VEGF(165) EXPRESSED BY A REPLICATION-DEFICIENT RECOMBINANT ADENOVIRUSVECTOR INDUCES ANGIOGENESIS IN-VIVO

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of isc hemic disorders, the replication-deficient adenovirus (Ad) vector AdCM V.VEGF(165) (where CMV is cytomegalovirus and VEGF is vascular endothe lial growth factor) containing the cDNA for human VEGF(165) secreted e ndothelial cell-specific angiogenic growth factor, was constructed. Hu man umbilical vein endothelial cells (HUVECs) and rat aorta smooth mus cle cells (RASMCs) infected with AdCMV.VEGF(165) (5 and 20 plaque-form ing units [pfu] per cell) demonstrated VEGF mRNA expression and protei n secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs a t 14 days demonstrated sixfold more cells for AdCMV. VEGF(165)-infecte d HUVECs (20 pfu per cell) than for either infected or uninfected cont rol cells. RASMC proliferation was unaffected by infection with AdCMV. VEGF(165). When plated in 2% serum on dishes precoated with reconstitu ted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF(165) (20 pfu per cell) differentiated into capillary-like structures. Unde r similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV .VEGF(165) to function in vivo, either AdCMV. VEGF(165) or AdCMV.beta gal (2x10(10) pfu) was resuspended in 0.5 mt Matrigel and injected sub cutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF, u p to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histo logical evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF(165) whereas no significant angiogenesi s was observed in response to AdCMV.beta gal. Furthermore, the Matrige l plugs with AdCMV.VEGF(165) demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro an d in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF(165) in the treatment of ischemic diseases.