Susan Jung

Pharmacologic stabilization of hypoxia-inducible transcription factors protects developing mouse brain from hypoxia-induced apoptotic cell death.

OBJECTIVE Accumulation of hypoxia-inducible transcription factors (HIFs) by prolyl-4-hydroxylase inhibitors (PHI) has been suggested to induce neuroprotection in the ischemic rodent brain. We aimed to investigate in vivo effects of a novel PHI on HIF-regulated neurotrophic and pro-apoptotic factors in the developing normoxic and hypoxic mouse brain. METHODS Neonatal mice (P7) were treated with PHI FG-4497 (30-100mg/kg, i.p.) followed by exposure to systemic hypoxia (8% O2, 6h) 4h later. Cerebral expression of HIFα-subunits, specific neurotrophic and vasoactive target genes (vascular endothelial growth factor (VEGF), adrenomedullin (ADM), erythropoietin (EPO), inducible nitric oxide synthase (iNOS)) as well as pro-apoptotic (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 gene (BNIP3), immediate early response 3 (IER3)) and migratory factors (chemokine receptor 4 (CXCR4), stromal cell-derived factor 1 (SDF-1)) was determined (quantitative real-time (RT)., Western blot analysis) in comparison to controls. Apoptotic cell death was analyzed by terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and cleaved caspase 3 (CC3) staining. RESULTS Under normoxic conditions, FG-4497 treatment significantly induced the accumulation of both HIF-1α and HIF-2α isoforms in developing mouse brain. In addition, there was a significant up-regulation of HIF target genes (VEGF, ADM, EPO, CXCR4, p<0.01) with FG-4497 treatment compared to controls supporting functional activation of the HIF proteins. Under hypoxia, differential target gene activation was observed in the developing brain including additive effects of FG-4497 and hypoxia on mRNA expression of VEGF and ADM as well as a dose-dependent down-regulation of iNOS. BNIP3 but not IER3 mRNA levels significantly increased in hypoxic brains pre-treated with high-dose FG-4497 compared to controls. Of special interest, FG-4497 treatment significantly diminished apoptotic cell death, quantified by TUNEL and CC3-positive cells, in hypoxic developing brains compared to controls. CONCLUSIONS PHI treatment modulates neurotrophic factors known to be crucially involved in hypoxia-induced cerebral adaptive mechanisms as well as early brain maturation. Pre-treatment with FG-4497 seems to protect the developing brain from hypoxia-induced apoptosis. Present observations provide basic information for further evaluation of neuroprotective properties of PHI treatment in hypoxic injury of the developing brain. However, potential effects on maturational processes need special attention in experimental research targeting HIF-dependent neuroprotective interventions during the very early stage of brain development.

Activin A regulation under global hypoxia in developing mouse brain

Activin A is a multifunctional growth and differentiation factor with pronounced neuroprotective properties that is strongly up-regulated in various forms of acute brain disorders and injuries including epilepsy, stroke and trauma. In a pediatric context, activin A has been advanced as a potential marker for the severity of perinatal hypoxic-ischemic brain injury. Here we investigated the regulation of activin A under global hypoxia without ischemia in primary cultures of cortical neurons and in neonatal and adult mice of two strains (C57BL/6 and CD-1). From birth to adulthood, activin βA subunit, activin receptors, and functional activin antagonists were all expressed at roughly similar mRNA levels in the brain of C57BL/6 mice. Independent of mouse line and age, we found both moderate (11% O2, 2h) and severe hypoxia (8%, 6h) to be consistently associated with normal or even reduced levels of activin βA (Inhba) mRNA. The surprising unresponsiveness of Inhba expression to hypoxia was confirmed at the protein level. In situ hybridization did not indicate regional, hypoxia-related differences in Inhba expression. Pharmacologic stabilization of hypoxia inducible factors with the prolyl hydroxylase inhibitor FG-4497 did not influence Inhba mRNA levels in neonatal mice. Our data indicate that pure hypoxia differs from other, more complex types of brain damage in that it appears not to recruit activin A as an endogenous neuroprotective agent.

