ABSTRACT
Advances in understanding the relationship between protein structure and DNA binding specificity have made it possible to engineer zinc finger protein (ZFP) transcription factors to specifically activate or repress virtually any gene. To evaluate the potential clinical utility of this approach for peripheral vascular disease, we investigated the ability of an engineered vascular endothelial growth factor (VEGFa)-activating ZFP (MVZ+426b) to induce angiogenesis and rescue hindlimb ischemia in a murine model. Hindlimb ischemia was surgically induced in advanced-age C57/BL6 mice. Adenovirus (Ad) encoding either MVZ+426b or the fluorescent marker dsRed was delivered to the adducter muscle of the ischemic hindlimb, and the effects on blood flow, limb salvage, and vascularization were assessed. Ad-MVZ+426b induced expression of VEGFa at the mRNA and protein levels and stimulated a significant increase in vessel counts in the ischemic limb. This was accompanied by significantly increased blood flow and limb salvage as measured serially for 4 wk. These data demonstrate that activation of the endogenous VEGFa gene by an engineered ZFP can induce angiogenesis in a clinically relevant model and further document the feasibility of designing ZFPs to therapeutically regulate gene expression in vivo.
Subject(s)
Gene Expression Regulation/genetics , Genetic Therapy , Genetic Vectors/therapeutic use , Ischemia/therapy , Neovascularization, Physiologic/genetics , Transcription Factors/physiology , Vascular Endothelial Growth Factor A/biosynthesis , Zinc Fingers/physiology , Adenoviridae/genetics , Aging , Amino Acid Sequence , Animals , Blood Flow Velocity , Feasibility Studies , Genes, Synthetic , Hindlimb/blood supply , Laser-Doppler Flowmetry , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Protein Engineering , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Proteins , Structure-Activity Relationship , Transcription Factors/genetics , Vascular Endothelial Growth Factor A/genetics , Zinc Fingers/geneticsABSTRACT
At a resting pulse rate the heart consumes almost twice-as much oxygen per gram tissue as the brain and more than 43 times more than resting skeletal muscle (1). Unlike skeletal muscle, cardiac muscle cannot sustain anaerobic metabolism. Balancing oxygen demand with availability is crucial to cardiac function and survival, and regulated gene expression is a critical element of maintaining this balance. We investigated the role of the hypoxia-inducible transcription factor HIF-1alpha in maintaining this balance under normoxic conditions. Cardiac myocyte-specific HIF-1alpha gene deletion in the hearts of genetically engineered mice caused reductions in contractility, vascularization, high-energy phosphate content, and lactate production. This was accompanied by altered calcium flux and altered expression of genes involved in calcium handling, angiogenesis, and glucose metabolism. These findings support a central role for HIF-1alpha in coordinating energy availability and utilization in the heart and have implications for disease states in which cardiac oxygen delivery is impaired. Heart muscle requires a constant supply of oxygen. When oxygen supply does not match myocardial demand cardiac contractile dysfunction occurs, and prolongation of this mismatch leads to apoptosis and necrosis. Coordination of oxygen supply and myocardial demand involves immediate adaptations, such as coronary vasodilatation, and longer-term adaptations that include altered patterns of gene expression (2-4). How the expression of multiple genes is coordinated with oxygen availability in the heart and the impact of oxygen-dependent gene expression on cardiac function are insufficiently understood. Further elucidating these relationships may help clarify the molecular pathology of various cardiovascular disease states, including ischemic cardiomyopathy and myocardial hibernation (5, 6).
Subject(s)
Calcium Signaling/physiology , Coronary Circulation/physiology , DNA-Binding Proteins/physiology , Myocardial Contraction/physiology , Myocardium/metabolism , Myocytes, Cardiac/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Energy Metabolism , Gene Deletion , Gene Expression Regulation/physiology , Heart Function Tests , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic/physiology , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Oxygen Consumption , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription, GeneticABSTRACT
Stimulating new blood vessel growth in ischemic hearts or limbs is a hopeful new approach for patients with advanced vascular disease. This approach is based generally upon the hypothesis that sufficient exposure of a vascular bed to an angiogenic protein will stimulate neovascularization. Most angiogenic proteins have a markedly short serum half-life. To overcome this, researchers have turned to gene therapy to ensure continuous expression of angiogenic proteins and prolonged exposure in the targeted vascular beds. This field is still evolving, and although early clinical trial results suggest angiogenic gene therapy can be successful, many questions remain. As we continue to learn more about the complex interplay and coordinated action of the various factors involved in regulating angiogenesis, it is likely that strategies for therapeutic angiogenesis will continue to change. This review addresses the current state of angiogenic gene therapy, contrasts gene therapy with angiogenic protein delivery, describes early and recent clinical trial data, and discusses potential new directions in the field.
