VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo

Judith Mühlhauser, Marsha J. Merrill, Roberto Pili, Hiroyuki Maeda, Mima Bacic, Burkhard Bewig, Antonino Passaniti, Nancy A. Edwards, Ronald G. Crystal, Maurizio C. Capogrossi

Research output: Contribution to journalArticle

Abstract

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HU-VECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein 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.βgal (where βgal is β-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.βgal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.βgal (2×1010 pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immuno-histochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.βgal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.βgal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.βgal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.

Original languageEnglish (US)
Pages (from-to)1077-1086
Number of pages10
JournalCirculation Research
Volume77
Issue number6
StatePublished - Dec 1995
Externally publishedYes

Keywords

  • Angiogenesis
  • Endothelium
  • Gene therapy
  • Vascular permeability factor
  • VEGF

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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