Vascular Regeneration in Ischemic Hindlimb by Adeno-Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor.

BACKGROUND: Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno-associated virus (AAV) and lentivirus have been tested in preclinical models. METHODS AND RESULTS: We present preclinical results of ischemia (hypoxia)-regulated conditionally silenced (CS) AAV-human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV-CS-hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra-arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV-CS-hVEGF. AAV-CS-hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch, SDF1, Angiopoietin, and Ephrin-B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. CONCLUSIONS: Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.