The Region-Specific Regulation of Complement in Diabetic Retinopathy
Lewis, Sean   (University of Michigan-Ann Arbor)
Mentor: Fort, Patrice
Diabetic retinopathy (DR) is the leading cause of blindness among adults in the United States. The disease is characterized by increased vascular permeability, tissue ischemia, neovascularization, neurodegeneration, and eventually, vision-loss. Recently, the complement system has been implicated in the multifactorial pathogenesis of DR. Complement serves an important role in the innate immune response by lysing pathogens and damaged host cells via three main pathways: classical, lectin, and alternative. Hyperactivation of complement, however, has the potential to cause a harmful inflammatory response and may damage particularly sensitive tissues such as the retina. The purpose of this study was to characterize the regionspecific expression of complement components and regulators in the retinas of diabetic human donors with or without retinopathy compared to nondiabetics. To test the hypothesis that complement activation is region-specific in the retina, donors (n = 30) were first divided into three groups based on their clinical status at the time of death: non-diabetics (ND), diabetics without retinopathy (D), and diabetics with retinopathy (DR). Retinal tissue was then dissected into central (CR) and peripheral (PR) regions. Deep sequencing was performed previously on RNA from human donors (n = 15, CR; n = 16, PR). As a means of probing for which complement components may be differentially expressed in DR, RNA-Seq data were analyzed for complement system players. Transcripts observed to be differentially expressed between groups were selected for further investigation by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from PR and CR tissues including but not restricted to donor samples used in the RNA-Seq and reverse transcribed. Samples were assayed in triplicate with ß-actin as the endogenous control. All primers used were premade TaqMan Gene Expression Assays (Applied Biosystems). qRT-PCR analysis of complement system players confirmed trends observed in the RNA-Seq data, demonstrating that complement activation is associated with retinopathy and not just diabetes. This trend was demonstrated most clearly by complement component 3, in which the expression of transcript is significantly higher for DR compared to D donors in both CR and PR (p<0.05). Region-specific differences in complement activation in DR were also observed, as early complement inhibitors SERPING-1 and CFH are more highly upregulated in CR and the late complement inhibitor CD59 is more highly upregulated in PR (both relative to D). These data illustrate that there is an association between complement activation and retinopathy in diabetic patients. Further studies are necessary to elucidate the implication of complement activation in the pathophysiology of DR.