Top of pageAbstract AAV vectors, allowing long term transgene expression in the absence of inflammation or immune response, represent a unique tool to elucidate gene function in vivo. By the use of this delivery system, we have developed an experimental model to investigate the molecular and cellular mechanisms leading to new blood vessel formation. Here we exploited the properties of AAV vectors to explore the interplay between Vascular Endothelial Growth Factor-A (VEGF) and Semaphorin 3A (Sema3A), two factors which are involved in the angiogenic process. VEGF is the main inducer of endothelial cell proliferation and migration during the early stages of angiogenesis and vasculogenesis; the coordinated action of several other cytokines is then required for the development of mature and functional vascular structures. Recent evidence indicates that Sema3A, originally discovered as a neuronal guidance mediator, might be also implicated in angiogenesis, since its main receptor Neuropilin-1 (NP-1) has been shown to represent a co-receptor for VEGF on endothelial and hematopoietic stem cells. Moreover, axon repulsion by Sema3A also requires the expression of the VEGF receptor-1 (Flt-1), suggesting that VEGF and Sema3A might use a series of shared receptors and thus cooperatively modulate the angiogenic process. Two AAV vectors expressing VEGF and Sema3A were thus injected, either alone or in combination, in the tibialis anterior muscle of mice. Morphological and functional evaluation was followed over time. At the earlier time points, muscles injected with AAV-VEGF were massively infiltrated by proliferating cells, which eventually led to the formation of an impressive set of new capillaries and small arteriolae. These findings correlated with an increased vascular volume within the injected areas, as well as with increased vascular leakiness. The appearance of new blood vessels and vascular lacunae was already detectable at 14 days post injection, but significantly increased over time and persisted up to 8 months, with a parallel reduction in the entity of the cellular infiltrate. Co-expression of VEGF and Sema3A resulted in a striking increase in the cellular infiltration of the injected muscles, whereas few structured vessels were still evident; in contrast the resident endothelial cells appeared to form small aggregates, suggesting the presence of a functional impairment during the early phase of angiogenesis. Strikingly, AAV-Sema3A alone also determined the appearance of a cellular infiltrate very similar to the one observed in VEGF-treated muscles, but did not increase the number of vessels. By the use of bone marrow transplantation and immunofluoresence experiments we showed that the cells infiltrating the muscles expressing either VEGF or Sema3A derive from the bone marrow and express a set of markers specific for progenitor and myeloid cells (including c-kit, VEGFR-2, Tie-2 and CD11b), suggesting a possible role for both factors in the local recruitment of bone marrow-derived progenitor cells, which in turn might have a relevant role during the angiogenic process.