Expression of proteolitic enzymes in induced angiogenesis

Angiogenesis (growth of new blood vessels) plays a key role in tissue repair, such as in cancer progression (Folkman 1995. New Engl. J. Med. 333, 1757-63). At the beginning of the process is observed the matrix degradation and endothelial cells (ECs) migration inside to the connective tissue in proximity of vessel walls. During angiogenesis several modifications occurs at plasma membrane level; a redistribution of cell-cell and cell-matrix adhesion molecules (Bazzoni et al 1999), such as an increased expression of several proteolytic enzymes, including MMPs and serine proteases, as plasminogen activators and SIMPs, were observed (Ghersi et al. 2006. Cancer Res. 66, 4652-61; Ghersi 2008 Front Biosci. 13, 2335-55). Angiogenesis “in vivo” is established by ephitelial-mesenchymal transition of endothelial cells; a similar phenotypic exchange can be induced “in vitro” by growing ECs to low density or by perturbing cell-cell contacts. Cell-cell contacts are mediated by several molecules, enclose: CD31/PECAM-1, CD144/VE-cadherins, N-cadherins and β1-integrin adhesion molecules. These molecules take contacts with actin cytoskeletal components to stabilize epithelial phenotype through specific cytoplasmatic mediators. In particular a cytoplasmic mediator of cadherin/cytoskeletal interaction is the β-catenin, that apart this function can acts as transcription factor in association with TCF/LEF members family when cell-cell contacts are not stable. During angiogenesis cadherin-cadherin contacts are modified and endothelial cells acquired mesenchymal phenotype; in this condition β-catenin dissociate from cadherin cytoplasmic tail and can acquire the capability to translocate, into the nucleus working as genes’ activator factor. Our experiments were focalised on the different expression/activation of angiogenic proteolytic enzymes when cell-cell contacts were perturbed in endothelial cells mechanically, in wound hedge system, or site specific, using antibodies against specific cadherin domains. We have investigated in this direction using different approaches: 1- Computational, on the 50.000 bp up-stream non translated sequences of several MMPs and SIMPs genes about the presence of just identify β-catenin/TCF-4 binding sequences hTBE-1 and hTBE-2, that was demonstrated to be MMP-7’s transcription regulation sequences (Li et al. 2005. World J.Gastroenterol. 11, 2117-23); 2- Biochemical, on different proteolytic enzymes mRNA (by rt-PCR), proteins and enzymatic activities (by immunoblotting and gelatin zymography) expression when cell-cell contacts were perturbed mechanically or by addition of IgGs and/or Fabs recognizing cadherins extracellular (Cac125) and cytoplasm (Pan-cadherin) domains; Preliminary results by computational approach, demonstrated that: the transcription binding sequence analysed hTBE-1 and hTBE-2 were present in all analysed non translated 50.000 bp sequences of thirteen proteolytic enzymes genes. Moreover, other sequences containing the “core” (CAAAG) specific for several transcription factors, with 1 or 2 mismatch in the “core” flanking sequences were also identified. The statistic z-score values determined for the hTBE-1 and hTBE-2 were 1,25 and 2,95 respectively. Suggesting the involvement of the hTBE-2 sequence as regulator of investigated enzyme genes. These results were also confirmed by direct biochemical/bimolecular experiments such as by experiments of specific competition.