Edwige Petit

Brain Genomic Response following Hypoxia and Re-oxygenation in the Neonatal Rat IDENTIFICATION OF GENES THAT MIGHT CONTRIBUTE TO HYPOXIA-INDUCED ISCHEMIC TOLERANCE

Hypoxic preconditioning (8% O2, 3 h) produces tolerance 24 h after hypoxic-ischemic brain injury in neonatal rats. To better understand the ischemic tolerance mechanisms induced by hypoxia, we used oligonucleotide microarrays to examine genomic responses in neonatal rat brain following 3 h of hypoxia (8% O2) and either 0, 6, 18, or 24 h of re-oxygenation. The results showed that hypoxia-inducible factor (HIF)-1- but not HIF-2-mediated gene expression may be involved in brain hypoxia-induced tolerance. Among the genes regulated by hypoxia, 12 genes were confirmed by real time reverse transcriptase-PCR as follows: VEGF,EPO, GLUT-1, adrenomedullin,propyl 4-hydroxylase α, MT-1,MKP-1, CELF, 12-lipoxygenase,t-PA, CAR-1, and an expressed sequence tag. Some genes, for example GLUT-1, MT-1,CELF, MKP-1, and t-PA did not show any hypoxic regulation in either astrocytes or neurons, suggesting that other cells are responsible for the up-regulation of these genes in the hypoxic brain. These genes were expressed in normal and hypoxic brain, heart, kidney, liver, and lung, with adrenomedullin,MT-1, and VEGF being prominently induced in brain by hypoxia. These results suggest that a number of endogenous molecular mechanisms may explain how hypoxic preconditioning protects against subsequent ischemia, and may provide novel therapeutic targets for treatment of cerebral ischemia.

Expression of Receptors for Complement Anaphylatoxins C3a and C5a Following Permanent Focal Cerebral Ischemia in the Mouse

In the present study, we have examined the expression of anaphylatoxin C3a and C5a receptors (C3aR and C5aR) at the mRNA and protein levels in ischemic brain tissues following permanent middle cerebral artery (MCA) occlusion in the mouse. C3aR and C5aR mRNAs were both detected by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR) and the cellular distribution of each receptor was analyzed by immunohistochemistry. Significant increases in the expression of C3aR and C5aR mRNAs in the ischemic cortex were observed; the expression of both reached a peak at 2 days after MCA occlusion (4.3- and 3.4-fold increases, respectively, compared with nonoperated control cortical samples; P < 0.00625 with Bonferronis correction, n = 3). C3aR and C5aR stainings were found constitutively on neurons and astrocytes. In ischemic tissues, we observed that C3aR and C5aR were expressed de novo on endothelial cells of blood vessels, at 6 h and 2 days after MCA occlusion, respectively. C3aR and C5aR immunostaining was increased in macrophage-like cells and reactive astrocytes 7 days postocclusion. C3a and C5a may play an important role in promoting inflammatory and/or repair processes in the ischemic brain by regulating glial cell activation and chemotaxis.

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.

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.

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.

Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner

The understanding of mechanisms involved in ischaemic brain tolerance may provide new therapeutical targets for stroke. In vivo genomic studies revealed an up‐regulation of adrenomedullin expression by hypoxic pre‐conditioning. Furthermore, adrenomedullin reduced ischaemia‐induced brain damage in rodents. However, whether adrenomedullin is involved in hypoxic pre‐conditioning‐induced tolerance and whether adrenomedullin protects directly neurons against ischaemia remain unknown. Using a neuronal model of hypoxic pre‐conditioning and oxygen glucose deprivation (OGD), we showed that 0.1% or 0.5% of O2 pre‐conditioning reduced the OGD‐induced neuronal death, whereas 1% or 2% of O2 pre‐treatment did not induce neuroprotection. Adrenomedullin expression increased following the hypoxic period, and following OGD only in pre‐conditioned (0.1% or 0.5% of O2) neurons. Adrenomedullin pre‐treatment and post‐treatment reduced the OGD‐induced neuronal death, partly through PI3kinase‐dependent pathway. However, adrenomedullin antagonism during hypoxic pre‐conditioning failed to inhibit the neuroprotection whereas adrenomedullin antagonism following OGD abolished the hypoxic pre‐conditioning‐induced neuroprotection. Finally, we showed that adrenomedullin is involved in neuroprotection induced by endothelial cells and microglia. In contrast, neuroprotection induced by astrocytes occurred through adrenomedullin‐independent mechanisms. Altogether, our results suggest that adrenomedullin is an effector of the hypoxic pre‐conditioning‐induced neuronal tolerance and a potent autocrine and paracrine neuroprotective factor during cerebral ischaemia.

