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

The Liver-Selective Thyromimetic T-0681 Influences Reverse Cholesterol Transport and Atherosclerosis Development in Mice

Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice.T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls.The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans

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. -

Secretogranin II; a Protein Increased in the Myocardium and Circulation in Heart Failure with Cardioprotective Properties

Background: Several beneficial effects have been demonstrated for secretogranin II (SgII) in non-cardiac tissue. As cardiac production of chromogranin A and B, two related proteins, is increased in heart failure (HF), we hypothesized that SgII could play a role in cardiovascular pathophysiology. Methodology/Principal Findings: SgII production was characterized in a post-myocardial infarction heart failure (HF) mouse model, functional properties explored in experimental models, and circulating levels measured in mice and patients with stable HF of moderate severity. SgII mRNA levels were 10.5 fold upregulated in the left ventricle (LV) of animals with myocardial infarction and HF (p<0.001 vs. sham-operated animals). SgII protein levels were also increased in the LV, but not in other organs investigated. SgII was produced in several cell types in the myocardium and cardiomyocyte synthesis of SgII was potently induced by transforming growth factor-beta and norepinephrine stimulation in vitro. Processing of SgII to shorter peptides was enhanced in the failing myocardium due to increased levels of the proteases PC1/3 and PC2 and circulating SgII levels were increased in mice with HF. Examining a pathophysiological role of SgII in the initial phase of post-infarction HF, the SgII fragment secretoneurin reduced myocardial ischemia-reperfusion injury and cardiomyocyte apoptosis by 30% and rapidly increased cardiomyocyte Erk1/2 and Stat3 phosphorylation. SgII levels were also higher in patients with stable, chronic HF compared to age-and gender-matched control subjects: median 0.16 (Q1-3 0.14-0.18) vs. 0.12 (0.10-0.14) nmol/L, p<0.001. Conclusions: We demonstrate increased myocardial SgII production and processing in the LV in animals with myocardial infarction and HF, which could be beneficial as the SgII fragment secretoneurin protects from ischemia-reperfusion injury and cardiomyocyte apoptosis. Circulating SgII levels are also increased in patients with chronic, stable HF and may represent a new cardiac biomarker

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