Effects of gene delivery on collateral development in chronic hypoperfusion
Diverse effects of angiopoietin-1 versus vascular endothelial growth factor
Yi Fu Zhou, MD*,
Eugenio Stabile, MD,
Jill Walker, MS,
Matie Shou, MD,
Richard Baffour, PhD,
Zuxi Yu, MD, PhD,
David Rott, MD,
George D. Yancopoulos, MD,
John S. Rudge, PhD and
Stephen E. Epstein, MD
Vascular Biology Laboratory, Cardiovascular Research Institute, MedStar Research Institute, Washington Hospital Center, Washington, DC, USA
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Figure 1 (a) Flow changes after ligation of the caudal and central arteries of the rabbit ear: flow recovery stabilizes after three weeks and is maintained at <70% of control for at least two months. (b) Western blot showing increased vascular endothelial growth factor (VEGF) expression in hypoperfused tissues two months after ligation. (c) Western blot showing angiopoietin-1 (Ang-1) expression in tissues where adenovirus-encoding Ang-1 gene (Ad.Ang-1) was injected.
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Figure 2 (a) Laser Doppler images eight weeks after ligation, before recombinant vector injection, and at indicated times after injection. In angiopoietin-1 (Ang-1)-treated animals, progressive recovery of perfusion occurs. After adenovirus-encoding vascular endothelial growth factor gene (Ad-VEGF) injection, a significant flow increase peaks at seven days and then gradually falls to below baseline by four weeks. (b) Effects of adenovirus-encoding Ang-1 gene (Ad-Ang-1), Ad-VEGF, and control adenovirus without transgene insert (Ad.Null) on tissue perfusion. The respective adenovirus vectors were injected intradermally eight weeks after ligation into both left and right ears of eight animals in each group. The flux index measured after injection was expressed as the percentage of the flux index immediately before injection. Values expressed as mean ± SD.
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Figure 3 (a) Effects on perfusion of adenovirus-encoding vascular endothelial growth factor gene (Ad.VEGF) or adenovirus-encoding angiopoietin-1 gene (Ad.Ang-1) injection in normal nonischemic ears; Ad.Ang-1 does not increase perfusion, while VEGF transiently increases flow, compatible with an inflammatory response. (b) The Ang-1-mediated increase in flow in ischemic ears is abolished when Ad.Ang-1 is injected concomitantly with adenovirus-encoding soluble VEGF receptor gene [Ad.Flt(1-3)-Fc], which encodes a soluble VEGF receptor that binds to and abolishes VEGF signaling. Ad.Null = control adenovirus without transgene insert.
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Figure 4 (a) Evens Blue (EB) concentration of rabbit ears 3 to 14 days after adenovirus-encoding angiopoietin-1 gene (Ad.Ang-1) or adenovirus-encoding vascular endothelial growth factor gene (Ad.VEGF). Because EB binds to albumin in vivo, leakage of this protein into the extravascular space is a measure of vascular permeability. Values recorded as EB: ng/mg tissue. Vascular endothelial growth factor induces vascular permeability peaking at seven days; (b) EM Section from Ad.VEGF-injected animals: left shows a small "gap" between endothelial cells (arrow); Right shows increased cytoplasmic vesicles (arrows). These were not found in Ang-1- or control adenovirus without transgene insert (Ad.Null)-treated animals. (c) Photographic images of rabbit ears seven days after injection of Ad.Ang-1 or Ad.VEGF showing bluish discoloration in VEGF-treated animals (top panel), and corresponding laser Doppler imaging (lower panel). Tissue perfusion increased in both; however, the vessel images of the VEGF-injected Ang-1-injected ears differed markedly (see text for details). (d) Bar graph showing mononuclear cell infiltration in the subcutaneous tissue after Ad.Ang-1, Ad.VEGF, or Ad.Null injection. Data are expressed as cells per high-power field (mean ± SD).
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