Intracerebroventricular infusion of secretoneurin inhibits neuronal NLRP3-Apoptosis pathway and preserves learning and memory after cerebral ischemia.

Transient global cerebral ischemia (GCI) results in delayed neuronal death, primarily apoptosis, in the hippocampal CA1 subregion, which leads to severe cognitive deficits. While therapeutic hypothermia is an approved treatment for patients following cardiac arrest, it is associated with various adverse effects. Secretoneurin (SN) is an evolutionarily conserved neuropeptide generated in the brain, adrenal medulla and other endocrine tissues. In this study, SN was infused into the rat brain by intracerebroventricular injection 1 day after GCI, and we demonstrated that SN could significantly preserve spatial learning and memory in the Barnes maze tasks examined on days 14-17 after GCI. To further investigate underlying pathways involved, we demonstrated that, on day 5 after GCI, SN could significantly inhibit GCI-induced expression levels of Apoptosis Inducing Factor (AIF) and cleaved-PARP1, as well as neuronal apoptosis and synaptic loss in the hippocampal CA1 region. Additionally, SN could attenuate GCI-induced activation of both caspase-1 and caspase-3, and the levels of pro-inflammatory cytokines IL-1ß and IL-18 in the CA1 region. Mechanically, we observed that treatment with SN effectively inhibited NLRP3 protein elevation and the bindings of NLRP3-ASC and ASC-caspase-1 in hippocampal neurons after GCI. In summary, our data indicate that SN could effectively attenuate NLRP3 inflammasome formation, as well as the activation of caspase-1 and -3, the production of pro-inflammatory cytokines, and ultimately the neuronal apoptotic loss induced by GCI. Potential neuronal pyroptosis, or caspase-1-dependent cell death, could also be involved in ischemic neuronal death, which needs further investigation.

Nanoparticular delivery system for a secretoneurin derivative induces angiogenesis in a hind limb ischemia model.

Common therapeutic strategies for peripheral arterial disease often fail to re-establish sufficient blood flow within legs and feet of patients for avoiding critical limb ischemia, what is characterized by a substantial risk for amputation. The neuropeptide secretoneurin induces angiogenesis in models of limb and myocardial ischemia and might be a promising tool in the treatment of patients without the option of revascularization therapy for severe ischemia. Within this manuscript, the biologically active part of secretoneurin was identified, modified by induction of a cysteine residue to gain higher stability against enzymatic degradation and further packed into S-protected thiolated chitosan nanoparticles, which enable intra-muscular application of secretoneurin. Secretoneurin nanoparticles restored blood flow in a mouse hind limb ischemia model within one week, whereas control particles did not. In vitro testing also revealed the angiogenic, antiapoptotic and proliferative effects of the new secretoneurin derivate, as tested in primary human umbilical vein endothelial cells. With the work from this study we provide a new promising tool for treatment of peripheral arterial disease.

A potential role for the secretogranin II-derived peptide EM66 in the hypothalamic regulation of feeding behaviour.

EM66 is a conserved 66-amino acid peptide derived from secretogranin II (SgII), a member of the granin protein family. EM66 is widely distributed in secretory granules of endocrine and neuroendocrine cells, as well as in hypothalamic neurones. Although EM66 is abundant in the hypothalamus, its physiological function remains to be determined. The present study aimed to investigate a possible involvement of EM66 in the hypothalamic regulation of feeding behaviour. We show that i.c.v. administration of EM66 induces a drastic dose-dependent inhibition of food intake in mice deprived of food for 18 hours, which is associated with an increase of hypothalamic pro-opiomelanocortin (POMC) and melanocortin-3 receptor mRNA levels and c-Fos immunoreactivity in the POMC neurones of the arcuate nucleus. By contrast, i.c.v. injection of EM66 does not alter the hypothalamic expression of neuropeptide Y (NPY), or that of its Y1 and Y5 receptors. A 3-month high-fat diet (HFD) leads to an important decrease of POMC and SgII mRNA levels in the hypothalamus, whereas NPY gene expression is not affected. Finally, we show that a 48 hours of fasting in HFD mice decreases the expression of POMC and SgII mRNA, which is not observed in mice fed a standard chow. Taken together, the present findings support the view that EM66 is a novel anorexigenic neuropeptide regulating hypothalamic feeding behaviour, at least in part, by activating the POMC neurones of the arcuate nucleus.

Manufacturing of a Secretoneurin Drug Delivery System with Self-Assembled Protamine Nanoparticles by Titration.

Since therapeutic peptides and oligonucleotides are gathering interests as active pharmaceutical ingredients (APIs), nanoparticulate drug delivery systems are becoming of great importance. Thereby, the possibility to design drug delivery systems according to the therapeutic needs of APIs enhances clinical implementation. Over the last years, the focus of our group was laid on protamine-oligonucleotide-nanoparticles (so called proticles), however, the possibility to modify the size, zeta potential or loading efficiencies was limited. Therefore, at the present study we integrated a stepwise addition of protamine (titration) into the formation process of proticles loaded with the angiogenic neuropeptide secretoneurin (SN). A particle size around 130 nm was determined when proticles were assembled by the commonly used protamine addition at once. Through application of the protamine titration process it was possible to modify and adjust the particle size between approx. 120 and 1200 nm (dependent on mass ratio) without influencing the SN loading capacity. Dynamic light scattering pointed out that the difference in particle size was most probably the result of a secondary aggregation. Initially-formed particles of early stages in the titration process aggregated towards bigger assemblies. Atomic-force-microscopy images also revealed differences in morphology along with different particle size. In contrast, the SN loading was only influenced by the applied mass ratio, where a slight saturation effect was observable. Up to 65% of deployed SN could be imbedded into the proticle matrix. An in-vivo biodistribution study (i.m.) showed a retarded distribution of SN from the site of injection after the application of a SN-proticle formulation. Further, it was demonstrated that SN loaded proticles can be successfully freeze-dried and resuspended afterwards. To conclude, the integration of the protamine titration process offers new possibilities for the formulation of proticles in order to address key parameters of drug delivery systems as size, API loading or modified drug release.

The angiogenic factor secretoneurin induces coronary angiogenesis in a model of myocardial infarction by stimulation of vascular endothelial growth factor signaling in endothelial cells.

BACKGROUND: Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is upregulated by hypoxia, and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. METHODS AND RESULTS: In vivo secretoneurin improved left ventricular function, inhibited remodeling, and reduced scar formation. In the infarct border zone, secretoneurin induced coronary angiogenesis, as shown by increased density of capillaries and arteries. In vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis, and activated Akt and extracellular signal-regulated kinase in coronary endothelial cells. Effects were abrogated by a vascular endothelial growth factor (VEGF) antibody, and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells, and binding was blocked by heparinase, indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its coreceptor neuropilin-1. In endothelial cells, secretoneurin also stimulated fibroblast growth factor receptor-3 and insulin-like growth factor-1 receptor, and in coronary vascular smooth muscle cells, we observed stimulation of VEGF receptor-1 and fibroblast growth factor receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin messenger RNA and protein. CONCLUSIONS: Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low-affinity binding sites and neuropilin-1 and stimulates further growth factor receptors like fibroblast growth factor receptor-3. Our in vivo findings indicate that secretoneurin may be a promising therapeutic tool in ischemic heart disease.