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


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
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.
Hughes
J M
JM
Ocular Angiogenesis Group, Department of Ophthalmology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands.
Kuiper
E J
EJ
Klaassen
I
I
Canning
P
P
Stitt
A W
AW
Van Bezu
J
J
Schalkwijk
C G
CG
Van Noorden
C J F
CJ
Schlingemann
R O
RO
D000818:Animals; D055513:Connective Tissue Growth Factor; D055515:Cysteine-Rich Protein 61; D003921:Diabetes Mellitus, Experimental / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D017127:Glycation End Products, Advanced / Q000502:physiology*; D017874:Immediate-Early Proteins / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D055514:Nephroblastoma Overexpressed Protein; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000503:physiopathology*
C491850:CCN1 protein, mouse; C526318:CCN2 protein, mouse; C526316:CCN2 protein, rat; C526320:Ccn3 protein, mouse; D055515:Cysteine-Rich Protein 61; D016326:Extracellular Matrix Proteins; D017127:Glycation End Products, Advanced; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D055514:Nephroblastoma Overexpressed Protein; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor