An engineered vascular endothelial growth factor-activating transcription factor induces therapeutic angiogenesis in ApoE knockout mice with hindlimb ischemia

ObjectiveAngiogenesis is the growth and proliferation of blood vessels from existing vascular structures, and therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) is a prototypic angiogenic agent that exists in vivo in multiple isoforms, and studies with VEGF to date had used single isoform therapy with disappointing results. We tested plasmid and adenoviral vectors encoding a zinc-finger DNA-binding transcription factor (ZFP-32E) that was designed to increase the expression of all major VEGF isoforms in a preclinical model of peripheral arterial obstructive disease (PAOD) in hypercholesterolemic (ApoE knock-out) mice.MethodsUnilateral femoral artery ligation/excision was performed in 117 mice. At 7 days postoperatively, the ischemic tibialis anterior (TA) and gastrocnemius (GAS) muscles received either ZFP-32E treatment (125 μg of plasmid, 2.5 × 1011 viral particle units [vpu] of adenovirus; some mice received a second plasmid injection 3 days later) or no-ZFP treatment (125 μg of β-galactosidase [β-gal], a plasmid-lacking insert, or an equal dose of adenoviral encoding β-gal; some mice received a second plasmid injection 3 days later). Group 1 mice (n = 31) were euthanized 3 days later, and VEGF messenger RNA (mRNA) and protein levels were measured. Group 2 mice (n = 38) were euthanized 7 days later, and measures of capillary density, cell proliferation, and apoptosis were quantified. Group 3 mice (n = 48) were euthanized 28 days later, and changes in lower limb blood flow perfusion were measured.ResultsIn group 1, VEGF mRNA and protein levels were significantly higher in those with ZFP-32E treatment vs β-gal. In group 2, capillary density and proliferating cells were significantly greater and apoptosis was significantly lower in those with ZFP-32E treatment vs β-gal. Finally, in group 3, changes in the perfusion ratio (ischemic/nonischemic limb) at 21 days after injection were significantly greater in those with ZFP-32E treatment vs no-ZFP treatment.ConclusionThe ability of this engineered zinc-finger VEGF-activating transcription factor to induce therapeutic angiogenesis in hypercholesterolemic mice suggests this approach warrants investigation as a novel approach to treat PAOD.Clinical RelevanceTherapeutic angiogenesis is an investigational method that seeks to promote the growth of blood vessels for the treatment of disorders of inadequate tissue perfusion. Patients with peripheral arterial occlusive disease (PAOD) are a major target population. This study used a novel approach to increase endogenous expression of vascular endothelial growth factor in a preclinical model of PAOD, where the capacity to induce therapeutic angiogenesis is known to be very difficult. This approach demonstrated beneficial effects in this model and will serve as a valuable link to advance this and related approaches, toward human investigation