Abstract 17622: Reversible Activation of Notch Signaling is Required for Arterial Regeneration of Hindlimb by Hypoxia-regulated AAV Gene Therapy
Background: Notch signaling downstream of VEGF is required to produce an organized vasculature during development, growth and following ischemic injury. The extent to which Notch intermediates are activated in the setting of therapeutic angiogenesis by pro-angiogenic stimuli is not known.
Hypothesis: Appropriate Notch regulation requires VEGF gradients and is achieved only when VEGF expression is confined to ischemia. Absence of precise Notch signaling may account for the clinical failure of therapeutic angiogenesis in trials such as TAMARIS where the delivery of unregulated gene vectors confers unrestricted gene expression with no gradients.
Methods: A Balb/C mouse model of critical limb ischemia was used to assess therapy and Notch signaling driven by unregulated (CMV) or hypoxia-regulated (HRE) human (h)VEGF gene delivery by intramuscular plasmid or AAV vectors. Limb perfusion was assessed by laser Doppler, DiI staining and immunohistology. Notch pathway intermediates were quantified by RT-PCR at intervals up to 4 weeks after gene therapy.
Results: Limb salvage and femoral regeneration were achieved only using HRE-vectors with AAV conferring 100% tissue salvage (p<0.01; n=10). hVEGF expression from HRE-vectors was extinguished within 2 weeks, coincident with tissue reperfusion (p<0.05; n=6). Notch pathway intermediates including Dll4, Hey2, Jag-1, and Notch-1 showed exquisite regulation by HRE-gene therapy with peak D-7 expression respectively of 8.5±1.5-fold; 3.5±0.6-fold, 6±1.8-fold and 12±2.5-fold relative to control no-surgery (all p<0.05; n=4). In each case expression declined almost to baseline (D-1) at 4 weeks. Similar trends were seen for angiogenic/vasculogenic intermediates SDF-1, Angiopoietin-1 and Ephrin-B2. Notch intermediates were not regulated in limbs treated with PBS or CMV-VEGF gene therapy but rather continued to increase during the time course or peaked and remained elevated. Re-arterialization and limb salvage by HRE-gene therapy were blocked by the selective γ-secretase inhibitor dibenzazepine confirming the essential role for Notch.
Conclusions: Gene therapy with VEGF under tight hypoxia-regulation recapitulates a fundamental angiogenesis/arteriogenesis program with essential Notch signaling.
- © 2013 by American Heart Association, Inc.