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Effect of recombinant human erythropoietin on endothelial cell apoptosis
R G Carlini1, E J Alonzo, J Dominguez
1Centro Nacional de Dialisis y Trasplante, Hospital Universitario de Caracas, Venezuela.
Background:
Recombinant human erythropoietin (rHuEPO) induces endothelial cell growth and angiogenesis in vitro. The mechanisms are unknown. Because an increase in endothelial cell survival could play a role in this process, we examined the effect of rHuEPO on lipopolysaccharide (LPS)-induced apoptosis in bovine pulmonary artery endothelial cells (BPAECs).
Methods:
Four groups of cells were studied. The first group was preincubated in serum-free medium followed by treatment with LPS. The second group was preincubated with rHuEPO followed by LPS. The third group was treated with only rHuEPO. Control cells were cultured in the absence of rHuEPO and LPS. Apoptosis was determined by flow cytometric DNA analysis, propidium iodide staining, cellular DNA fragmentation by ELISA, and gel electrophoresis.
Results:
LPS-treated cells showed an increase in hypodiploid DNA (36.4 +/- 6.1%) compared with controls (9.8 +/- 3.3%, P < 0.001). Preincubation with rHuEPO decreased this effect to 14.7 +/- 5.1% (P < 0.001). Apoptosis determined by propidium iodide was observed in 33 +/- 8% of LPS-treated cells, but in only 9 +/- 3% of cells preincubated with rHuEPO cells (P < 0.001). Similarly, DNA fragmentation was decreased in rHuEPO pretreated cells compared with LPS alone (0.155 OD +/- 0.02 vs. 0.538 +/- 0.09 OD, P < 0.001). DNA breakdown was observed in only LPS-treated cells.
Conclusions:
These results suggest that rHuEPO prevents LPS-induced apoptosis in endothelial cells. This protective effect could be an important factor in the action of rHuEPO on vascular endothelium.