Regulation of Lymphatic GM-CSF Expression by the E3 Ubiquitin Ligase Cbl-b

Genome-wide association studies as well as lymphatic expression analyses have linked both Cbl-b and GM-CSF to human multiple sclerosis as well as other autoimmune diseases. Both Cbl-b and GM-CSF have been shown to play a prominent role in the development of murine encephalomyelitis; however, no functional connection between the two has yet been established. In this study, we show that Cblb knockout mice demonstrated significantly exacerbated severity of experimental autoimmune encephalomyelitis (EAE), augmented T cell infiltration into the central nervous system (CNS) and strongly increased production of GM-CSF in T cells in vitro and in vivo.GM-CSF neutralization demonstrated that the increased susceptibility of Cblb−/− mice to EAE was dependent on GM-CSF. Mechanistically, p50 binding to the GM-CSF promoter and the IL-3/GM-CSF enhancer element “CNSa” was strongly increased in nuclear extracts from Cbl-b-deficient T cells. This study suggests that Cbl-b limits autoimmunity by preventing the pathogenic effects of GM-CSF overproduction in T cells

Toll-like receptor 3 signalling mediates angiogenic response upon shock wave treatment of ischaemic muscle

Aims Shock wave therapy (SWT) represents a clinically widely used angiogenic and thus regenerative approach for the treatment of ischaemic heart or limb disease. Despite promising results in preclinical and clinical trials, the exact mechanism of action remains unknown. Toll-like receptor 3, which is part of the innate immunity, is activated by binding doublestranded (ds) RNA. It plays a key role in inflammation, a process that is needed also for angiogenesis. We hypothesize that SWT causes cellular cavitation without damaging the target cells, thus liberating cytoplasmic RNA that in turn activates TLR3. Methods and results SWT induces TLR3 and IFN-b1 gene expression as well as RNA liberation from endothelial cells in a time-dependant manner. Conditioned medium from SWT-treated HUVECs induced TLR3 signalling in reporter cells. The response was lost when the medium was treated with RNase III to abolish dsRNAs or when TLR3 was silenced using siRNAs. In a mouse hind limb ischaemia model using wt and TLR3 2/2 mice (n ¼ 6), SWT induced angiogenesis and arteriogenesis only in wt animals. These effects were accompanied by improved blood perfusion of treated limbs. Analysis of main molecules of the TLR3 pathways confirmed TLR3 signalling in vivo following SWT. Conclusion Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT. -

Secretoneurin Gene Therapy Improves Blood Flow in an Ischemia Model in Type 1 Diabetic Mice by Enhancing Therapeutic Neovascularization

<div><p>Deficient angiogenesis after ischemia may contribute to worse outcome of peripheral arterial disease in patients with diabetes mellitus. Based on our previous work where we demonstrated that Secretoneurin (SN) is up-regulated under hypoxic conditions and enhances angiogenesis, we analyzed the therapeutic potential of SN gene therapy using a model of severe hind limb ischemia in streptozotocin-induced diabetic mice (STZ-DM). After induction of hind limb ischemia, blood flow was assessed by means of laser Doppler perfusion imaging (LDPI) and increased blood perfusion in the SN-treated animal group was observed. These results were complemented by the clinical observation of reduced necrosis and by an increased number of capillaries and arterioles in the SN-treated animal group. <i>In vitro</i>, we found that SN is capable of promoting proliferation and chemotaxis and reduces apoptosis in HUVECs cultured under hyperglycemic conditions. Additionally, SN activated ERK, eNOS and especially AKT as well as EGF-receptor in hyperglycemic HUVECs. In conclusion, we show that SN gene therapy improves post-ischemic neovascularization in diabetic mice through stimulation of angiogenesis and arteriogenesis indicating a possible therapeutic role of this factor in ischemia-related diseases in diabetic patients.</p></div

Effects of SN on metabolic parameters.

<p>No significant changes in (A) blood glucose level and (B) body weight were found between p-SN and p-CTR treated animals after 28 days.</p

Secretoneurin gene therapy improves blood flow monitored <i>in vivo</i> by LDPI in diabetic mice.

<p>(A) Time course of hind limb blood flow measured by LDPI is expressed as ischemic-to-control leg ratio. Treatment with p-SN significantly improved perfusion 7, 14 and 28 days after induction of ischemia compared to p-CTR: #, p<0.05. (B) Representative pictures of the ischemic (left) and non ischemic (right) hind limbs on day 28 after ischemia induction. In color-coded images treatment with p-SN improves perfusion where red indicates normal perfusion and blue indicates a marked reduction in blood flow in the ischemic hind limb.</p

Secretoneurin gene therapy rescues ischemic tissue defects in a diabetic PAD model.

<p>(A) Necrosis score at day 28 is shown as % mice with saved limbs (white), % mice with skin necrosis (grey) and % mice with amputations (black). Treatment with p-SN significantly inhibited ischemic tissue loss (necrosis and amputation). p<0.05. (B) Representative pictures of the ischemic (left) and non ischemic (right) limbs on day 28 after ischemia induction and treatment with p-SN or p-CTR.</p

SN stimulates proliferation and reduces apoptosis of HUVECs under hyperglycemic conditions.

<p>(A) HUVECs were cultured with medium containing mannitol or glucose and stimulated with SN 10 ng/ml, SN 100 ng/ml and VEGF 50 ng/ml. Proliferation was determined using a BrdU assay. Proliferation was stimulated by SN and VEGF to a comparable extent even under hyperglycemic conditions. *, p<0.001 vs. mannitol or glucose respectively. (B) Apoptotic cells were identified as TUNEL positive cells and are shown as percentage of the total cell number (obtained by DAPI staining). SN and VEGF significantly inhibited apoptotic reactions even under hyperglycemic conditions. *, p<0.001; #, p<0.05; §, p<0.005 vs. mannitol or glucose respectively.</p

SN induces outgrowth of ECs from aortic rings.

<p>Incubation of aortic rings with SN resulted in a significant outgrowth of EC from explanted aortic rings under hyperglycemic conditions (A) and with mannitol as osmotic control (B). The observed effects in our study were comparable or even superior to VEGF used as positive control. +, p<0.01 and #, p<0.05 vs. CTR.</p

SN induces HUVEC migration and induces MAPK, AKT, eNOS and EGF receptor activation under hyperglycemic conditions.

<p>(A) HUVECs were cultured with medium containing mannitol or glucose and stimulated with SN 10 ng/ml and VEGF 50 ng/ml. Migration was determined as movement of cells within a membrane towards attracting substances and is given as relative (to control) chemotactic index. *, p<0.001 vs. mannitol or glucose respectively. (B) As shown in this representative western blot hyperglycemia didn't affect activation of the MAPK-ERK1/2 signaling pathway by SN. (C) HUVECs stimulation with SN 10 ng/ml for 40 minutes activated eNOS under high glucose as well as under mannitol control. AKT activation mediated by SN is shown after 5 and 10 minutes under diabetic and control conditions. Under high glucose, AKT stimulation by SN is even stronger compared to mannitol control. (D) SN 10 ng/ml stimulation for 20 minutes revealed phosphorylation of EGF receptor (EGFR) under high glucose as well as under mannitol osmotic control conditions.</p