Hypoxia Potentiates LPS-Mediated Cytotoxicity of BV2 Microglial Cells In Vitro by Synergistic Effects on Glial Cytokine and Nitric Oxide System.

BACKGROUND Microglial activation due to a variety of stimuli induces secretion of neurotoxic substances including inflammatory cytokines and nitric oxide (NO). Clinical studies indicate a cross-link between inflammatory and hypoxia-regulated pathways suggesting that bacterial infections markedly sensitize the immature brain to hypoxic injury. METHODS The impact of inflammation and hypoxia on interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), and NO secretion and microglia-induced cytotoxicity was investigated exposing BV2 cells to lipopolysaccharides (LPS) and hypoxia (1% O2). Cytotoxicity, NO, and cytokine release was quantified by MTS and Griess assays and by enzyme-linked immunosorbent assays, respectively. RESULTS LPS exposure of BV2 cells induced a significant, persistent production of NO, IL-1β, IL-6, and TNF-α. Even after LPS removal, ongoing NO and cytokine secretion was observed. Hypoxia mediated exclusively a significant, short-term IL-1β increase, but enhanced LPS-induced cytokine and NO secretion significantly. In addition, LPS-induced supernatants exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition (p < 0.001). Hypoxia potentiated LPS-induced cytotoxicity. CONCLUSION Present data prove that LPS-induced soluble factors rather than LPS exposure mediate microglial toxicity under conditions of hypoxia in vitro. Apart from potential protective effects of the hypoxia-inducible transcription factor (HIF)-1α system, activation of proinflammatory pathways may markedly sensitize microglial cells to promote hypoxia-induced injuries of the developing brain.

Differential regulation of angiogenesis in the developing mouse brain in response to exogenous activation of the hypoxia-inducible transcription factor system

Angiogenesis due to hypoxic-ischemic (HI) injury represents a crucial compensatory mechanism of the developing brain that is mainly regulated by hypoxia-inducible transcription factors (HIF). Pharmacological stimulation of HIF is suggested as a neuroprotective option, however, studies of its effects on vascular development are limited. We analyzed the influence of the prolyl-4-hydroxylase inhibitor (PHI), FG-4497, and erythropoietin (rhEPO) on post-hypoxic angiogenesis (angiogenic growth factors, vessel structures) in the developing mouse brain (P7) assessed after a regeneration period of 72 h. Exposure to systemic hypoxia (8% O2, 6 h) was followed by treatment (i.p.) with rhEPO (2500/5000 IU/kg) at 0, 24 and 48 h or FG-4497 (60/100 mg/kg) compared to controls. In response to FG-4497 treatment cortical and hippocampal vessel area and branching were significantly increased compared to controls. This was associated with elevated ANGPT-2 as well as decreased ANGPT-1 and TIE-2 mRNA levels. In response to rhEPO, mildly increased angiogenesis was associated with elevated ANGPT-2 but also TIE-2 mRNA levels in comparison to controls. In conclusion, present data demonstrate a differential regulation of the angiopoietin/TIE-2 system in response to PHI and rhEPO in the post-hypoxic developing brain pointing to potential functional consequences for vascular regeneration and vessel development.

Oxygen-sensitive regulation and neuroprotective effects of growth hormone-dependent growth factors during early postnatal development

