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Objective Astrocytes participate in the local innate immune response of the central nervous system. In response to stress such as ischemia, activated cells release endogenous factors known as damage-associated molecular patterns (DAMPs). Self-extracellular RNA (eRNA) is such a ubiquitous alarm signal. However, it is unclear whether eRNA is involved in the early acute phase of cerebral ischemia and is sufficient to sensitize astrocytes towards a DAMP or PAMP (pathogen-associated molecular pattern) reaction. Methods Pro-inflammatory activation upon eRNA stimulation was characterized in primary murine astrocyte cultures. In vivo, an experimental stroke model was used to localize and quantify eRNA in murine brain sections. Using primary cortical neurons and the mouse hippocampal neuronal cell line HT-22, neuronal RNA release upon stress conditions related to cerebral hypoxia/ischemia was analyzed. Results While low-dose eRNA alone did not promote pro-inflammatory activation of astrocytes in culture, it strongly enhanced the expression of pro-inflammatory cytokines in the presence of either Pam2CSK4, a synthetic PAMP molecule that mimics bacterial infection, or high mobility group box 1 (HMGB1), a prominent DAMP. Synergism of eRNA/Pam2CSK4 and eRNA/HMGB1 was prevented by blockage of the astroglial toll-like receptor (TLR)-2. Inhibition of NF-κB- and mitogen-activated protein kinase-dependent signaling pathways hampered eRNA/Pam2CSK4-mediated pro-inflammatory activation of astrocytes. In vivo, the amount of non-nuclear, presumably extracellular ribosomal RNA in close proximity to neurons significantly accumulated across the infarct core and peri-infarct areas that was accompanied by transcriptional up-regulation of various pro-inflammatory factors. Accordingly, the exposure of neurons to hypoxic/ischemic stress in vitro resulted in the release of eRNA, partly mediated by active cellular processes dependent on the cytosolic calcium level. Conclusion The DAMP signal eRNA can sensitize astrocytes as active players in cerebral innate immunity towards exogenous and endogenous activators of inflammation (PAMPs and DAMPs) in a synergistic manner via TLR2-NF-κB-dependent signaling mechanisms. These findings provide new insights into the pathogenesis of ischemic stroke and other inflammatory neurological disorders. Further studies will clarify whether administration of RNase in vivo may serve as an effective treatment for inflammatory brain pathologies.

ZUSAMMENFASSUNGNeozytolyse bezeichnet die bevorzugte Zerstörung von jungen roten Blutzellen (Neozyten), wenn zu viele Erythrozyten vorhanden sind. Das kommt in der Raumfahrt oder beim Abstieg nach langem Höhenaufenthalt vor. Mit dem Ziel, die Existenz von Neozytolyse erstmals zu beweisen, haben wir in der Forschungsstation Jungfraujoch (3450 Hm) eine prospektive Studie durchgeführt. Neu gebildete Erythrozyten von 12 männlichen Probanden wurden durch Einnahme von nichtradioaktivem Glyzin mit seltenen Isotopen markiert: in Heidelberg (110 Hm) und während eines 19-tägigen Aufenthalts (3450 Hm). Die Eliminationsdynamik für Erythrozyten, die in Normoxie und in großer Höhe produziert wurden, unterschied sich nicht. Zusammen mit Änderungen der Retikulozytenzahlen konnte damit das Konzept der Neozytolyse nicht bestätigt werden.

Aims Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). Methods Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2- and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. Results Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C- (normoxia) and 15 N- (high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. Conclusion We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

