In Mice With Type 2 Diabetes, a Vascular Endothelial Growth Factor (VEGF)-Activating Transcription Factor Modulates VEGF Signaling and Induces Therapeutic Angiogenesis After Hindlimb Ischemia

In Mice With Type 2 Diabetes, a Vascular Endothelial Growth Factor (VEGF)-Activating Transcription Factor Modulates VEGF Signaling and Induces Therapeutic Angiogenesis After Hindlimb Ischemia Yongjun Li , Surovi Hazarika , Donghua Xie , Anne M. Pippen , Christopher D. Kontos and Brian H. Annex From the Division of Cardiology, Department of Medicine, Durham VA and Duke University Medical Center, Durham, North Carolina Address correspondence and reprint requests to Brian H. Annex, MD, Division of Cardiology, Durham Veterans Affairs and Duke University Medical Center, 508 Fulton St., Box 111A, Durham, NC 27710. E-mail: annex001{at}mc.duke.edu Abstract Peripheral arterial disease is a major complication of diabetes. The ability to promote therapeutic angiogenesis may be limited in diabetes. Type 2 diabetes was induced by high-fat feeding C57BL/6 mice ( n = 60). Normal chow–fed mice ( n = 20) had no diabetes. Mice underwent unilateral femoral artery ligation and excision. A plasmid DNA encoded an engineered transcription factor designed to increase vascular endothelial growth factor expression (ZFP-VEGF). On day 10 after the operation, the ischemic limbs received 125 μg ZFP-VEGF plasmid or control. Mice were killed 3, 10, or 20 days after injection ( n = 10/group, at each time point). Limb blood flow was measured by laser Doppler perfusion imaging. VEGF mRNA expression was examined by real-time PCR. VEGF, Akt, and phospho-Akt protein were measured by enzyme-linked immunosorbent assay. Capillary density, proliferation, and apoptosis were assessed histologically. Compared with normal mice, mice with diabetes had greater VEGF protein, reduced phospho-Akt–to–Akt ratio before ligation, and an impaired perfusion recovery after ligation. At 3 and 10 days after injection, in mice with diabetes, gene transfer increased VEGF expression and signaling. At later time points, gene transfer resulted in better perfusion recovery. Gene transfer with ZFP-VEGF was able to promote therapeutic angiogenesis mice with type 2 diabetes. Cox-2, cyclooxygenase 2 eNOS, endothelial NO synthase iNOS, inducible NO synthase PAD, peripheral arterial disease PAOD, peripheral arterial obstructive disease TUNEL, transferase-mediated dUTP nick-end labeling VEGF, vascular endothelial growth factor VEGFR, VEGF receptor Footnotes B.H.A.’s laboratory has received educational grants from Edwards Life Sciences, and B.H.A. has received consulting fees from Edwards Life Sciences. The costs of publication