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J. Mark Petrash, Biehouy Shieh, Kun-Che Chang, Kenneth L Jones; Lens-enriched transgenic expression of human aldose reductase (AKR1B1) upregulates inflammatory mediators in the eye. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3175. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Aldose reductase (AR), a key enzyme in the polyol pathway, is upregulated in the diabetic eye. Given that inflammatory markers are also increased in diabetes, we designed this study to test a possible connection between AR expression and inflammatory signaling.
Global expression profiles were determined for transgenic mice designed for lens-directed over-expression of human AR and age and sex-matched nontransgenic controls. Total RNA was extracted from lenses of p14 pups (n=4 for each strain) and used for cDNA library construction. Eight independently and uniquely indexed libraries were pooled and analyzed using an Illumina HiSeq 2000 flow cell yielding a 50 cycle single end run. The resulting sequences from these cDNA libraries were analyzed to remove of low-quality bases [Phred score <15] using Python. The remaining sequences were mapped to Mus musculus using GSNAP. Transcript abundance was measured as fragments per kilobase of exon per million mapped reads (FPKM) using CUFFLINKS. FPKM values from all 8 libraries were analyzed using principal components analysis (PCA) to visualize the differences between samples. FPKM values were calculated to rank gene expression using ANOVA. Biological pathways and molecular networks affected by AR over-expression were investigated using Ingenuity Pathway Analysis (IPA).
Genes relating to inflammatory response and immune cell trafficking were among the most highly represented transcripts that were differentially expressed in the AR transgenic lens. Prominent examples included Ccl2 and Ccl7 (monocyte chemotactic protein-1 and 3, resp.) increased 9.5- and 44-fold (p=1.7x10-6, 3.7x10-6, resp.), Cx3cl1 (a monocyte attractant) increased 4.5-fold (p=8x10-7), and Egr1 (a transcription factor) increased 6-fold (p=3.5x10-4). qRT-PCR results are consistent with RNA-seq data in fold changes of transcript level. The functional significance of this transcription profile was further confirmed by ELISA measurements of increased levels of Cx3CL-1 (2-fold; p<0.05), MCP-1 (4-fold; p<0.05) and VEGF (4.5-fold; p<0.05) in whole eye extracts from AR transgenic mice.
AR over-expression induces the proinflammatory signaling molecules in the eye
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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