Samuel Valable

Autocrine and Paracrine Angiopoietin 1/Tie-2 Signaling Promotes Muscle Satellite Cell Self-Renewal

Mechanisms governing muscle satellite cell withdrawal from cell cycle to enter into quiescence remain poorly understood. We studied the role of angiopoietin 1 (Ang1) and its receptor Tie-2 in the regulation of myogenic precursor cell (mpc) fate. In human and mouse, Tie-2 was preferentially expressed by quiescent satellite cells in vivo and reserve cells (RCs) in vitro. Ang1/Tie-2 signaling, through ERK1/2 pathway, decreased mpc proliferation and differentiation, increased the number of cells in G0, increased expression of RC-associated markers (p130, Pax7, Myf-5, M-cadherin), and downregulated expression of differentiation-associated markers. Silencing Tie-2 had opposite effects. Cells located in the satellite cell neighborhood (smooth muscle cells, fibroblasts) upregulated RC-associated markers by secreting Ang1 in vitro. In vivo, Tie-2 blockade and Ang1 overexpression increased the number of cycling and quiescent satellite cells, respectively. We propose that Ang1/Tie-2 signaling regulates mpc self-renewal by controlling the return to quiescence of a subset of satellite cells.

Synergistic effects of CoCl2 and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells

Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl2 on MSCs. CoCl2 treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1α and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl2 effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl2-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl2, an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.

Inactivation of the HIF-1α/PDK3 Signaling Axis Drives Melanoma toward Mitochondrial Oxidative Metabolism and Potentiates the Therapeutic Activity of Pro-Oxidants

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Time- and cell type-specific induction of platelet-derived growth factor receptor-β during cerebral ischemia

During cerebral ischemia, angiogenesis occurs inside and around the infarcted area. The growth of new blood vessels may contribute to a better outcome after stroke due to accelerated and increased delivery of nutrients and oxygen to the ischemic tissue. The platelet-derived growth factor (PDGF)-B/PDGF receptor (PDGFR)-beta system, hitherto thought to contribute mainly to neuroprotection, may also support angiogenesis and vascular remodeling by mediating interactions of endothelial cells with pericytes after cerebral ischemia. While platelet-derived growth factor (PDGF)-B and its receptor PDGFR-beta are essential factors for the recruitment of pericytes to brain capillaries during embryonic development, their role in blood vessel maturation during cerebral ischemia is not clear. The aim of the present study was to investigate the time course and location of PDGF-B and PDGFR-beta expression in a mouse model of focal cerebral ischemia. In contrast to the early and continuous induction of PDGF-B, PDGFR-beta mRNA was specifically upregulated in vascular structures in the infarcted area 48 h after occlusion of the middle cerebral artery. Immunohistology and confocal microscopy analysis revealed the specific upregulation of PDGFR-beta on blood vessels and suggested expression mainly on pericytes. Our results imply PDGFR-beta as a key factor in vascular remodeling during stroke and suggest that the pleiotropic functions of PDGF-B may be regulated via the expression of its receptor. Influencing the PDGF system therapeutically might improve angiogenesis, cellular protection, and edema inhibition.

FLIPL Protects Neurons against In Vivo Ischemia and In Vitro Glucose Deprivation-Induced Cell Death

