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

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

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

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

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

RGS18-/- mice present a defective thrombopoiesis after acute thrombocytopenia.

<p>(A) Complement-mediated immune thrombocytopenia. 10-week-old WT (n = 8; black circle) and RGS18-/- mice (n = 8; open circle) were given a sterile intraperitoneal injection of anti-mouse αGpIb antibody (2 µg/g of mouse) at T0 (Arrow). Platelet counts were measured at 4, 48, 72, 96, and 168 hours after injection. (B) Busulfan-induced thrombocytopenia. Busulfan was injected to 9/13-week-old WT (n = 15; black circle) and RGS18-/- mice (n = 14; open circle) intraperitoneally at 30 mg/kg (day 0; arrow). Platelet counts were measured at 7, 11, 15, and 24 days after injection. *P<.05; **P<.01; ***P<.001.</p

RGS18-/- mice present a defective megakaryopoiesis.

<p>(A) <i>In vitro</i> MK maturation. Lin^- bone marrow cells (5×10⁴ cells/chamber) from 9/10-week-old WT and RGS18-/- mice (n = 5 per group) were cultured in Mega-Cult-C media containing 50 ng/mL rhTPO, 10 ng/ml rmIL3, and 3 ng/ml rmIL6 in double-chamber slides at 37°C for 11 days. Slides were then dehydrated and stained with CD41. Slides were scored microscopically, and MK colonies (CFU-MKs) were defined as colonies containing at least 3-4MKs. (B) <i>In vivo</i> MK maturation. Bone marrow cells from 9-week-old WT and RGS18-/- mice (n = 6 per group) were stained for CD42b, and MKs were identified by flow cytometry on the basis of size and CD42b positivity. Percentages of total bone marrow cell population are shown. (C) MK ploidy analysis. Bone marrow cells from 9-week-old WT and RGS18-/- mice (n = 6 per group) were stained for CD41 in presence of propidium iodide (PI). MKs were identified by flow cytometry on the basis of size and CD41 positivity. DNA cell content was evaluated on the basis of PI fluorescence intensity. Percentages of total bone marrow cell population with DNA content >2N are shown. *P<.05; **P<.01; ***P<.001.</p