Korean Diabetes J.  2008 Feb;32(1):44-52. 10.4093/kdj.2008.32.1.44.

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

• Kim YS
• Sohn EJ
• Kim CS
• Lee YM
• Jung DH
• Kim NH
• Lee HY
• Kim JY
• Kim JS

• KMID: 2298109
• DOI: http://doi.org/10.4093/kdj.2008.32.1.44

Abstract

BACKGROUND: 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.

Keyword

Diabetic retinopathy; Vascular endothelial growth factor (VEGF); Advanced glycation end products (AGEs); Lens opacity

MeSH Terms

Animals
Apoptosis
Cataract
Cell Death
Diabetic Retinopathy
Enzyme-Linked Immunosorbent Assay
Glycosylation End Products, Advanced
In Situ Nick-End Labeling
Models, Theoretical
Rats
Retina
Retinal Ganglion Cells
Streptozocin
Vascular Endothelial Growth Factor A
Glycosylation End Products, Advanced
Streptozocin
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Expression of VEGF in the retina and the expression of VEGF in the serum of STZ-induced diabetic rats. A. Hematoxylin and eosin staining (a, normal rats; b, diabetic rats). B. VEGF staining: The brown product of the peroxidase reaction is present in the GCL (a' and b') and in the INL (a'' and b'') after VEGF staining of the retinal sections. (n = 4 retinas per group). Scale bar = 50 µm. C. Distribution of VEGF immunostaining score in the retina: Each sample was scored from 0 to 5 based on the intensity of the positive area with VEGF immunostaining. Results are given as mean ± S.E.M. D. Expression of VEGF mRNA in the retina was quantified by a RT-PCR method. Results are given as mean ± S.E.M. (n = 6). E. Serum VEGF levels: Each dot represents the serum VEGF levels determined by ELISA for each animal. N, normal (n = 12); STZ-DM, STZ-induced diabetic rats (n = 12).

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  Fig. 2 Localization of AGEs and RAGE in the retina and expression of AGEs in the serum of STZ-induced diabetic rats. A. AGEs and RAGE double-immunofluorescence staining of STZ-induced diabetic rat retina. The sections were immunolabeled with anti-AGE antibody (a and b) and then RAGE antibody (a' and b'). Fluorescein (a and b) and Texas red (a' and b') show identical fields, respectively. Cell bodies showing AGEs immunoreactivity are present in the GCL and INL; The retinas of normal rats (a, a', and a'') and of STZ-induced diabetic rats (b, b', and b''). Scale bar = 50 µm. B. Serum AGEs levels. Each dot represents the serum AGEs levels determined by ELISA for each animal. The serum AGEs levels of STZ-induced diabetic rats are significantly higher than those of normal control rats. N, normal (n = 12); STZ-DM, STZ-induced diabetic rats (n = 12).

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  Fig. 3 Correlation between serum VEGF and AGEs levels. A strong correlation (r = 0.504) between serum VEGF levels measured by ELISA and serum AGEs levels was found in both normal rats (n = 12) and STZ-induced diabetic rats (n = 12).

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  Fig. 4 Retinal cell apoptosis detected by TUNEL and DAPI staining. (A) The retinas of normal control rats (a, a', and a'') and STZ-induced diabetic rats (b, b', and b''): apoptotic retinal ganglion cell death was detected by TUNEL (green), and the nuclei (blue) were stained with DAPI. Merged images (a'' and b'') of TUNEL and DAPI. Scale bar = 50 µm. The number of TUNEL-positive cells was increased compared to that of normal control retinas (B) as indicated in the graph.

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  Fig. 5 Cataractogenesis in the lenses of STZ-induced diabetic rats. A. Images of rat lenses in normal and STZ-induced diabetic rats. B. Lens opacities. All lens opacities were analyzed. N, normal rats; DM, STZ-induced diabetic rats. Since the opacity changes associated with diabetes are usually asymmetrical, the eyes from each animal were always analyzed independently. All data are expressed as means ± S.E.M. (n = 14).

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