Episodes of cerebral hypoxia/ischemia increase the risk of dementia, which is associated with impaired learning and memory. Previous studies in rodent models of dementia indicated a favorable effect of the hypoxia-inducible factor (HIF) targets VEGF (vascular endothelial growth factor) and erythropoietin (Epo). In the present study we thus investigated whether activation of the entire adaptive HIF pathway in neurons by cell-specific deletion of the HIF suppressor prolyl-4-hydroxylase 2 (PHD2) improves cognitive abilities in young (3months) and old (18-28months) mice suffering from chronic brain hypoperfusion. Mice underwent permanent occlusion of the left common carotid artery, and cognitive function was assessed using the Morris water navigation task. Under conditions of both normal and decreased brain perfusion, neuronal PHD2 deficiency resulted in improved and faster spatial learning in young mice, which was preserved to some extent also in old animals. The loss of PHD2 in neurons resulted in enhanced hippocampal mRNA and protein levels of Epo and VEGF, but did not alter local microvascular density, dendritic spine morphology, or expression of synaptic plasticity-related genes in the hippocampus. Instead, better cognitive function in PHD2 deficient animals was accompanied by an increased number of neuronal precursor cells along the subgranular zone of the dentate gyrus. Overall, our current pre-clinical findings indicate an important role for the endogenous oxygen sensing machinery, encompassing PHDs, HIFs and HIF target genes, for proper cognitive function. Thus, pharmacological compounds affecting the PHD-HIF axis might well be suited to treat cognitive dysfunction and neurodegenerative processes.

The contribution of neurons to growth and refinement of the microvasculature during postnatal brain development is only partially understood. Tissue hypoxia is the physiologic stimulus for angiogenesis by enhancing angiogenic mediators partly through activation of hypoxia-inducible factors (HIFs). Hence, we investigated the HIF oxygen-sensing pathway in postmitotic neurons for physiologic angiogenesis in the murine forebrain during postnatal development by using mice lacking the HIF suppressing enzyme prolyl-4-hydroxylase domain (PHD)2 and/or HIF-1/2α in postmitotic neurons. Perinatal activation or inactivation of the HIF pathway in neurons inversely modulated brain vascularization, including endothelial cell number and proliferation, density of total and perfused microvessels, and vascular branching. Accordingly, several angiogenesis-related genes were up-regulated in vivo and in primary neurons derived from PHD2-deficient mice. Among them, only VEGF and adrenomedullin (Adm) promoted angiogenic sprouting of brain endothelial cells. VEGF and Adm additively enhanced endothelial sprouting through activation of multiple pathways. PHD2 deficiency in neurons caused HIF-α stabilization and increased VEGF mRNA levels not only in neurons but unexpectedly also in astrocytes, suggesting a new mechanism of neuron-to-astrocyte signaling. Collectively, our results identify the PHD-HIF pathway in neurons as an important determinant for vascularization of the brain during postnatal development.-Nasyrov, E., Nolan, K. A., Wenger, R. H., Marti, H. H., Kunze, R. The neuronal oxygen-sensing pathway controls postnatal vascularization of the murine brain.

Gliosis in Niemann-Pick type C (NP-C) disease is characterized by marked changes in microglia and astrocytes. However, the gliosis onset and progression in NP-C has not been systematically studied, nor has the mechanism underlying this finding. Here, we found early gliosis in the subventricular zone (SVZ) of NP-C mice. Neural progenitor damage by Npc1 mutation suppressed vascular endothelial growth factor (VEGF) expression and further induced microglia activation followed by astrogliosis. Interestingly, excessive astrogliosis in the SVZ induced neural progenitor retention and/or migration into thalamus via astrocyte-derived VEGF, resulting in acceleration of thalamic and cortical gliosis through thalamo-cortical pathways. Transplantation of VEGF-overexpressing neural stem cells into the SVZ improved whole-brain pathology of NP-C mice. Overall, our data provide a new pathological perspective on NP-C neural pathology, revealing abnormalities in the subventricular-thalamo-cortical circuit of NP-C mouse brain and highlighting the importance of the SVZ microenvironment as a therapeutic target for NP-C disease.

The loss of blood-brain barrier (BBB) integrity leading to vasogenic edema and brain swelling is a common feature of hypoxic/ischemic brain diseases such as stroke, but is also central to the etiology of other CNS disorders. In the past decades, numerous proteins, belonging to the family of angioneurins, have gained increasing attention as potential therapeutic targets for ischemic stroke, but also other CNS diseases attributed to BBB dysfunction. Angioneurins encompass mediators that affect both neuronal and vascular function. Recently, increasing evidence has been accumulated that certain angioneurins critically determine disease progression and outcome in stroke among others through multifaceted effects on the compromised BBB. Here, we will give a concise overview about the family of angioneurins. We further describe the most important cellular and molecular components that contribute to structural integrity and low permeability of the BBB under steady-state conditions. We then discuss BBB alterations in ischemic stroke, and highlight underlying cellular and molecular mechanisms. For the most prominent angioneurin family members including vascular endothelial growth factors, angiopoietins, platelet-derived growth factors and erythropoietin, we will summarize current scientific literature from experimental studies in animal models, and if available from clinical trials, on the following points: (i) spatiotemporal expression of these factors in the healthy and hypoxic/ischemic CNS, (ii) impact of loss- or gain-of-function during cerebral hypoxia/ischemia for BBB integrity and beyond, and (iii) potential underlying molecular mechanisms. Moreover, we will highlight novel therapeutic strategies based on the activation of endogenous angioneurins that might improve BBB dysfuntion during ischemic stroke.

