An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice.
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.
Article: Engineered zinc finger protein-mediated VEGF-a activation restores deficient VEGF-a in sensory neurons in experimental diabetes.[show abstract] [hide abstract]
ABSTRACT: The objectives of the study were to evaluate retrograde axonal transport of vascular endothelial growth factor A (VEGF-A) protein to sensory neurons after intramuscular administration of an engineered zinc finger protein activator of endogenous VEGF-A (VZ+434) in an experimental model of diabetes, and to characterize the VEGF-A target neurons. We compared the expression of VEGF-A in lumbar (L)4/5 dorsal root ganglia (DRG) of control rats and VZ+434-treated and untreated streptozotocin (STZ)-induced diabetic rats. In addition, axonal transport of VEGF-A, activation of signal transduction pathways in the DRG, and mechanical sensitivity were assessed. VEGF-A immunoreactivity (IR) was detected in small- to medium-diameter neurons in DRG of control rats. Fewer VEGF-A-IR neurons were observed in DRG from STZ-induced diabetic rats; this decrease was confirmed and quantified by Western blotting. VZ+434 administration resulted in a significant increase in VEGF-A protein expression in ipsilateral DRG, 24 h after injection. VEGF-A was axonally transported to the DRG via the sciatic nerve. VZ+434 administration resulted in significant activation of AKT in the ipsilateral DRG by 48 h that was sustained for 1 week after injection. VZ+434 protected against mechanical allodynia 8 weeks after STZ injection. Intramuscular administration of VZ+434 increases VEGF-A protein levels in L4/5 DRG, correcting the deficit observed after induction of diabetes, and protects against mechanical allodynia. Elevated VEGF-A levels result from retrograde axonal transport and are associated with altered signal transduction, via the phosphatidylinositol 3'-kinase pathway. These data support a neuroprotective role for VEGF-A in the therapeutic actions of VZ+434 and suggest a mechanism by which VEGF-A exerts this activity.Diabetes 11/2009; 59(2):509-18. · 8.29 Impact Factor
Article: Gene expression profiling of peripheral blood mononuclear cells in the setting of peripheral arterial disease.[show abstract] [hide abstract]
ABSTRACT: Peripheral arterial disease (PAD) is a relatively common manifestation of systemic atherosclerosis that leads to progressive narrowing of the lumen of leg arteries. Circulating monocytes are in contact with the arterial wall and can serve as reporters of vascular pathology in the setting of PAD. We performed gene expression analysis of peripheral blood mononuclear cells (PBMC) in patients with PAD and controls without PAD to identify differentially regulated genes. PAD was defined as an ankle brachial index (ABI) ≤0.9 (n = 19) while age and gender matched controls had an ABI > 1.0 (n = 18). Microarray analysis was performed using Affymetrix HG-U133 plus 2.0 gene chips and analyzed using GeneSpring GX 11.0. Gene expression data was normalized using Robust Multichip Analysis (RMA) normalization method, differential expression was defined as a fold change ≥1.5, followed by unpaired Mann-Whitney test (P < 0.05) and correction for multiple testing by Benjamini and Hochberg False Discovery Rate. Meta-analysis of differentially expressed genes was performed using an integrated bioinformatics pipeline with tools for enrichment analysis using Gene Ontology (GO) terms, pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), molecular event enrichment using Reactome annotations and network analysis using Ingenuity Pathway Analysis suite. Extensive biocuration was also performed to understand the functional context of genes. We identified 87 genes differentially expressed in the setting of PAD; 40 genes were upregulated and 47 genes were downregulated. We employed an integrated bioinformatics pipeline coupled with literature curation to characterize the functional coherence of differentially regulated genes. Notably, upregulated genes mediate immune response, inflammation, apoptosis, stress response, phosphorylation, hemostasis, platelet activation and platelet aggregation. Downregulated genes included several genes from the zinc finger family that are involved in transcriptional regulation. These results provide insights into molecular mechanisms relevant to the pathophysiology of PAD.Journal of clinical bioinformatics. 03/2012; 2:6.