Secretoneurin gene-therapy and therapeutic angiogenesis

Therapeutic angiogenesis, the exogenously induced growth of new blood vessels in order to enhance collateral development after arterial obstruction, represents a promising therapy for patients with coronary heart disease and peripheral artery disease after failure of other therapeutic options. The most promising molecules which mediate this angiogenic response are vascular endothelial growth factor (VEGF) and members of the fibroblast growth factor (FGF) family of molecules. VEGF however, just showed modest effects in large randomized clinical trials whereas gene therapy with FGF-1 reduced amputation rate in patients with severe peripheral artery disease. We could recently show, that the neuropeptide secretoneurin (SN), which exerts effects on vascular cells and is located in neurons around blood vessels, induces angiogenesis in vivo and in vitro and exerts proliferative and anti- apoptotic effects on endothelial cells. Furthermore, SN also induces postnatal vasculogenesis, the growth of new blood vessels mediated by endothelial progenitor cells. We also could demonstrate that SN expression is up regulated by hypoxia specifically in skeletal muscle cells, indicating a role of this peptide in ischemic tissue repair. We also were able to generate a SN plasmid gene therapy vector, characterized the recombinant SN molecule and were able to show beneficial effects of SN gene therapy in the mouse hindlimb ischemia model, a commonly used animal model of angiogenesis in first experiments. In the current project we would like to follow up our data on SN plasmid gene therapy and its effects on tissue perfusion, clinical outcome and functional recovery in the hindlimb ischemia model of the mouse. The project will contain 2 specific aims: 1. Secretoneurin Plasmid Gene Therapy in the Hindlimb Ischemia Model in Diabetic, Hyperlipidemic and Old Mice 2. Effect of Secretoneurin gene therapy on blood vessel growth mediated by endothelial progenitor cells (Vasculogenesis) in the Hindlimb Ischemia Model In the first specific aim we will evaluate the effect of SN gene therapy in the hindlimb ischemia model with intra- muscular plasmid application in mice at high vascular risk, like diabetic, old or hyperlipidemic mice. In the second specific point we would like to investigate the effects of SN gene therapy on vasculogenesis mediated by endothelial progenitor cells in the hindlimb ischemia model. We are planning to finish this project within 24 months.

Peripheral arterial disease represents a common disease and eventually might lead to critical limb ischemia (CLI) which is characterized by a bad prognosis for the life of the patient and for the fate of the limb. A significant proportion of CLI patients have no option of revascularization and this group of patients would profit from novel therapeutic approaches like therapeutic angiogenesis. Angiogenesis represents the growth of new blood vessels due to action of several cytokines known to moderate this effect. Vascular endothelial growth factor and fibroblast growth factor (FGF) are the best characterized angiogenic cytokines. We recently described a novel angiogenic factor, the neuropeptide secretoneurin (SN) and tested the hypothesis that gene therapy with this factor improves outcome in the hindlimb ischemia model in mice with vascular risk factors like aged mice, hypercholesterolemic and diabetic mice. After induction of hindlimb ischemia in aged mice SN gene therapy improved blood perfusion as measured by laser Doppler perfusion imaging and reduced necrosis of toes and limbs. When muscles were analyzed for density of blood vessels SN gene therapy induced significant increase of capillaries and arteries indicating induction of angiogenesis and arteriogenesis by this factor. When we investigated the effect of SN gene therapy on endothelial progenitor cells we observed increase of these cells in ischemic muscles by confocal microscopy and FACS analysis. We further observed that beneficial effects of SN were dependent on nitric oxide and that SN upregulated other angiogenic cytokines like FGF or platelet-derived growth factor in endothelial cells. The results of this work were published recently in Circulation Research (Schgoer et al., 2009). We also investigated the effect of SN gene therapy in the hindlimb ischemia model in mice with diabetes and hypercholesterolemia, other vascular risk factors. We found that SN gene therapy improved blood perfusion and reduced necrosis of toes and limbs also in these mice. Additionally density of capillaries and arteries were increased by SN gene therapy. In-vitro SN inhibited apoptosis of endothelial cells induced by high glucose exposure. In summary, our data indicate that SN gene therapy improves blood perfusion and clinical outcome in the hindlimb ischemia model of critical limb ischemia by induction of angiogenesis, arteriogenesis and vasculogenesis. Future studies will determine potential mechanisms to improve transfection efficacy of SN gene therapy vectors to apply this therapy in patients suffering from this disease.