Perinatal hypoxia severely disrupts metabolic and somatotrophic development, as well as cerebral maturational programs. Hypoxia-inducible transcription factors (HIFs) represent the most important endogenous adaptive mechanisms to hypoxia, activating a broad spectrum of growth factors that contribute to cell survival and energy homeostasis. To analyze effects of systemic hypoxia and growth hormone (GH) therapy (rhGH) on HIF-dependent growth factors during early postnatal development, we compared protein (using ELISA) and mRNA (using quantitative RT PCR) levels of growth factors in plasma and brain between normoxic and hypoxic mice (8% O2, 6 h; postnatal day 7, P7) at P14. Exposure to hypoxia led to reduced body weight (P < 0.001) and length (P < 0.04) compared with controls and was associated with significantly reduced plasma levels of mouse GH (P < 0.01) and IGF-1 (P < 0.01). RhGH abrogated these hypoxia-induced changes of the GH/IGF-1 axis associated with normalization of weight and length gain until P14 compared with controls. In addition, rhGH treatment increased cerebral IGF-1, IGF-2, IGFBP-2, and erythropoietin mRNA levels, resulting in significantly reduced apoptotic cell death in the hypoxic, developing mouse brain. These data indicate that rhGH may functionally restore hypoxia-induced systemic dysregulation of the GH/IGF-1 axis and induce upregulation of neuroprotective, HIF-dependent growth factors in the hypoxic developing brain.

Hypoxia potentiates cytotoxicity of LPS-activated microglial BV2 cells in vitro by synergistic effects on cytokine and nitric oxide secretion

Results Activation of BV2 microglial cells by LPS exposure stimulated significant and persistent production of NO, IL-1b, IL-6, and TNF-a. Even after LPS removal, ongoing NO and cytokine secretion could be observed. While hypoxia alone mediated exclusively a significant, shortterm increase of IL-1b, oxygen deprivation enhanced LPS-induced secretion of NO, IL-1b, IL-6, and TNF-a significantly. Surprisingly, pre-stimulation of BV2 cells by hypoxia prior LPS exposure abolished microglial activation suppressing LPS-induced NO production. Hereby, cell-free supernatants derived from LPS-activated microglial cells exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition per se (P < 0.001). Again, hypoxia potentiated LPS-induced cytotoxicity. Conclusion Present data prove that i) the outcome of hypoxia is determined by the microglial activation status and that ii) LPS-induced soluble factors rather than LPS are mediators of microglial neurotoxicity under conditions of hypoxia in vitro. Activation of pro-inflammatory pathways may sensitize microglial cells to promote hypoxia-induced injury of the developing brain. Consequently, our findings may promote neuroprotective therapeutic strategies in the field of perinatal brain injury.

The Impact of Timing of Microglial Activation by Inflammation and Hypoxia Regarding Additive Neurotoxic Effects

Introduction: Perinatal cerebral hypoxia and endotoxins are main risk factors for acquired brain injury and neurological disabilities. Here, we investigated the impact of lipopolysaccharide stimulated microglia on inflammation and cytotoxicity in vitro in the context of hypoxia. Methods: BV2 cells were exposed to LPS and hypoxia (1% O2). Expression of the transcription factor hypoxia-inducible factor (HIF)-1α as well as of iNOS, IL-1b, IL-6, and tumor necrosis factor (TNF)-α were analyzed by real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. NO production was quantified by the Griess assay. Results: LPS exposition prior hypoxia increased mRNA and protein expression of HIF-1α, iNOS, and inflammatory cytokines suggesting synergistic regulation of hypoxia and inflammatory signaling pathways in microglial cells. Whereas HIF-1α accumulation was transient depending on the presence of LPS, ongoing cytokine and NO secretion was observed for at least 3 days after LPS removal. Notably, hypoxia potentiated the LPS-induced secretion of IL-1b (22-fold), IL-6 (threefold), TNF-α (threefold), and increased NO production by approximately 60%. Cell-free supernatants derived from LPS exposed BV2 cells were highly cytotoxic. Again, hypoxia enhanced the cytotoxic effect significantly. In contrast, stimulation of BV2 cells by hypoxia prior LPS abolished additive inflammatory neurotoxic effects compared with controls (p < 0.01). Conclusion: Present in vitro data indicate that LPS induces a dose-dependent and long-lasting sensitization of BV2 cells to hypoxia. Enhanced cytokine and NO secretion may contribute to delayed glial damage following cerebral hypoxic insults during the perinatal period. As hypoxia induced accumulation of the neurotrophic transcription factor HIF-1α abolishes cytotoxic inflammatory effects, present observations may have implications for neuroprotective pharmacological strategies.