O203	Shp-2 is involved in bFGF dependent endothelial cell proliferation and prevents apoptosis.
1H.Bridell, 2Chr.Plank, 1T.Gloe, 1N.Hellwig, 2H-Y.Sohn, 1U.Pohl, 2F.Krötz
1Institute of Physiology, Ludwig-Maximilians-University, München, DE; 2Institute of Cardiology, Medical Policlinic, Ludwig-Maximilians-University, München, DE.

The Src homology-2 domain containing tyrosine phosphatase (Shp-2) can be activated by bFGF which is a potent stimulator of angiogenesis. It is unknown, however, whether Shp-2 has a role in the control of endothelial cell proliferation and survival, both preconditions for angiogenesis. Using antisense oligonucleotide (AS ODN) magnetofection, where AS ODNs coupled to nanoparticles are rapidly delivered to cells upon application of an external magnetic field, we investigated the role of Shp-2 in endothelial cell proliferation, survival and capillary formation. Data are presented as means ± SEM. Students t-test was used for analysis and results were considered significant when p<0,05. AS-ODN magnetofection against Shp-2 in human microvascular- (HMEC) or umbilical vein endothelial cells (HUVEC) led to a virtually complete knock-down of Shp-2 protein as assessed by western blotting. Basal proliferation of HMECs, as measured by MTT reduction, was inhibited to 41% (±5%, p<0.05, n=12) in Shp-2 AS ODN treated cells as compared to nonsense oligonucleotide (NS ODN) treated cells. In addition, bFGF (10ng/ml) dependent proliferation following Shp-2 knock-down was impeded to a similar extent (by 57±13%; p<0.05, n=12). This decrease appeared to be due to an enhanced apoptosis, since cell cycle analysis by flow cytometric propidium iodide and subsequent Annexin V staining revealed a 2.4 fold (±0,5; p<0.05, n=6) increase in cells detected in the subG0 fraction and a significant rise in Annexin V positive cells (23±7%, p<0,01, n=9) following Shp-2 AS ODN transfection compared to NS ODN treated cells. Shp-2 knock-down was also associated with a decreased phosphorylation of the PI3-Kinase regulatory subunit p85 (n=3) and its downstream target Akt (n=4). A diminished phosphorylation of the MAP Kinase ERK 42/44 was observed despite bFGF stimulation (n=4) as well, but not after a control stimulus (Phorbol-myristate-acetate). Finally, endothelial cells embedded in a proangiogenic matrix formed a reduced number of capillary like structures upon application of a Shp-2 inhibitor. Our results indicate that Shp-2 protects human endothelial cells from apoptosis and favours proliferation by affecting bFGF dependent activation of PI3-K and/or MAP kinase. Moreover, Shp-2 seems to be necessary for the formation of capillary like structures. These observations suggest Shp-2 as a key enzyme in the control of angiogenesis.

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