Aim: To investigate the effect of VEGF on TNF-alpha- or H2O2-induced apoptosis in cultured bovine aortic endothelial cells (BAEC) and the underlied signal transduction mechanisms related to Ca2+-calmodulin dependent protein kinase (CCDPK).
Methods: BAEC were cultured and passaged in DMEM. Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope with Hoechst 33258 staining. Cell viability was detected with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p42/p44 CCDPK was measured by Western blotting.
Results: TNF-alpha 5000 kU/L and H2O2 300 micromol/L elicited DNA fragmentation in BAEC. Vascular endothelial growth factor (VEGF) 100 microg/L significantly protected BAEC from apoptosis induced by TNF-alpha or H2O2, as shown in cell viability assay and apoptotic cell counting. DNA fragmentation induced by TNF-alpha or H2O2 was also reduced by VEGF 100 microg/L. VEGF enhanced TNF-alpha and H2O2 stimulated expression of phospho-p42/p44 CCDPK, simultaneously inhibited TNF-alpha- and H2O2-induced activation of phospho-p38 CCDPK. Both the VEGF-induced up-regulation of phospho-p42/p44 CCDPK and its anti-apoptotic action were prevented by the specific p42/p44 CCDPK inhibitor U0126.
Conclusion: VEGF protects BAEC from apoptosis induced by TNF-alpha and H2O2, and its co-modulatory effects by activation of p42/p44 CCDPK signaling together with inhibition of p38 CCDPK signaling appear to be an important mechanism for its survival effect on endothelial cells.
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