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used as a standard in this assay. Produced EPO protein was found to be 35 times
more biologically active than recombinant protein (Eprex; Table 1, p = 0.0006).
The rhEPO production level in transduced cultures was 11.5 ± 0.8 mIU/24 h/10
6
cells.
Table 1.
EPO biological activity in vitro
To generate viable cardiac cell culture producing rhEPO for subsequent trans-
plantation, we used rat neonatal cardiac fibroblasts. EPO activity parameters of
transduced cardiac fibroblasts are presented in Table 1. The produced protein was
found to be 12 times more active than recombinant protein (Eprex; p = 0.03).
The rhEPO production level in transduced cells was 14.2 ± 3.1 mIU/24 h/106
cells.
Anti-Apoptotic effect of ePo in isolated cardiomyocytes
The rate of apoptosis was significantly lower in cultured cardiomyocytes treat-
ed with supernants derived from rhEPO-tranduced fibroblasts. To determine
the protective effect of rhEPO produced from genetically modified cardiac
fibroblasts on cardiomyocyte apoptosis, we used an H
2
O
2
-induced oxidative
stress model [24]. Isolated rat neonatal cardiomyocytes were treated with 150
µ
M H
2
O
2
for 60 minutes, with or without 24 h preconditioning with super-
nants containing rhEPO (12 mIU/ml) from transduced cardiac fibroblasts.
The degree of apoptosis was assayed by Annexin V/PI staining and morpho-
logically evaluated using a DAPI nuclear stain. FACS analysis of Annexin V/
PI staining showed that the percentage of apoptotic cells was significantly
lower in cardiomyocytes pretreated with rhEPO-containing supernants com-
pared with control (Figure 1A): 17.8 ± 2.3% versus 32.1 ± 3.7% apoptotic
cells (p < 0.05).
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