Effects of aging and chronic cigarette exposure to circulatory microRNAs in the Rhesus macaque

Abstract

Purpose : AMD is a blinding disease having both genetic and environmental components, with cigarette smoke(CS) exposure as the leading environmental risk factor. The purpose of this study was to identify age-related circulatory microRNA(miRNA) changes in the plasma and ocular fluids of the Rhesus macaque and compare them to CS induced changes, with intent is to develop an animal model of dry AMD.

Methods : All Rhesus macaques were housed at the California National Primate Research Center(CNPRC) at UC Davis. We used 6 animals of the same gender per group: young (2-4 years old), old (20-25 years old) and middle aged (9-12 years old)-4 of which were exposed to smoke for 1 month. These 4 macaques were clinically assessed prior to and following CS exposure using Fourier-domain optical coherence tomography(FD-OCT) and fundus photography. Data was analyzed using a combination of Stata software and paired t tests. Ocular fluids and plasma samples were collected, miRNA isolated, and expression data obtained from Affymetrix miRNA GeneTitan Array Plates 4.0, followed by bioinformatics analysis on Affymetrix Expression Console(EC) and Transcriptome Analysis Software(TAS), and using ANOVA. Immunohistochemistry was performed on paraffin embedded ocular tissue sections.

Results : Retinal imaging using OCT, showed no significant changes in retinal thickness. Statistically significant changes were seen in miRNA populations in ocular fluids and plasma. In the plasma samples, 45 miRNAs were strongly upregulated (Fold Change >+/-1.5, p<0.05) upon CS exposure, while in aging monkeys a different miRNAs in plasma were affected, and downregulated. In the vitreous, 3 miRNAs were downregulated in animals exposed to CS, intriguing enough two of them (miR-6794 and miR-6790) were the same ones downregulated with age. Cotinine assays showed that animals received on average dose of 50-60 ng/ml cotinine, a marker for CS exposure. Immunohistochemistry of Sirt1, a marker of aging, showed decreased intensity of staining in retina with age as well as with CS exposure.

Conclusions : One month of CS exposure of Rhesus macaques resulted in significant changes of expression of circulatory miRNAs. Healthy aging changes of miRNAs populations were different from the CS exposure induced changes in plasma, while the ones in vitreous were similar. This data will be utilized to develop an animal model of dry AMD, using a monkey model exposed to CS.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.