Hypoxia-inducible factors mediate adaptive responses to ischemia, among others, by induction of anti- and pro-survival genes. Thus, the impact of HIF on neuronal survival upon stroke is controversial. Therefore, neuron-specific knockout mice deficient for Hif1a and Hif2a were exposed to inspiratory hypoxia or ischemia–reperfusion injury. Both Hif1a- and Hif2a-deficient mice showed no altered infarct and edema size, suggesting that both HIF-α subunits might compensate for each other. Accordingly, hypoxic HIF-target gene regulation was marginally affected with exception of anti-survival Bnip3 and pro-survival erythropoietin. In the early acute stage upon stroke, Hif1a/Hif2a double knockout mice exhibited significantly reduced expression of the anti-survival Bnip3, Bnip3L, and Pmaip1. Accordingly, global cell death and edema were significantly reduced upon 24 h but not 72 h reperfusion. Behavioral assessment indicated that Hif1a/Hif2a-deficient mice initially performed better, but became significantly more impaired after 72 h accompanied by increased apoptosis and reduced angiogenesis. Our findings suggest that in neurons HIF-1 and HIF-2 have redundant functions for cellular survival under ischemic conditions. By contrast, lack of anti-survival factors in Hif1a/Hif2a-deficient mice might protect from early acute neuronal cell death and neurological impairment, indicating a benefit of HIF-pathway inhibition in neurons in the very acute phase after ischemic stroke.

Hypoxia inducible factors (HIFs) mediate the endogenous adaptive responses to hypoxia. HIF prolyl 4-hydroxylase domain proteins (PHD) are important suppressors of the HIF pathway. Recently, we demonstrated that neuron-specific deletion of Phd2 reduces cerebral tissue damage in the very acute phase of ischemic stroke. In the present study, we investigated whether neuronal Phd2 ablation is likewise beneficial for stroke recovery, and aimed to identify underlying cellular mechanisms. Mice underwent permanent occlusion of the distal middle cerebral artery (pdMCAO) for either 7 days (sub-acute stage) or 30 days (chronic stage). One week after pdMCAO the infarct size of Phd2-deficient mice was significantly reduced as compared to wild-type (WT) mice. Accordingly, Phd2-deficient animals showed less impaired sensorimotor function. Neuronal loss of Phd2 upregulated vascular endothelial growth factor (VEGF) and significantly increased microvascular density along the infarct border in the sub-acute stage of stroke. Phd2-deficient mice showed reduced expression of pro-inflammatory cytokines and increased numbers of resting microglia/macrophages and reactive astrocytes within peri-infarct regions in comparison to WT littermates. Finally, brain tissue protection and increased angiogenesis upon sub-acute ischemic stroke was completely absent in Phd2 knockout mice that were additionally deficient for both Hif1a and Hif2a. Our findings suggest that lack of PHD2 in neurons improves histological and functional long-term outcome from ischemic stroke at least partly by amplifying endogenous adaptive neovascularization through activation of the HIF-VEGF axis.

Intrauterine or perinatal complications constitute a major risk for psychiatric diseases. Infants who suffered from hypoxia–ischemia (HI) are at twofold risk to develop schizophrenia in later life. Several animal models attempt to reproduce these complications to study the yet unknown steps between an insult in early life and outbreak of the disease decades later. However, it is very challenging to find the right type and severity of insult leading to a disease-like phenotype in the animal, but not causing necrosis and focal neurological deficits. By contrast, too mild, repetitive insults may even be protective via conditioning effects. Thus, it is not surprising that animal models of hypoxia lead to mixed results. To achieve clinically translatable findings, better protocols are urgently needed. Therefore, we compare widely used models of hypoxia and HI and propose future directions for the field.

