Figure 9.

Requirement of Notch signaling for limb salvage. Gene therapy and femoral artery excision were implemented as described in Methods. Mice were treated or not with (S)‐2‐[2‐(3,5‐difluoro‐phenyl)‐acetylamino]N‐((S)‐5‐methyl‐6‐oxo‐6,7‐dihydro‐5H‐dibenzo[b,d]azepin‐7‐yl)‐propionamide (DBZ; Axon Medchem BV) delivered via intraperitoneal injection (10 μg/kg) immediately after ischemia surgery and daily thereafter for 7 days. Then, 10¹¹ VP of adeno‐associated virus (AAV) was delivered intramuscularly to each treatment limb 5 days before surgery. A, Representative photographs of limbs 1 week after treatments with AAV9‐CS [conditionally silenced]‐hVEGF [human vascular endothelial growth factor]±DBZ. B, Doppler ratios confirm recovery of perfusion in the AAV9‐CS‐hVEGF treatment group without DBZ (squares) but not with DBZ treatment (circles). C, Composite DiI confocal images of the femoral tract reveal uncontrolled sprouting and vessel leakiness in DBZ‐treated mice. The magnified area highlights an area of vascular sprouting in a DBZ limb. Red dashed lines indicate the location of the femoral nerve; red arrows show suture locations. White and yellow arrows highlight differences in vascular morphology between treatment groups. *P<0.05; n=4 by Student t test comparing AAV treatment groups with or without DBZ at each time point. DBZ indicates dibenzazepine.