טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRoguin Ariel
SubjectMouse Hindlimb Chronic Ischemia Model and Genetic
Treatment Using Continuous Infusion of Different
VEGF Vectors
DepartmentDepartment of Medicine
Supervisor Professor Andrew Peter Levy


Abstract

 Background: The optimal vector, regulatory sequences and method of delivery of angiogenic gene therapy to promote collateral formation are a matter of considerable interest and debate. We set out to determine if continuous perimuscular administration of plasmid DNA for vascular endothelial growth factor (VEGF) containing its endogenous internal ribosome entry sites (IRES), compared to intra-muscular injections, could augment collateral formation and increase tissue perfusion in a normal and in a diabetic mouse unilateral ischemic hindlimb-model.

Methods: The left-iliac artery of C57-mice was ligated in two places and cut. An osmotic-infusion pump containing placebo or tested vector was implanted in the abdominal cavity with an outlet tube fenestrated in the quadriceps muscle. Ischemic/normal limb blood flow was serially measured by laser-Doppler. The ischemic and non-ischemic limbs were quantitatively analyzed for the expression of smooth muscle actin (SMA) and factor VIII-related antigen. Diabetes was induced with Streptozotocin.

Results:  In normal mice VEGF administration resulted in a significantly more rapid and complete restoration of blood flow as compared to placebo and was faster than intra-muscular. The vector without the IRES was intermediate between the placebo and the VEGF vector. None of the VEGF-treated group developed grossly visible necrosis versus 33% of the placebo group. Vessel density using SMA was higher in the proximal limb area in the VEGF groups compared to the placebo. Vessel density, using factor VIII was higher both proximally and distally in the VEGF groups. In contrast, in the setting of diabetes, VEGF had no effect in the rate or adequacy of flow restoration. However, there was a significant increase in vessel density, both in placebo and the VEGF treated arms.

Conclusion: Continuous perimuscular administration of angiogenic gene-therapy offers a new approach to restore blood flow to an ischemic limb and results in muscle salvage. Incorporation of an IRES-element may assist in the expression of transgenes delivered to ischemic tissues. However, angiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.