Abstract
Purpose :
Early stages of human diabetic retinopathy (DR) are characterized by the loss of human retinal pericytes (HRP), which lead to the development of advanced-stage pathology including angiogenesis. Although much is known about the diagnosis of DR, the apoptotic pathway that exhibits retinal pericyte loss and apoptosis remains unclear. It is known that α3β1 integrin plays a role in the mediation of apoptosis in pericytes through angiopoietin and the activation of caspases 3, 8, 10 and 14 have been reported to be involved in pericyte apoptosis (Park, S.W et al., 2014). TGFβ-Induced Gene Human Clone 3 (BIGH3) is a downstream target molecule of TGFβ, which is known to contribute to HRP apoptosis under hyperglycemic conditions. In this present study, we examined the effect of BIGH3 on HRP caspase 3 activation.
Methods :
HRP were cultured with 10% FBS, 1% Antibiotic-Antimycotic in Complete Classic Medium (CCM) in a humidified 5% CO2 incubator with a temperature of 37°C. Cells were harvested from passages 5-8 until reaching confluency. Cells were treated with 5 ug/mL of BIGH3 for 24, 48 and 72 hr. An enzymatic colorimetric assay quantified activated Caspase 3 activity. Western blots of cell lysates were probed with an anti-BIGH3 polyclonal antibody; an in-house generated rabbit antibody against full-length recombinant BIGH3 and commercially acquired Cleaved Caspase 3. Treatment with Camptothecin and H2O2 served as positive controls; cells in CCM served as negative controls for both experiments.
Results :
Immunoblotting identified cleaved caspase 3 in cells treated with BIGH3 (but not in vehicle controls). The level of cleaved caspase 3 increased at the 48 hr and 72 hr data points. An increase in cleaved caspase 3 was not evident in negative control conditions. Consistent with this observation (based on protein expression), the caspase 3 enzymatic colorimetric assay also identified an increase in apoptosis in HRP cells within 24 hr of treatments. (caspase 3 enzyme activity for the control was 0.0125 Unit; BIGH3 treatment resulted in 0.0285 Unit, and H2O2 in 0.0415 Unit).
Conclusions :
These results strongly suggest that BIGH3 treatment results in the activation of a caspase dependent activity that induces HRP apoptosis
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.