In vitro ASC withstand hyperglycemic stress with minimal apoptosis and increase retinal endothelial survival.
(A). Number of viable cells at normal (5.5 mM) or high glucose concentrations did not change at 72hrs as evidenced from the quantity of formazan product measured at 490 nm, is directly proportional to the number of living cells in culture. On the other hand, normal human fibroblasts (HDF) demonstrated a significant decrease (**p<0.01) in viable cells in comparison to ASC. Mannitol (Man) used as osmolality control did not affect ASC. (B). ASC treated with increasing doses of glucose or mannitol were fixed in Prefer fixative. Cells in each well were stained using an active caspase-3 antibody (Promega Corp). The % apoptotic cells were calculated based on total fluorescence intensity of caspase-3 stained cells relative to total cell count. As expected staurosporine (ST, 1 µM) caused massive cell death (**p<0.01) at low glucose (5.5 mM), whereas both high glucose and mannitol did not show any significant (p>0.05) apoptosis compared to low glucose. (C). HREC co-cultured with ASC and exposed to high glucose (HG; 25 mM) were protected from apoptosis as shown by decrease in active caspase-3 staining in co-cultures compared to HREC cultured alone with HG (arrows). (D). Quantification of total fluorescence intensity of caspase-3 stained cells relative to total cell count by MetaMorph show a significant decrease in caspase-3 staining, **p<0.01. Mannitol (MAN) at 25 mM served as an osmolality control and had no effect.