How to achieve functional therapeutic angiogenesis: lessons from in vivo gene transfer using AAV vectors

The work described in this thesis has been m.ainly focused on the dissection of some of the fundamental mechanisms underlining new blood vessel formation in vivo. More precisely, a gene transfer approach, based on Adena-Associated Virus (AAV) vectors, has been exploited for the sustained and prolonged expression of several factors involved in angiogenesis and arteriogenesis. Bearing in mind the key role of Vascular Endothelial Growth Factor (VEGF) in the angiogenic process, we decided to thoroughly investigate the morphology and the functional performance of the VEGF-induced vasculature, by taking advantage of molecular imaging techniques, such as PET and SPECT, which allowed a non invasive assessment of vascular function over time. Starting from the observation that VEGF single gene transfer is not sufficient to drive the formation of functional blood vessels, we showed that the simultaneous delivery of Angiopoietin-1 (Angl) allowed an improved remodeling and maturation of the newly formed vasculature. This finding is of particular interest in a clinical perspective, suggesting that the delivery of a proper combination of growth factors, which is feasible and particularly straightforward with the use of AAV vectors, is probably required to fulfil! the goal of therapeutic angiogenesis. By studying the in viva effect of VEGF, particular emphasis was attributed to the distinct biological effect specifically exerted by the two main VEGF isoforms, composed of 121 and 165 aminoacids, respectively. Since the main structural difference between the two isoforms consists of their diverse ability to bind the coreceptor NP-1, we also assessed the effects induced by the overexpression of Semaphorin 3A (Sema3A), a well known NP-1 ligand in the developing nervous system. Collectively, these data provided convincing evidence of a massive recruitment of mononuclear cells from the bone marrow to the sites of neoangiogensis through NP-1. Although these cells did not seem to directly participate to new blood vessel formation through transdlfferentiation, they turned up to have a key role in promoting arteriogenesis. A schematic representation of the molecules studied in this work, and their ability to bind to the relative receptors, is shown in Figure 1.1.