Extensive Sub-RPE Complement Deposition in a Nonhuman Primate Model of Early Stage Diabetic Retinopathy

Purpose: This study aims to reveal retinal abnormities in a spontaneous diabetic nonhuman primate (NHP) model and explore the mechanism of featured injuries. Methods: Twenty-eight cynomolgus monkeys were identified to suffer from spontaneous Type 2 diabetes from a colony of more than eight hundred aged monkeys and twenty-six age-matched ones were used as controls. Their blood biochemistry profiles were determined and the retinal changes were examined by multimodal imaging, hematoxylin-eosin (H&E) staining and immunofluorescence. Retinal pigment epithelium (RPE) cells were isolated and determined by RNA-sequencing and computational analyses. Results: Diabetic monkeys were characterized by early vascular and neural damage in the retina and dyslipidemia. The typical acellular capillaries and pericyte ghost were found in the diabetic retina, which also exhibited reduced retinal nerve fiber layer (RNFL) thickness compared to the controls. Of note, distinct sub-RPE drusenoid lesions were extensively observed in these diabetic monkeys and complements deposition including C3 and C5b-9 were accumulated in these lesions. The RNA-seq data of RPE cells showed complement activation, AGE/RAGE activation and 18 inflammatory response at the setting of diabetes. Consistently, the plasma levels of C3 and C4 were also increased in the diabetic monkeys. Conclusions: The spontaneous Type 2 diabetic cynomolgus monkeys featured with early-stage retinopathy including not only typical vascular and neural damage, but also a distinct sub-RPE deposition. The complement activation and metabolic stress of RPE cells in response to hyperglycemia might contribute to the deposition, revealing an unrecognized role of RPE cells in the early-stage pathological process of diabetic retinopathy (DR). Overall design: RPE cells were collected from 3 diabetic and 3 control subjects using an ophthalmic spatula. Total RNA was purified using a TRIzol (Thermo Fisher Scientific) in accordance with the manufacturer's protocol. The RNA sample quality was confirmed by the Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were constructed using the TruSeq RNA-Seq kit (Illumina) following the manufacturer's instructions. After evaluating the quality by Agilent 2100 Bioanalyzer, libraries were pooled and sequenced on Illumina Hiseq X10.