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Ann. N.Y. Acad. Sci., Annals PrePrint, published online ahead of print May 10, 2007 doi: 10.1196/annals.1415.019 Copyright © 2007 by the New York Academy of Sciences description
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1 Biophysics, Medical University, 6/8 Mazowiecka Street, Lodz, 92-215, Poland 2 Molecular and Medical Biophysics, Medical University in Lodz, Lodz, Lodz, Poland 3 Cellular Proteomics, Center of Medical Biology, Lodz, Lodz, Poland 4 Medical and Molecular Biophysics, Medical University in Lodz, Lodz, Lodz, Poland
* To whom correspondence should be addressed. E-mail: cciern{at}zdn.am.lodz.pl. PrePrint Abstract
Recent data show that formation of new vessels may be stimulated by thymosin beta4 (TB4), but it is still not clear whether TB4 alone is angiogenic or the angiogenic potential of TB4 is mediated by VEGF. In this report to further characterize TB4 angiogenic activity, we produced its mutants that were deprived of the N-terminal tetrapeptide AcSDKP (TB4(AcSDKPT/4A)), the actin binding sequence KLKKTET (TB4(KLKKTET/7A)) and with the nuclear localization sequence damaged by a point mutation Lys16Ala (TB4(K16A)). Then we tested their activity to induce expression and release of MMPs as well as plasminogen activators inhibitor type-1 (PAI-1). We also analyzed their effect on migration and proliferation of endothelial cells in three-dimensional fibrin matrix as well as on their ability to stimulate the outgrowth of human endothelial cells in capillary-like tubular structures. Our data demonstrate that increased intracellular expression of TB4 and its mutants is necessary and sufficient to induce PAI-1 gene expression in endothelial cells. Similarly, they stimulate expression and release of MMP-1, -2, and -3. As evaluated by using specific inhibitors to these MMPs, they modified specifically the structure of fibrin and thus facilitated migration of endothelial cells. To sum up, our data show that the mechanism by which TB4 induced transition of endothelial cells from quiescent to proangiogenic phenotype characterized by increased expression of PAI-1 and MMPs did not require presence of the N-terminal sequence AcSDKP, and depended only partially on its ability to bind G actin or enter the nucleus. Key Words:
Thymosin ß4, Thymosin ß4 mutants, metaloproteinases, fibrin matrix, angiogenesis
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