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Narsing A. Rao, Sindhu Saraswathy; Hsa-mir-let 7i Treatment Suppresses Tlr4 Mediated Mitochondrial Oxidative Stress In Giant Cell Arteritis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6243.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous findings have shown downregulation of 15 miRNAs related to TLR4, its signaling molecules, and mitochondrial oxidative stress in giant cell arteritis (GCA). One of the 15 miRNAs, hsa-let-7i is validated to target TLR4. In this study, we investigate the role of hsa-let-7i in the modulation of TLR4 and mitochondrial oxidative stress in GCA that lead to amplification of the inflammatory process.
Non-GCA temporal artery biopsy specimens were exposed to LPS and subjected to total RNA isolation and immunohistochemical studies to detect TLR4, CD209 (dendritic cells) and CD31 (endothelial cells). qPCR analysis was performed to detect TLR4 and CD83 gene expression. miRNA PCR Array (SABiosciences, Frederick, MD) was performed using specific primers for hsa-mir-let 7i, mir-let 7a, mir-10a, mir-106b, mir-221. Another group of similar specimens were treated with miRNA hsa-let-7i mimic and LPS. Scrambled miRNA and samples without LPS treatment were used as controls. TLR4 expression was studied by qPCR analysis. Another set of specimens were exposed to LPS and then treated with the TLR4 inhibitor OXPAPC or miRNA hsa-let-7i mimic or scrambled microRNA. DNA damage marker 8-OHdG was colocalized with the mitochondrial marker COX IV by immunohistochemistry.
TLR4 was localized in the dendritic cells in the adventitia region of the temporal artery after LPS exposure whereas it was absent on the endothelial layer. miRNA-hsa-let 7i in LPS treated temporal artery specimens were significantly downregulated compared to non-treated controls.TLR4 was markedly upregulated in the temporal artery exposed to LPS. Such overexpression was suppressed with miRNA hsa-let-7i mimic treatment whereas it was not altered with scrambled microRNA treatment. 8-OHdG was colocalized with COX-IV in the adventitia of the temporal artery exposed to LPS. Such damage was absent in the artery treated with the TLR4 inhibitor or with miRNA hsa-let-7i mimic.
The increased TLR4 expression on the dendritic cells of adventitia indicates that the initial inflammatory insult in GCA could begin in the adventitia. This study reveals that TLR4 activation from LPS leads to mitochondrial oxidative stress, and such TLR4 mediated damage can be prevented by the miRNA hsa-let-7i mimic treatment.
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