Knowledge of the molecular mechanisms that underlie neuron death after stroke is important to allow the development of effective neuroprotective strategies. In this study, we investigated the contribution of death receptor signaling pathways to neuronal death after ischemia using in vitro and in vivo models of ischemic injury and transgenic mice that are deficient in tumor necrosis factor receptor I (TNFRI KO) or show neuron-specific overexpression of the long isoform of cellular Fas-associated death domain-like interleukin-1-β-converting enzyme-inhibitory protein (FLIPL). Caspase 8 was activated in brain lesions after permanent middle cerebral artery occlusion (pMCAO) and in cortical neurons subjected to glucose deprivation (GD) and was necessary for GD-induced neuron death. Thus, neurons treated with zIETD-FMK peptide or overexpressing a dominant-negative caspase 8 mutant were fully protected against GD-induced death. The presence of the neuroprotective TNFRI was necessary for selectively sustaining p50/p65NF-κB activity and the expression of the p43 cleavage form of FLIPL, FLIP(p43), an endogenous inhibitor of caspase 8, in pMCAO lesions and GD-treated neurons. Moreover, TNF pretreatment further upregulated p50/p65NF-κB activity and FLIP(p43) expression in neurons after GD. The knock-down of FLIP in wild-type (WT) neurons using a short hairpin RNA revealed that FLIPL is essential for TNF/TNFRI-mediated neuroprotection after GD. Furthermore, the overexpression of FLIPL was sufficient to rescue TNFRI KO neurons from GD-induced death and to enhance TNF neuroprotection in WT neurons, and neuron-specific expression of FLIPL in transgenic mice significantly reduced lesion volume after pMCAO. Our results identify a novel role for the TNFRI–NF-κB–FLIPL pathway in neuroprotection after ischemia and identify potential new targets for stroke therapy.

Reduced brain edema and functional deficits after treatment of diffuse traumatic brain injury by carbamylated erythropoietin derivative.

Objective:To investigate the effects of carbamylated erythropoietin, a modified erythropoietin lacking erythropoietic activity, on brain edema and functional recovery in a model of diffuse traumatic brain injury. Design:Adult male Wistar rats. Setting:Neurosciences and physiology laboratories. Interventions:Thirty minutes after diffuse traumatic brain injury (impact-acceleration model), rats were intravenously administered with either a saline solution (traumatic brain injury-saline) or carbamylated erythropoietin (50 &mgr;g/kg; traumatic brain injury-carbamylated erythropoietin). A third group received no traumatic brain injury insult (sham-operated). Measurements and Main Results:Three series of experiments were conducted to investigate: 1) the effect of carbamylated erythropoietin on brain edema before and 1 hr after traumatic brain injury using diffusion-weighted magnetic resonance imaging and measurements of apparent diffusion coefficient (n = 10 rats per group), and the phosphorylation level of brain extracellular-regulated kinase-1/-2 was also determined to indicate the presence of an activated cell signaling pathway; 2) the time course of brain edema using magnetic resonance imaging between 4 and 6 hrs postinjury and the gravimetric technique at 6 hrs (n = 10 rats per group); and 3) motor and cognitive function over 10 days post traumatic brain injury, testing acute somatomotor reflexes, adhesive paper removal, and two-way active avoidance (n = 8 rats per group). Compared to traumatic brain injury-saline rats, rats receiving traumatic brain injury-carbamylated erythropoietin showed a significant reduction in brain edema formation at 1 hr that was sustained until 6 hrs when results were comparable with sham-operated rats. This antiedematous effect of carbamylated erythropoietin was possibly mediated through an early inhibition of extracellular-regulated kinase-1/-2 phosphorylation. Compared to traumatic brain injury-saline rats, traumatic brain injury-carbamylated erythropoietin rats showed improved functional recovery of the acute somatomotor reflexes post traumatic brain injury, took less time to remove adhesive from the forelimbs, and showed higher percentages of correct avoidance responses. Conclusion:Our findings indicate that early posttraumatic administration of carbamylated erythropoietin reduces brain edema development until at least 6 hrs postinjury and improves neurologic recovery. Carbamylated erythropoietin can thus be considered as a potential agent in the treatment of traumatic brain injury-induced diffuse edema.