Increased HIF-1α expression correlates with cell proliferation and vascular markers CD31 and VEGF-A in uveal melanoma.

PURPOSE Overexpression of hypoxia inducible factor-1 α (HIF-1α) has been found in several cancers and is thought to correlate with aggressive disease. The purpose of our study was to investigate the influence of HIF-1α on clinical outcome in uveal melanoma (UM) along with proliferative (MIB-1) and vascular (CD31, VEGF-A) markers. METHODS A retrospective analysis was carried out on UM tumors from 88 patients. HIF-1α, MIB-1, CD31, and VEGF-A expression, as well as necrosis, were assessed by immunohistochemistry and hematoxylin/eosin on paraffin-embedded UM tumor sections by using a tissue microarray. The bivariate analysis involving HIF-1α expression and clinicopathologic covariates was performed by using the χ(2) test. The association of clinicopathologic covariates and HIF-1α expression with patient survival was evaluated by using the Kaplan-Meier approach and Cox proportional-hazards regression analysis. RESULTS Among our study population, 56 patients (63.6%) had high levels of HIF-1α expression. High expression of HIF-1α was associated with high expression of MIB-1 (P = 0.04), CD31 (P = 0.03), and VEGF-A (P < 0.0001), as well as necrosis (P = 0.04). However, high HIF-1α expression was not correlated with cell type, largest macroscopic tumor dimension or thickness, anterior margin, pigmentation, or mitotic figures. Patients with high HIF-1α expression did not show a reduced survival when compared to patients with low HIF-1α expression (P = 0.92). Finally, HIF-1α expression was not increased after irradiation. CONCLUSIONS An increase in HIF-1α expression was significantly associated with proliferative (MIB-1) and vascular (CD31 and VEGF-A) markers, as well as necrosis, in UM. However, there was no correlation between high HIF-1α expression and patient survival.

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.

Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models

Hypoxia is a common feature of solid tumors, particularly in glioblastoma (GBM), and known to be a poor prognosis factor in GBM patients. The growth of GBM is also associated with a marked inflammation partially characterized by an accumulation of macrophage (MΦ) of the M2 phenotype. However, the transition between M1 MΦ (antitumoral) and M2 MΦ (protumoral) phenotypes is a dynamic process. We made the assumption that oxygen (O2) availability could be a major regulator of this transition and that the intratumoral O2 gradient is of importance. We evaluated, in vivo, the impact of hypoxia on MΦ tropism and polarization in two models of human GBM, well differentiated by their degree of hypoxia. MΦ migration in the tumor was more pronounced in the more hypoxic tumor of the two GBM models. In the more hypoxic of the models, we have shown that MΦ migrated at the tumor site only when hypoxia takes place. We also demonstrated that the acquisition of the M2 phenotype was clearly an evolving phenomenon with hypoxia as the major trigger for this transition. In support of these in vivo finding, M0 but also M1 MΦ cultured in moderate or severe hypoxia displayed a phenotype close to that of M2 MΦ whose phenotype was further reinforced by severe hypoxia. These results highlight the role of hypoxia in the aggressiveness of GBM, in part, by transforming MΦ such that a protumoral activity is expressed.

MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model

Despite treatment efforts, the median survival in patients with glioblastoma multiforme, the most aggressive form of glioma, does not extend beyond 12-15 months. One of the major pathophysiological characteristics of these tumors is their ability to induce active angiogenesis. Thus, based on the lack of efficient therapies, agents that inhibit angiogenesis are particularly attractive as a therapeutic option. However, it has been recently proposed that although specifically targeting vascular endothelial growth factor, the main angiogenic factor, certainly leads to significant tumor regression, it could also be followed by tumor relapses. In this case, angiogenesis is driven by alternate pathways that include other angiogenic factors. One possible strategy to overcome this therapeutic obstacle is to overexpress antivascular factors such as angiopoietin-2 (Ang2). Here, by using MRI and histological analysis, we studied the vascular events involved in glioma growth impairment induced by Ang2 overexpression. Our results show that an increase in Ang2 expression, during the tumor growth, leads to a significant decrease in tumor growth ( approximately 86%) along with an increase in the survival median ( approximately 70%) but does not modify the tumor vascular area or cerebral blood volume. However, tumor Ang2-derived blood vessels display an abnormal, enlarged morphology. We show that the presence of Ang2 leads to an enhancement of tumor perfusion and a decrease in tumor vessel permeability. Based on our MR image evaluations of hemodynamic tumor vessel changes, we propose that Ang2-derived tumor vessels lead to an inadequate oxygenation of the tumor tissue, leading to impairment in tumor growth.