Angiogenesis is required for the expansion of primary tumor mass, and new blood vessels penetrating the tumor are frequent sites for tumor cell entry into the circulation. In this complex process are involved cytokines and growth factors, proteinases of endothelial origin and integrins which act as receptors for the extracellular matrix. We recently demonstrated that tumor necrosis factor-alpha (TNF)-induced angiogenesis depends on in situ platelet activating factor (PAF) biosynthesis. PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation, embryogenesis and cell differentiation. PAF is considered a mediator of cell to cell communication which may function both as intercellular and intracellular messenger. PAF is produced after appropriate stimulation by a variety of cells that may participate to the development of inflammatory reaction such as monocytes/macrophages, polymorphonuclear neutrophils, basophils platelets, and the neoplastic cells. In addition, human endothelial cells (EC) were also found to produce PAF.

We recently demonstrated that PAF has a direct angiogenic effect in vivo and stimulates in vitro the migration of EC but not their proliferation.

The in vivo angiogenic effect of PAF is modulated in autocrine manner by interleukin-8 (IL-8). Moreover, PAF stimulates the endothelial synthesis of t-PA and the production of plasmin, which are known to be produced in vivo during angiogenesis triggered by TNF, bFGF, HGF. It was suggested that endothelial-derived proteinases play a role in the migration of EC in the extracellular matrix. Recently, we found that t-PA and plasmin stimulate the synthesis of PAF by EC. Moreover, other EC-derived proteinases such as metalloproteinases (gelatinase, collagenase and stromelysin) as well as the metalloproteinase-inhibitors (TIMP-1 and 2) can be instrumental in the migration of EC. An imbalance between proteinase activity and inhibitors may occurs during angiogenesis. Moreover, cell interaction with matrix via integrins may modulate both migration and proliferation of EC.

The aim of the present project is to evaluate in vitro and in vivo:

1) the relative contribution of PAF, t-PA, plasmin, metalloproteinases, integrins and cytokines in tumoral angiogenesis;

2) the possibility to interfere with the angiogenic process with PAF receptor antagonist, plasmin and metalloproteinase inhibitors and neutralizing antibodies to t-PA, integrins and several cytokines.

3) the ability of normal breast epithelial cells and mammary carcinoma to produce PAF, plasmin, metalloproteinases and inhibitors of metalloproteinases. The production of these factors will be correlated with that of cytokines (IL-8, Il-6, TNF, TGF-beta) and with the ability of cell supernatants to stimulate the migration of EC.

In vivo, the angiogenic properties of normal breast epithelial cells and of cells of mammary carcinoma in relation to the pattern of cytokine-, plasmin-, metalloproteinase- and PAF-production will be studied and the inhibitory effect of PAF receptor antagonists, alpha 2-antiplasmin, metalloproteinase-inhibitors and neutralizing antibodies to t-PA and selected cytokines will be tested.

In conclusion, these studies may contribute to better understand an important physiological phenomenon, the neoangiogenesis, which is critical for several pathological events, such as tumor growth and metastatization, and inflammation.