Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina

Abstract

Aims/hypothesis: Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, -2 and -3; also known as CCN4, -5 and -6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression.

Materials and methods: CCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12 weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo.

Results: After 6 weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12 weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina.

Conclusions/interpretation: CTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.

Fig. 1

Plasma glucose (a) and CML (b) levels in control and diabetic rats at 6 and 12 weeks of streptozotocin-induced diabetes. White bars, control rats; black bars, diabetic rats; cross-hatched bars, diabetic rats treated with aminoguanidine. *p < 0.05 and *** p < 0.001 for difference between experimental and control group. The aminoguanidine group was only significantly different from the groups with diabetes at 12 weeks (^†p < 0.05). The error bars show the standard deviation for each group

Fig. 2

Gene expression of CCN family members. Fold change, compared with control values, in retinal mRNA levels of CCN family members in streptozotocin-induced diabetic rats at 6 (white bars) and 12 weeks (cross-hatched bars) after streptozotocin-induction and in aminoguanidine-treated, streptozotocin-induced diabetic rats at 6 (black bars) and 12 weeks (grey bars) of diabetes. *p < 0.05 for difference between experimental group and control group; ^†p < 0.05 for difference between aminoguanidine-treated diabetic group and diabetes-only group

Fig. 3

Immunohistochemical staining patterns of a CYR61, b CTGF and c TIMP1 in control rat retinas. Intense staining of CYR61 and CTGF was present in large cell bodies of the ganglion cell layer (GCL) and weak staining in the inner plexiform layer (IPL). Intense uniform immunostaining of TIMP1 was found in the GCL and weak staining in the IPL. INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, RCL rod and cones layer, RPE retinal pigment epithelium. Magnification: ×150

Fig. 4

CTGF protein levels in retina of control and diabetic rats. a Western blots of CTGF and GAPDH as loading control. Samples were pooled for each group. A prominent band of CTGF protein is present in the 12-week diabetic group (12D; n = 8), whereas protein bands were similar in all other groups (control rats at 6 [6C; n = 6] and 12 [12C; n = 8] weeks, diabetic rats at 6 [6D; n = 8] weeks and diabetic rats treated with aminoguanidine at 6 [6AG; n = 7] and 12 [12AG; n = 7] weeks). b The blots were quantified by densitometry and expressed as a ratio of CTGF:GAPDH

Fig. 5

Fold change of Tgfb1 and Tgfb2 expression in diabetic rats at 6 (white bars) and 12 weeks (cross-hatched bars) after streptozotocin-induction and in aminoguanidine-treated diabetic rats at 6 (black bars) and 12 weeks (grey bars) after streptozotocin-induction. Tgfb1 was significantly decreased in the diabetic rats after 6 weeks (*p < 0.05 vs control). This difference was not observed in the aminoguanidine-treated group at 6 weeks. Tgfb2 expression was not significantly altered at either time point

Fig. 6

Immunohistochemical staining patterns of TGFB1 (a, d), laminin (b, e) and fibronectin (c, f) in retina of control rats (a–c) and rats 12 weeks after streptozotocin-induced diabetes (STZ) (d– f). Immunostaining of TGFB1, laminin and fibronectin was confined to the retinal microvasculature and did not notably differ between control and streptozotocin sections. Magnification: × 150

Fig. 7

Gene expression of extracellular matrix components. Fold change of extracellular matrix gene expression as indicated in diabetic rats at 6 (white bars) and 12 weeks (cross-hatched bars) after streptozotocin-induction and in aminoguanidine-treated diabetic rats at 6 (black bars) and 12 weeks (grey bars) after streptozotocin-induction. *p < 0.05 for difference between experimental and control group; ^†p < 0.05 for difference between aminoguanidine-treated diabetic group and diabetes-only group

Fig. 8

Relative mRNA levels of Cyr61 and Ctgf in retinas of control (white bars) and AGE-treated (black bars) mice, depicted as fold change in comparison with control mice. *p < 0.05 for effect of AGE treatment on Cyr61 and Ctgf mRNA levels