Use of vascular endothelial cell growth factor gene transfer to enhance implantable sensor function in vivo.
In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.
Klueh
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Center for Molecular Tissue Engineering, University of Connecticut, School of Medicine, Farmington 06030, USA.
Dorsky
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Kreutzer
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D000082:Acetaminophen / Q000378:metabolism; D018712:Analgesics, Non-Narcotic / Q000378:metabolism; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D002460:Cell Line; D048429:Cell Size; D002642:Chick Embryo; D005321:Extraembryonic Membranes / Q000098:blood supply / Q000166:cytology / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D020007:Implants, Experimental; D051379:Mice; D018919:Neovascularization, Physiologic; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D018712:Analgesics, Non-Narcotic; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; C413374:p27 antigen; D000082:Acetaminophen