Brain edema is a hallmark of various neuropathologies, but the underlying mechanisms are poorly understood. We aim to characterize how tissue hypoxia, together with oxidative stress and inflammation, leads to capillary dysfunction and breakdown of the blood-brain barrier (BBB). In a mouse stroke model we show that systemic treatment with dimethyl fumarate (DMF), an antioxidant drug clinically used for psoriasis and multiple sclerosis, significantly prevented edema formation in vivo. Indeed, DMF stabilized the BBB by preventing disruption of interendothelial tight junctions and gap formation, and decreased matrix metalloproteinase activity in brain tissue. In vitro, DMF directly sustained endothelial tight junctions, inhibited inflammatory cytokine expression, and attenuated leukocyte transmigration. We also demonstrate that these effects are mediated via activation of the redox sensitive transcription factor NF-E2 related factor 2 (Nrf2). DMF activated the Nrf2 pathway as shown by up-regulation of several Nrf2 target genes in the brain in vivo, as well as in cerebral endothelial cells and astrocytes in vitro, where DMF also increased protein abundance of nuclear Nrf2. Finally, Nrf2 knockdown in endothelial cells aggravated subcellular delocalization of tight junction proteins during ischemic conditions, and attenuated the protective effect exerted by DMF. Overall, our data suggest that DMF protects from cerebral edema formation during ischemic stroke by targeting interendothelial junctions in an Nrf2-dependent manner, and provide the basis for a completely new approach to treat brain edema.

Background and Purpose— Detection and localization of the early phase of blood–brain barrier disruption (BBBD) in vivo during cerebral ischemia/reperfusion injury remain a major challenge but may be a relevant outcome parameter in stroke. Methods— We studied early BBBD in mice after transient middle cerebral artery occlusion by multimodal, high-field (9.4T) in vivo magnetic resonance imaging, including the contrast agent gadofluorineM as an albumin-binding tracer. GadofluorineM contrast-enhanced magnetic resonance imaging was performed to determine BBBD at 2, 6, and 24 hours after reperfusion. BBBD was confirmed and localized along the microvascular tree by using fluorescent gadofluorineM and immunofluorescence stainings (cluster of differentiation 31, ephrin type-B receptor 4, alpha smooth muscle actin, ionized calcium binding adaptor molecule 1). Results— GadofluorineM contrast-enhanced magnetic resonance imaging revealed a multifocal spatial distribution of early BBBD and its close association with the microvasculature at a resolution of 40 μm. GadofluorineM leakage was closely associated with ephrin type-B receptor 4-positive but not alpha smooth muscle actin-positive vessels. The multifocal pattern of early BBBD (already at 2 hours after reperfusion) thus occurred in the distal capillary and venular microvascular bed. These multifocal zones showed distinct imaging signs indicative of early vasogenic edema. The total volume of multifocal early BBBD accurately predicted infarct size at 24 hours after reperfusion. Conclusions— Early BBBD in focal cerebral ischemia initiates multifocally in the distal capillary and venular bed of the cerebral microvasculature. It is closely associated with perimicrovascular vasogenic edema and microglial activation and predicts the extent of final infarction.

Formation of a precise vascular network within the central nervous system is of critical importance to assure delivery of oxygen and nutrients and for accurate functionality of neuronal networks. Vascularization of the spinal cord is a highly stereotypical process. However, the guidance cues controlling blood vessel patterning in this organ remain largely unknown. Here we describe a new neuro-vascular communication mechanism that controls vessel guidance in the developing spinal cord. We show that motor neuron columns remain avascular during a developmental time window, despite expressing high levels of the pro-angiogenic vascular endothelial growth factor (VEGF). We describe that motor neurons express the VEGF trapping receptor sFlt1 via a Neuropilin-1-dependent mechanism. Using a VEGF gain-of-function approach in mice and a motor neuron-specific sFlt1 loss-of-function approach in chicken, we show that motor neurons control blood vessel patterning by an autocrine mechanism that titrates motor neuron-derived VEGF via their own expression of sFlt1., The guidance cues regulating blood vessel patterning in the central nervous system remain unclear. Here, the authors show in mice and chicken developing spinal cord that motor neurons control blood vessel patterning by an autocrine mechanism titrating VEGF via the expression of its trapping receptor sFlt1.

