinine were independently correlated with the urinary ACR. CONCLUSIONS: The excretion of urinary VEGF increased at a relatively earlier stage in diabetic nephropathy and was significantly correlated with the excretion of urinary albumin. These results suggested the possibility of urinary VEGF as a sensitive marker or the detection of diabetic nephropathy and in predicting disease progression.

Effect of Angiotensin II Receptor Blockade on VEGF Expression in Diabetic Nephropathy.: Myoungsook Shim, Mijin Kim, Munkyu Kim, Hyunjin Chang, Younggoo Shin, Junam Kim, Jaeman Song, Hosuk Kang, Eunyoung Lee, Kihak Song, Choonhee Chung; Korean Diabetes J. 2003;27(2):106-114. Published online April 1, 2003

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Abstract PDF: BACKGROUND
Diabetic nephropathy is one of the most serious complications of diabetes, and is the leading cause of chronic renal failure. Vascular endothelial growth factor (VEGF) plays an important role in the pathophysiology of diabetic retinopathy, which can be blocked by ACE inhibitors, but its precise role in diabetic nephropathy is uncertain. METHODS: 32 eight week-old Sprague-Dawley male rats were prepared, of which 16 were chosen for injection with streptozotocin (60 mg/kg) into the peritoneal cavity, with the goal of inducing diabetes. One week later, the peripheral blood sugar, taken from the tail vein was checked. A glucose level exceeding 200 mg/dL was taken as evidence of diabetes. The rats were divided into 4 groups of 8. Group I served as a control. Group II was treated with angiotensin II receptor blockade (L-158,809, 5 mg/kg/day, in drinking water). Group III consisted of diabetic rats and group IV diabetic rats treated with the same angiotensin II receptor blockade (L-158,809). At the beginning of the experiment and on 8th and 12th weeks, 24-hour urine protein and body weight checks were performed. At the end of the study, I extracted kidney and the glomerular volumes and optical densities of the VEGF expression in the glomeruli compared. RESULTS: The basal characteristics were initially the same. However, on weeks 8 and 12 the amount of 24-hour urine protein had increased in groups III and IV (p<0.05). By week 12, it was noticeably greater in group III than in group IV (p<0.05). The glomerular volume was also greater in groups III and IV (p<0.05). Optical density of the VEGF in the glomeruli had increased more in group III than in groups I, II and IV (p<0.05). CONCLUSION: VEGF plays a precise role in diabetic nephropathy, and angiotensin II receptor blockade can reduce diabetic nephropathy by suppressing the expression of VEGF.

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