ABSTRACT
The Nε-(carboxymethyl)lysine (CML), the predominant advanced glycation end products (AGEs) in diabetes and its RAGE induced cytokine release has been well explored. But the CML mediated multiple AGEs receptor expression is still not understood and the role played by RAGE silencing in modulating CML generated pro-inflammatory cytokines in micro and macrovascular endothelial cells is yet to be studied. HUVEC and HREC cells were exposed with CML for 24 h. RAGE, AGER1, AGER2, Gal-3, TLR4, TLR2, CD36, FEEL-1, FEEL-2, and chemokine HMGB1 were quantified by either qPCR/western blotting. The receptor's expression was also determined in control vs diabetic retina. Expression of pro-inflammatory genes, ROS, and mitochondrial membrane potential change were assessed using ELISA, DCFDA, and JC-1 method respectively. RAGE expression was silenced either by Si-RAGE or neutralising antibody with anti-RAGE and expression of other AGE receptors, adaptors, and signalling pathway were studied compared with Si-Control. CML activated RAGE, TLR4, HMGB1(p < 0.001) and Gal-3 (p < 0.05) expression in both micro and macro vascular cells. Cadaveric diabetic retinal tissues also showed increased expression of RAGE, TLR4 and HMGB1 (p < 0.05). RAGE silencing significantly reduced TLR4, HMGB1 (p < 0.05) expression and inhibited the phosphorylation of NFκB and ERK1/2 in both these cells. The TLR4 adaptors MyD88 and TIRAP (p < 0.05) showed down regulation on RAGE silencing. This study shows CML induces AGE receptors expression as observed in diabetic retina and RAGE silencing down regulated TLR4 signalling and cytokine release by partly modulating TLR4 adaptors which needs further validation. From this study we speculate targeting the TLR4 adaptors like MyD88 and TIRAP can be a potential therapeutic target for reducing diabetic induced vascular complications.
Subject(s)
Antigens, Neoplasm/genetics , Diabetic Retinopathy/genetics , Endothelial Cells/metabolism , Gene Expression Regulation , Mitogen-Activated Protein Kinases/genetics , RNA/genetics , Toll-Like Receptor 4/genetics , Antigens, Neoplasm/metabolism , Cells, Cultured , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Endothelial Cells/pathology , Humans , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Toll-Like Receptor 4/biosynthesisABSTRACT
BACKGROUND & OBJECTIVES: The enzyme paraoxonase (PON), an antioxidant enzyme that has both arylesterase and thiolactonase activity, is well studied in cardiovascular diseases. Although a few studies have shown altered PON activity in ocular diseases such as age-related macular degeneration and diabetic retinopathy, but the tissue-wise expression of PON in its three gene forms has not been studied. This study was conducted to see the ocular distribution of PON for any altered expression in ocular pathologies such as in cataract and diabetes mellitus. METHODS: Immunohistochemistry (IHC) of the ocular tissues was done for localizing all three forms of the PON in the human donor eyeballs. The PON arylesterase (PON-AREase) and thiolactonase (PON-HCTLase) activities were determined by spectrophotometry in kinetic mode, and the mRNA expression of the PON genes (PON1-3) was determined by reverse transcription-polymerase chain reaction. RESULTS: IHC showed the presence of both PON1 and 2 in all the ocular tissues and PON3 was seen only in retina. The mRNA expression analysis showed that PON2 and PON3 were present in all the tissues, whereas PON1 was seen only in ciliary and retina. Both the PON-AREase and PON-HCTLase activities were detected in all ocular tissues and was in the order of lens>retina>choroid>ciliary body>iris. The expression and activity were studied in cataractous lens and in diabetic retina of the donor eyes. A significant decrease in PON-AREase activity was seen in cataractous lens (P<0.05) but not in diabetic retina, and there was an increase in PON- HCTLase activity (P<0.05) only in diabetic retina. Bioinformatic studies and in vitro experiments indicated that advanced glycation end products (AGE) such as carboxymethyl -lysine might decrease the PON- AREase activity of the PON. INTERPRETATION & CONCLUSIONS: Distribution of PON enzyme and its activity in ocular tissues is reported here. The study revealed maximal PON activity in lens and retina, which are prone to higher oxidative stress. Differential activities of PON were observed in the lens and retinal tissues from cataractous and diabetic patients, respectively.