Hypoxia-inducible factors (HIFs) are key transcriptional regulators that play a major role in oxygen homeostasis. HIF activity is tightly regulated by oxygen-dependent hydroxylases, which additionally require iron and 2-oxoglutarate as cofactors. Inhibition of these enzymes has become a novel target to modulate the hypoxic response for therapeutic benefit. Inhibition of prolyl-4-hydroxylase domains (PHDs) have been shown to delay neuronal cell death and protect against ischemic injury in the hippocampus. In this study we have examined the effects of prolyl hydroxylase inhibition on synaptic transmission and plasticity in the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) and excitatory postsynaptic currents (EPSCs) were elicited by stimulation of the Schaffer collateral pathway in the CA1 region of the hippocampus. Treatment of rat hippocampal slices with low concentrations (10 µM) of the iron chelator deferosoxamine (DFO) or the 2-oxoglutarate analogue dimethyloxalyl glycine (DMOG) had no effect on fEPSP. In contrast, application of 1 mM DMOG resulted in a significant decrease in fEPSP slope. Antagonism of the NMDA receptor attenuated the effects of DMOG on baseline synaptic signalling. In rat hippocampal slices pretreated with DMOG and DFO the induction of long-term potentiation (LTP) by tetanic stimulation was strongly impaired. Similarly, neuronal knockout of the single PHD family member PHD2 prevented murine hippocampal LTP. Preconditioning of PHD2 deficient hippocampi with either DMOG, DFO, or the PHD specific inhibitor JNJ-42041935, did not further decrease LTP suggesting that DMOG and DFO influences synaptic plasticity primarily by inhibiting PHDs rather than unspecific effects. These findings provide striking evidence for a modulatory role of PHD proteins on synaptic plasticity in the hippocampus. © 2013 Wiley Periodicals, Inc.

Presenilin (PSEN) 1 and 2 are the catalytic components of the γ-secretase complex, which cleaves a variety of proteins, including the amyloid precursor protein (APP). Proteolysis of APP leads to the formation of the APP intracellular domain (AICD) and amyloid β that is crucially involved in the pathogenesis of Alzheimer's disease. Prolyl-4-hydroxylase-domain (PHD) proteins regulate the hypoxia-inducible factors (HIFs), the master regulators of the hypoxic response. We previously identified the FK506 binding protein 38 (FKBP38) as a negative regulator of PHD2. Genetic ablation of PSEN1/2 has been shown to increase FKBP38 protein levels. Therefore, we investigated the role of PSEN1/2 in the oxygen sensing pathway using a variety of genetically modified cell and mouse lines. Increased FKBP38 protein levels and decreased PHD2 protein levels were found in PSEN1/2-deficient mouse embryonic fibroblasts and in the cortex of forebrain-specific PSEN1/2 conditional double knock-out mice. Hypoxic HIF-1α protein accumulation and transcriptional activity were decreased, despite reduced PHD2 protein levels. Proteolytic γ-secretase function of PSEN1/2 was needed for proper HIF activation. Intriguingly, PSEN1/2 mutations identified in Alzheimer patients differentially affected the hypoxic response, involving the generation of AICD. Together, our results suggest a direct role for PSEN in the regulation of the oxygen sensing pathway via the APP/AICD cleavage cascade.

