Published Online
on October 11, 2004

A more recent version of this article appeared on October 19, 2004

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Submitted on April 6, 2004
Revised on August 13, 2004
Accepted on August 17, 2004

Engineered Zinc Finger-Activating Vascular Endothelial Growth Factor Transcription Factor Plasmid DNA Induces Therapeutic Angiogenesis in Rabbits With Hindlimb Ischemia

Qunsheng Dai MD, Jianhua Huang MD, Bruce Klitzman PhD, Chunming Dong MD, Pascal J. Goldschmidt-Clermont MD, PhD, Keith L. March MD, PhD, Joseph Rokovich PhD, Brian Johnstone PhD, Edward J. Rebar PhD, S. Kaye Spratt PhD, Casey C. Case PhD, Christopher D. Kontos MD, and Brian H. Annex MD^*

From the Division of Cardiology (Q.D., J.H., C.D., P.J.G.-C., C.D.K., B.H.A.), Department of Medicine, Durham VA and Duke University Medical Centers, Durham, NC; the Division of Plastic Surgery (B.K.), Department of Surgery, Duke University Medical Center, Durham, NC; the Division of Cardiology (K.L.M.), Department of Medicine, University of Indiana, Indianapolis; Edwards Lifesciences LLC (J.R.), Irvine, Calif; and Sangamo BioSciences, Inc (B.J., E.J.R., S.K.S., C.C.C.), Point Richmond, Calif.

^* To whom correspondence should be addressed. E-mail: annex001{at}mc.duke.edu.

Background--Therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) exists in multiple isoforms. We investigated an engineered zinc finger-containing transcription factor plasmid designed to activate the endogenous VEGF gene (ZFP-VEGF).

Methods and Results--New Zealand White rabbits (n=56) underwent unilateral femoral artery ligation and excision. At day 10 postoperatively, the ischemic muscle received ZFP treatment (500 µg ZFP-VEGF plasmid) or no ZFP treatment (-galactosidase, empty, or no plasmid). Group 1 (n=13) was harvested 3 days after injection to examine VEGF mRNA by real-time polymerase chain reaction and protein by ELISA. Groups 2 (n=13) and 3 (n=10) were harvested 11 days after injection. Group 2 was studied by histology and group 3, by histology and changes in blood flow. Groups 4 and 5 (n=10 each) were harvested 22 and 32 days after injection, respectively, and studied for changes in blood flow. In group 1, VEGF mRNA copy numbers were significantly higher for VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and protein in the ZFP-VEGF-treatment versus no-ZFP-treatment arms. In groups 2 and 3, capillary density and proliferating cells were significantly greater and apoptosis significantly lower in the treatment versus no-treatment arms. Changes in the blood flow ratio of the ischemic to the nonischemic limb were significantly greater in the treatment versus no-ZFP-treatment groups (6.57±1.52% versus 3.38±0.87%, P<0.005; 13.15±1.77% versus 6.13±1.55%, P<0.001; and 20.16±2.84% versus 13.88±3.14%, P<0.01, for groups 3, 4, and 5, respectively).

Conclusions--This engineered ZFP-VEGF-activating transcription factor may provide a novel approach to treat peripheral arterial disease.

Key words: muscle • angiogenesis • growth substances • apoptosis • endothelium-derived factors

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