Transplantation of Genetically Engineered Cardiac Fibroblasts Producing Recombinant Human Erythropoietin to Repair the Infarcted Myocardium - Genetic Engineering: Recent Developments in Applications - page 195

Figure 1. RhEPO inhibits H 2 O 2 -induced apoptosis in isolated cardiomyocytes. Neonatal rat cardiomyocytes
were treated with PBS as a control or with H 2 O 2 (150 µ M) for 60 minutes in the 24 h presence or absence
of rhEPO produced from transduced fibroblasts (12 mIU/ml). (A) Annexin V/PI staining: (a) Representative
density profile (percentage of cells in each quadrant is indicated); (b) Quantitative analysis, showing that EPO
attenuated cardiomyocyte apoptosis. (B) DAPI staining: (a) representative photomicrographs (arrows show
apoptotic cardiomyocyte nuclei chromatin condensation and nuclei fragmentation); (b) quantitative analysis, as
represented by relative apoptotic nuclei counts.
Morphological changes in cells that were exposed to oxidative stress were eval-
uated after DAPI staining. Apoptotic nuclei counts showed a significant reduc-
tion in cell death in cells treated with rhEPO-containing supernants, compared to
untreated cells: 13.5 ± 0.6% versus 19.0 ± 0.7%, p < 0.001 (Figure 1B).
in situ expression of erythropoietin Promotes cytoprotection
and Vascularization
There was no difference among the groups in the levels of hematocrit at all time
points (Table 2). Four weeks after cell transplantation, heart tissue sections were
stained with anti-rhEPO monoclonal antibodies to detect rhEPO expression from
implanted cells. Human kidney tissue was used as positive control (Figure 2A).
RhEPO was detected only in hearts treated with transduced cardiac fibroblasts 5
weeks post MI (Figure 2B-D). The staining was identified in all animals treated
with transduced cells (n = 5), towards the center of the infarct zone.