Background and Purpose— Stroke is triggered by several risk factors, including influenza and other respiratory tract infections. However, it is unknown how and in which way influenza infection affects stroke outcome. Methods— We infected mice intranasally with human influenza A (H1N1) virus and occluded the middle cerebral artery to induce ischemic strokes. Infarct volume and intracerebral hemorrhage were determined by histology. To evaluate the integrity of the blood–brain barrier and inflammation, we measured various cytokines in vivo and in vitro and performed immunohistochemistry of leukocyte markers, collagen IV, immunoglobulins, and matrix metalloproteinase-9. Results— Influenza virus infection increased infarct size. Whereas changes in cardiovascular parameters did not explain this effect, we found evidence for an inflammatory mechanism. In influenza virus infection, the respiratory tract released cytokines into the blood, such as RANTES that induced macrophage inflammatory protein-2 and other inflammatory mediators in the ischemic brain. In infected mice, there was an increased number of neutrophils expressing the matrix metalloproteinase-9 in the ischemic brain. This was accompanied by severe disruption of the blood–brain barrier and an increased rate of intracerebral hemorrhages after tissue plasminogen activator treatment. To investigate the role of cytokines, we blocked cytokine release by using GTS-21, a selective agonist of the α7 nicotinic acetylcholine receptor. GTS-21 ameliorated ischemic brain damage and improved survival. Conclusions— Influenza virus infection triggers a cytokine cascade that aggravates ischemic brain damage and increases the risk of intracerebral hemorrhage after tissue plasminogen activator treatment. Blockade of cytokine production by α7 nicotinic acetylcholine receptor agonists is a novel therapeutic option to treat stroke in a proinflammatory context.

Hyperbaric oxygen (HBO) and normobaric hyperoxia (NBO) protect the brain parenchyma and the cerebral microcirculation against ischemia. We studied their effect on secondary hemorrhage after thrombolysis in two thromboembolic middle cerebral artery occlusion (MCAO) (tMCAO) models. Beginning 60 minutes after tMCAO with either thrombin-induced thromboemboli (TT) or calcium-induced thromboemboli (CT), spontaneously hypertensive rats ( n=96) breathed either air, 100% O2 (NBO), or 100% O2 at 3 bar (HBO) for 1 hour. Immediately thereafter, recombinant tissue plasminogen activator (rt-PA, 9 mg/kg) was injected. Although significant reperfusion was observed after thrombolysis in TT-tMCAO, vascular occlusion persisted in CT-tMCAO. In TT-tMCAO, NBO and HBO significantly reduced diffusion-weighted imaging–magnetic resonance imaging (MRI) lesion volume and postischemic blood–brain barrier (BBB) permeability on postcontrast T1-weighted images. NBO and, significantly more potently, HBO reduced macroscopic hemorrhage on T2* MRI and on corresponding postmortem cryosections. Oxygen therapy lowered hemoglobin content and attenuated activation of matrix metalloproteinases in the ischemic hemisphere. In contrast, NBO and HBO failed to reduce infarct size in CT but both decreased BBB damage and microscopic hemorrhagic transformation. Only HBO reduced hemoglobin extravasation in the ischemic hemisphere. In conclusion, NBO and HBO decrease infarct size after thromboembolic ischemia only if recanalization is successful. As NBO and HBO also reduce postthrombolytic intracerebral hemorrhage, combining the two with thrombolysis seems promising.

Reducing post-stroke disability is the major goal of stroke therapy. Consequently, functional testing is essential in experimental stroke studies to increase the predictive value of animal models. We used several sensory and motor tests to assess functional disability in a mouse model of permanent distal middle cerebral artery occlusion (pdMCAO) that induced mainly cortical infarcts. Gait dynamics were transiently disturbed after pdMCAO as measured by different analysis techniques. Stance and brake duration were shorter after pdMCAO. Consistent with sensory and motor deficits the latency to move was prolonged up to 14 days after pdMCAO and the performance in the corner test and handedness were affected on day 1 or 2 after pdMCAO. Heart rate was decreased and heart rate variability were increased after pdMCAO indicating sympathetic-parasympathetic imbalance. In summary, pdMCAO-induced cortical infarcts lead to clinically relevant sensory, motor and cardiac autonomic dysfunction in mice. The present study provides a basis to explore the potential of functional testing for neuroprotection and neuroregeneration after stroke.

