Abstract
Purpose :
Diabetic retinopathy (DR) is the leading cause of new cases of blindness among working age adults. Inflammatory pathways are considered as the major cause of DR pathogenesis. Recent findings from our lab showed upregulation of S100A proteins in ocular inflammatory diseases. This study aims to characterize the expression and localization of S100 proteins in DR mice models.
Methods :
Eight-weeks old Ins2Akita (spontaneous dominant mutation in the insulin 2 gene), Kimba (huVEGF165 overexpression in photoreceptors) and Akimba (double transgenic progeny of Ins2Akita x Kimba) mice were chosen to represent the DR phenotypes. Age-matched wild type mice with C57BL/6J background were used as control. Eyes were enucleated and neural retina was isolated. Tissues were homogenized and RNA isolated for global profiling of all known S100 proteins by real-time PCR. Protein quantification by western blot and ELISA were used to confirm expression profiles. Whole eye fixed in 4% PFA, followed by cryopreservation by sucrose gradient, was embedded in OCT for immunostaining of S100 proteins.
Results :
Expression of S100A1, A3, A4, A5, A6, A7, A8, A9, A10, A11, A13, A14, A16, B, G, and Z were seen in mice retinas. Ins2Akita showed moderate increase of most S100 genes, whereas both Kimba and Akimba retina showed higher expression levels. S100A8 and S100A9 was most significantly elevated in Ins2Akita, Kimba and Akimba retina, with the largest increase in the double transgenic mouse. This was confirmed by protein expression assays using ELISA and western blot. S100A8 and S100A9 proteins were seen to be localized in the inner retina, predominantly in the outer plexiform layer (OPL) and ganglion cell layers (GCL). Akimba mice retina showed maximal expression compared to Ins2Akita and Kimba mice.
Conclusions :
S100 proteins are constitutively present in the mice retina. Elevated S100A8 and A9 at gene and protein levels suggest their role in the pathogenesis of DR.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.