A novel inhibitor of p75-neurotrophin receptor improves functional outcomes in two models of traumatic brain injury.

The p75 neurotrophin receptor is important in multiple physiological actions including neuronal survival and neurite outgrowth during development, and after central nervous system injury. We have discovered a novel piperazine-derived compound, EVT901, which interferes with p75 neurotrophin receptor oligomerization through direct interaction with the first cysteine-rich domain of the extracellular region. Using ligand binding assays with cysteine-rich domains-fused p75 neurotrophin receptor, we confirmed that EVT901 interferes with oligomerization of full-length p75 neurotrophin receptor in a dose-dependent manner. Here we report that EVT901 reduces binding of pro-nerve growth factor to p75 neurotrophin receptor, blocks pro-nerve growth factor induced apoptosis in cells expressing p75 neurotrophin receptor, and enhances neurite outgrowth in vitro Furthermore, we demonstrate that EVT901 abrogates p75 neurotrophin receptor signalling by other ligands, such as prion peptide and amyloid-β. To test the efficacy of EVT901 in vivo, we evaluated the outcome in two models of traumatic brain injury. We generated controlled cortical impacts in adult rats. Using unbiased stereological analysis, we found that EVT901 delivered intravenously daily for 1 week after injury, reduced lesion size, protected cortical neurons and oligodendrocytes, and had a positive effect on neurological function. After lateral fluid percussion injury in adult rats, oral treatment with EVT901 reduced neuronal death in the hippocampus and thalamus, reduced long-term cognitive deficits, and reduced the occurrence of post-traumatic seizure activity. Together, these studies provide a new reagent for altering p75 neurotrophin receptor actions after injury and suggest that EVT901 may be useful in treatment of central nervous system trauma and other neurological disorders where p75 neurotrophin receptor signalling is affected

Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2.

The effects of somatostatin analogues RC-160 and SMS-201-995 on tyrosine phosphatase and cell proliferation were investigated in COS-7 and NIH 3T3 cells expressing human somatostatin receptor subtype 1 or 2 (SSTR1 or SSTR2). Binding experiments were performed on membranes from COS-7 cells expressing human SSTR1 or SSTR2 using 125I-labeled [Tyr11]S-14 or [Tyr3]SMS-201-995, respectively. The somatostatin analogues RC-160 and SMS-201-995 exhibited low affinity for SSTR1 (IC50 of 0.43 and 1.5 microM, respectively) and high affinity for SSTR2 (IC50 of 0.27 and 0.19 nM). Addition of these analogues to cells expressing either SSTR1 or SSTR2 did not result in an inhibition of adenylate cyclase activity. In SSTR2-expressing cells, both analogues induced a rapid stimulation of a tyrosine phosphatase activity (EC50: RC-160, 2 pM; SMS-201-995, 6 pM) and an inhibition of serum-stimulated proliferation (EC50: RC-160, 6.3 pM; SMS-201-995, 12 pM). In SSTR1-expressing cells, only RC-160 induced stimulation of a tyrosine phosphatase activity. Both analogues caused an inhibition of cell proliferation at a concentration higher than 10 nM in accordance with their affinities for the SSTR1 receptor subtype. A good correlation between the affinities of RC-160 and SMS-201-995 for each receptor subtype and their potencies to inhibit cell proliferation suggests the involvement of these receptors in cell growth regulation. Tyrosine phosphatase was stimulated by both these analogues in SSTR2 and by RC-160 in SSTR1 at affinities similar to their ability to inhibit growth and bind to receptors, implicating tyrosine phosphatase as a transducer of the growth inhibition signal. We also found that mRNAs of receptor subtypes were variably expressed in different pancreatic and colon cancer cell lines, indicating the necessity of a precise analysis of receptor subtypes in target tissues before therapy with analogues

Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCK(A) receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCK(A) and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor

A New Synthetic FGF Receptor Antagonist Inhibits Arteriosclerosis in a Mouse Vein Graft Model and Atherosclerosis in Apolipoprotein E-Deficient Mice

<div><p>Objective</p><p>The role of fibroblast growth factors (FGFs) in the development of vascular diseases remains incompletely understood. The objective of this study was to examine the effects of a new small-molecule multi-FGF receptor blocker with allosteric properties, SSR128129E, on neointimal proliferation after a vein graft procedure in mice and on the development of atherosclerosis in atherosclerosis-prone apolipoprotein E (apoE)-deficient mice.</p> <p>Methods and Results</p><p>Vein grafts were performed in 3 month-old male C57BL6 mice. Segments of the vena cava were interposed at the level of the carotid artery. In SSR128129E (50 mg/kg/d)-treated animals, a dramatic decrease in neointimal proliferation was observed 2 and 8 weeks after the graft (72.5 %, p<0.01, and 47.8 %, p<0.05, respectively). Four-week old male apoE-deficient mice were treated with SSR128129E (50 mg/kg/d) for 3 and 5 months in comparison with a control group. SSR128129E treatment resulted in a reduction of lesion size in the aortic sinus (16.4 % (ns) at 3 months and 42.9 % (p<0.01) at 5 months, without any change in serum lipids. SSR128129 significantly reduced FGFR2 mRNA levels in the aortic sinus (p<0.05, n=5-6), but did not affect the mRNA expression levels of other FGF receptors or ligands.</p> <p>Conclusion</p><p>These studies indicate that FGFs have an important role in the development of vascular diseases like atherosclerosis and graft arteriosclerosis. These data suggest that inhibition of FGF receptors by compounds like SSR128129E might be useful as a new therapeutic approach for these vascular pathologies.</p> </div

mRNA expression levels of FGF receptors and ligands in the aortic sinus.

<p>mRNA levels were determined by quantitative PCR of extracts of the aortic sinus of 6 month-old normal mice (C57BL/6, white bars), apoE-deficient mice (light green bars) and SSR128129-treated apoE-deficient mice (light blue bars). Bars represent the mean±SEM of the data, n=5-6. (*: p<0.05 , **: p<0.01).</p

Effect of SSR128129E on neointimal proliferation in the vein graft model.

<div><p>A-C: Effect on lesion macrophage content 2 weeks after surgery</p> <p>Representative sections of vein grafts immunostained for macrophages (mac3) in control (A) and SSR128129E-treated mice (B). The size of the regions showing macrophage infiltration was determined by image analysis (C, n= 4 per group).</p> <p>D-I: Effect on neointimal proliferation 8 weeks after surgery.</p> <p>Representative sections of vein grafts stained by Masson’s trichrome in control (D) and SSR128129E-treated mice (E). The area of neointimal proliferation was determined by image analysis (F, n= 8-11 per group). Smooth muscle content is shown in representative sections of vein grafts labelled for α-actin in control (G) and SSR128129E-treated mice (H). Smooth muscle content as determined by the area of α-actin staining was determined by image analysis (I, n= 8-11 per group).</p></div

Lesion morphology and size in the aortic sinus of 6 month old apoE-deficient mice.

<p>Lesion morphology as shown in representative slices of aortic sinus stained by Masson’s trichrome in 6 month-old control apoE-KO (A) and SSR128129E-treated mice (B). Aortic sinus lesion size was determined by image analysis in 6 month-old apoE-deficient mice (C, n= 8 per group). </p

Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity

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Regulator of G-Protein Signaling 18 Controls Both Platelet Generation and Function

<div><p>RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/- and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation <i>in vitro</i>. RGS18 deficiency markedly increased thrombus formation <i>in vivo</i>. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation <i>in vitro</i> and <i>in vivo</i> revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery <i>in vivo</i> under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18.</p></div