Detection of glioblastoma response to temozolomide combined with bevacizumab based on µMRI and µPET imaging reveals [18F]-fluoro-l-thymidine as an early and robust predictive marker for treatment efficacy

The individualized care of glioma patients ought to benefit from imaging biomarkers as precocious predictors of therapeutic efficacy. Contrast enhanced MRI and [(18)F]-fluorodeoxyglucose (FDG)-PET are routinely used in clinical settings; their ability to forecast the therapeutic response is controversial. The objectives of our preclinical study were to analyze sensitive µMRI and/or µPET imaging biomarkers to predict the efficacy of anti-angiogenic and/or chemotherapeutic regimens. Human U87 and U251 orthotopic glioma models were implanted in nude rats. Temozolomide and/or bevacizumab were administered. µMRI (anatomical, diffusion, and microrheological parameters) and µPET ([(18)F]-FDG and [(18)F]-fluoro-l-thymidine [FLT]-PET) studies were undertaken soon (t(1)) after treatment initiation compared with late anatomical µMRI evaluation of tumor volume (t(2)) and overall survival. In both models, FDG and FLT uptakes were attenuated at t(1) in response to temozolomide alone or with bevacizumab. The distribution of FLT, reflecting intratumoral heterogeneity, was also modified. FDG was less predictive for treatment efficacy than was FLT (also highly correlated with outcome, P < .001 for both models). Cerebral blood volume was significantly decreased by temozolomide + bevacizumab and was correlated with survival for rats with U87 implants. While FLT was highly predictive of treatment efficacy, a combination of imaging biomarkers was superior to any one alone (P < .0001 in both tumors with outcome). Our results indicate that FLT is a sensitive predictor of treatment efficacy and that predictability is enhanced by a combination of imaging biomarkers. These findings may translate clinically in that individualized glioma treatments could be decided in given patients after PET/MRI examinations.

Magnetic Resonance Imaging and Fluorescence Labeling of Clinical-Grade Mesenchymal Stem Cells Without Impacting Their Phenotype: Study in a Rat Model of Stroke

Human mesenchymal stem cells (hMSCs) have strong potential for cell therapy after stroke. Tracking stem cells in vivo following a graft can provide insight into many issues regarding optimal route and/or dosing. hMSCs were labeled for magnetic resonance imaging (MRI) and histology with micrometer‐sized superparamagnetic iron oxides (M‐SPIOs) that contained a fluorophore. We assessed whether M‐SPIO labeling obtained without the use of a transfection agent induced any cell damage in clinical‐grade hMSCs and whether it may be useful for in vivo MRI studies after stroke. M‐SPIOs provided efficient intracellular hMSC labeling and did not modify cell viability, phenotype, or in vitro differentiation capacity. Following grafting in a rat model of stroke, labeled hMSCs could be detected using both in vivo MRI and fluorescent microscopy until 4 weeks following transplantation. However, whereas good label stability and unaffected hMSC viability were observed in vitro, grafted hMSCs may die and release iron particles in vivo.

Noninvasive assessment of hypoxia with 3-[18F]-fluoro-1-(2-nitro-1-imidazolyl)-2-propanol ([18F]-FMISO): a PET study in two experimental models of human glioma.

Abstract Despite multiple advances in cancer therapies, patients with glioblastoma (GBM) still have a poor prognosis. Numerous glioma models are used not only for the development of innovative therapies but also to optimize conventional ones. Given the significance of hypoxia in drug and radiation resistance and that hypoxia is widely observed among GBM, the establishment of a reliable method to map hypoxia in preclinical human models may contribute to the discovery and translation of future and more targeted therapies. The aim of this study was to compare the hypoxic status of two commonly used human orthotopic glioma models (U87 and U251) developed in rats and studied by noninvasive hypoxia imaging with 3-[18F]fluoro-1-(2-nitro-1-imidazolyl)-2-propanol-micro-positron emission tomography ([18F]-FMISO-μPET). In parallel, because of the relationships between angiogenesis and hypoxia, we used magnetic resonance imaging (MRI), histology, and immunohistochemistry to characterize the tumoral vasculature. Although all tumors were detectable in T2-weighted MRI and 2-deoxy-2-[18F]fluoro-d-glucose-μPET, only the U251 model exhibited [18F]-FMISO uptake. Additionally, the U251 tumors were less densely vascularized than U87 tumors. Our study demonstrates the benefits of noninvasive imaging of hypoxia in preclinical models to define the most reliable one for translation of future therapies to clinic based on the importance of intratumoral oxygen tension for the efficacy of chemotherapy and radiotherapy.