Abstract
Purpose:
Diabetic Retinopathy (DR) is the leading cause of blindness in working age Americans. Current therapies for DR address only the latest stages of the disease, are invasive and are of limited effectiveness. Retinal pericyte death is one of the earliest pathologic features of DR. Though early pericyte apoptosis has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. High glucose has been shown to cause nuclear accumulation of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting in death of several cell types. A novel pro-apoptotic pathway initiated by the interaction between GAPDH and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified. The goal of the present study was to determine if the Siah1/GAPDH complex is involved in pericyte apoptosis under diabetes-relevant conditions.
Methods:
Annexin V immunostaining combined with Fluorescence Activated Cell Sorting (FACS) was used to quantify apoptosis levels in human retinal pericytes (HRP) exposed to LPS (10μg/mL), TNFα (10ng/mL) or D-glucose (30mM). In the letter case, medium supplemented with mannitol (30mM) was used as an osmotic control. For Western blot analysis, HRPs were lysed and nuclear and cytoplasmic fractions were isolated. The following primary antibodies were used in western blot and immunocytochemical analysis: anti-Siah1, anti-GAPDH and anti-β-actin. Western blots were quantified using Image J.
Results:
FACS analysis revealed that LPS, TNFα and high glucose caused HRP apoptosis. Growth medium supplemented with high glucose led to significant upregulation of Siah1 total protein levels when compared to control conditions (p<0.05). TNFα also caused an upregulation of Siah1 total protein when compared to control groups (p<0.05). In addition, high glucose stimulation of GAPDH nuclear translocation was confirmed by both immunocytochemistry and western blotting.
Conclusions:
To our knowledge, this is the first study identifying a role for the GAPDH/Siah1 complex in HRP apoptosis. Our findings suggest that GAPDH/Siah1 may provide a novel cell death mechanism linking, HRP apoptosis and diabetes-relevant retinal conditions. Future experiments will examine the role of GAPDH nuclear translocation and HRP death in an animal model of DR.
Keywords: 426 apoptosis/cell death •
499 diabetic retinopathy