Secretoneurin gene-therapy and diabetic neuropathy

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 (PAD) 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. Additionally, VEGF and other angiogenic cytokines have been reported to improve diabetic neuropathy in several animal models by increase of vascular blood supply of nerves though vasa nervorum, which is impaired in diabetic nerves. Additionally, VEGF was shown to directly exert effects on Schwann cells, cells important for peripheral nerve regeneration. Furthermore, VEGF gene therapy administered because of severe PAD also improved neuropathy in patients including diabetics. Currently the effect of VEGF gene therapy on diabetic neuropathy is investigated in a controlled, randomized trial. We recently could 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 post-natal vasculogenesis, the growth of new blood vessels mediated by endothelial progenitor cells. Recent data also revealed that SN expression is up- regulated by hypoxia specifically in skeletal muscle cells. We further were able to generate a SN plasmid gene therapy vector, characterized the recombinant SN molecule and data on the effect of SN gene therapy in the mouse hindlimb ischemia model indicate that SN gene therapy is capable of inducing therapeutic angiogenesis. SN was also reported to act on neuronal cells by inducing neurite outgrowth and preliminary data form our experiments suggest that SN inhibits apoptosis of Schwann cells. In the current project we would like to investigate the effects of SN on Schwann cells and to test the hypothesis that SN gene therapy improves diabetic neuropathy in animal models of type I and type II diabetes. The project will contain 2 specific aims: 1. To evaluate effects of SN on Schwann cells (proliferation, migration, apoptosis) 2. To test the hypothesis that SN gene therapy exerts beneficial effects on diabetic neuropathy in animal models of type I and II diabetes. Effects of SN gene therapy on neuro-physiological parameters of this neuropathy and the effects on nerve vasculature and blood perfusion will be studied. Additionally, possible underlying mechanisms like effects on neuronal nitric oxide synthase will be investigated. We are planning to finish this project within 24 months.

Diabetic neuropathy represents a severe clinical problem but the pathogenesis of this disease still remains enigmatic. Beside metabolic changes also defects in the microvascular supply of nerves might play are role in the development of this disease. Consistent with this hypothesis treatment with cytokines which induce the growth of new blood vessels (angiogenesis) like vascular endothelial growth factor leads to improvement of this neuropathy in animal models and in human clinical trials. We recently described a novel angiogenic cytokine, the neuropeptide secretoneurin and could show that gene therapy with this factor improves outcome in the hindlimb ischemia model. We tested the hypothesis that secretoneurin improves nerve function in an animal model of diabetic neuropathy. Db/db mice (a model of type 2 diabetes developing neuropathy) were treated by secretoneurin gene therapy. Before treatment diabetic mice revealed significant lower values for sciatic motor and sensory nerve conduction velocities compared to heterozygous mice consistent with neuropathy. After secretoneurin gene therapy but not after injection of control plasmids nerve conduction velocities significantly increased up to nine weeks after therapy indicating long lasting improvement of neuropathy. We also found that secretoneurin gene therapy improved thermal nociception as indicated by decreased temperature in the tailflick test. Injection of fluorescent lectin to label nerve vascularity showed reduced vasa nervorum in diabetic nerves and rescue by secretoneurin therapy. To study effects of secretoneurin on Schwann cells of peripheral nerves primary rat Schwann cells were isolated from the sciatic nerve, cultured and studied. Secretoneurin stimulated cell proliferation and inhibited apoptosis. In summary, our data show that secretoneurin gene therapy exerts beneficial effects in an animal model of diabetic neuropathy in-vivo and on Schwann cells in-vitro. Future studies have to show the exact mechanisms of secretoneurin action on nerve endothelial and Schwann cells.