Angiogenesis and neurogenesis are adaptive responses protecting cerebral tissue from hypoxic–ischemic injury. Both processes seem to be governed by hypoxia-induced growth factors, of which vascular endothelial growth factor (VEGF) is a prominent example. The aim of this study was to investigate the influence of VEGF overexpression (V1 mice) on mice cognitive function and cerebral structure under moderate cerebral oligemia. In 33 V1 and wild-type (wt) mice, the left common carotid artery was permanently occluded (CCAO) under acute (48 h) and subchronic (12 days) conditions. Sham operation was performed in 35 mice (controls). Psychometric testing was done using holeboard test and Morris Water Maze system, immunohistochemistry was performed for detection of cerebral apoptosis, nestin and CD31 expression. The results show that under control conditions V1 mice showed better spatial cognitive abilities as compared to their wt littermates. During CCAO, time and distance to reach a hidden platform in Water Maze were shorter in V1 mice as compared to wt animals, indicative of faster learning and better spatial memory processes. While no signs of necrosis or apoptosis were detected, immunohistochemistry showed that VEGF transgenity was related to higher number of nestin-positive precursor cells. Finally, acute CCAO was paralleled by a reduction of CD31 staining in wt but not V1 mice. We conclude that VEGF overexpression led to a protective effect on cognitive function, because V1 mice showed evidence for faster spatial learning and better memory, as well as an increased number of neuronal precursor cells and a prevention of endothelial cell loss after CCAO.

To adequately supply tissues with oxygen and nutrients, the formation of functional vascular networks requires generation of normal, healthy vessels and their arrangement into an effective network architecture. While our knowledge about the development of single vessels significantly increased during the last years, mechanisms responsible for network formation are still poorly understood. This is probably due to the lack of suitable methods for quantification of structural properties of microvascular networks. Previously we showed that cerebral blood flow is not increased in mice exhibiting a 2- to 3-fold higher density of normal and perfused capillaries as a result of transgenic overexpression of the human vascular endothelial growth factor (VEGF(165)). Here we used vascular corrosion casting and hierarchical micro-computed tomography combined with a new network analysis tool to characterize the vascular architecture in gray and white matter of these mice. Our results indicate that VEGF overexpression leads to formation of additional micro-networks connected to higher order vessels rather than insertion of individual capillaries into the existing vessel structure. This implies that the smallest "angiogenic quantum", i.e. the final, stable result of angiogenesis and subsequent remodeling, is not a single microvessel, but a complete micro-network. In conclusion, high-resolution 3D imaging combined with network analysis can substantially improve our understanding of vascular architecture, beneficial for the development of therapeutic angiogenesis as a clinical tool for applications such as wound healing or treatment of ischemic diseases.

Objective Insufficient angiogenesis with tissue ischemia and accumulation of extracellular matrix are hallmarks of systemic sclerosis (SSc). Based on the severely decreased oxygen levels in the skin of patients with SSc, we aimed to investigate the role of hypoxia in the pathogenesis of SSc. Methods Subtractive hybridization was used to compare gene expression in dermal fibroblasts under hypoxic and normoxic conditions. Dermal fibroblasts were further characterized by exposure to different concentrations of oxygen and for different time periods as well as by interference with hypoxia-inducible factor 1α (HIF-1α). The systemic normobaric hypoxia model in mice was used for in vivo analyses. Results Several extracellular matrix proteins and genes involved in extracellular matrix turnover, such as thrombospondin 1, proα2(I) collagen, fibronectin 1, insulin-like growth factor binding protein 3, and transforming growth factor β–induced protein, were induced by hypoxia in SSc and healthy dermal fibroblasts. The induction of these genes was time- and dose-dependent. Experiments with HIF-1α–knockout mouse embryonic fibroblasts, deferoxamine/cobalt ions as chemical stabilizers of HIF-1α, and HIF-1α small interfering RNA consistently showed that extracellular matrix genes are induced in dermal fibroblasts by HIF-1α–dependent, as well as HIF-1α–independent, mechanisms. Using the systemic normobaric hypoxia mouse model, we demonstrated that dermal hypoxia leads to the induction of the identified extracellular matrix genes in vivo after both short exposure and prolonged exposure to hypoxia. Conclusion These data show that hypoxia contributes directly to the progression of fibrosis in patients with SSc by increasing the release of major extracellular matrix proteins. Targeting of hypoxia pathways might therefore be of therapeutic value in patients with SSc.

Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.

We sought genetic evidence for the involvement of neuronal vascular endothelial growth factor (VEGF) in amyotrophic lateral sclerosis (ALS). Mice expressing human ALS mutant superoxide dismutase-1 (SOD1) were crossed with mice that overexpress VEGF in neurons (VEGF+/+). We report that SOD1G93A/VEGF+/+double-transgenic mice show delayed motor neuron loss, delayed motor impairment, and prolonged survival compared with SOD1G93Asingle transgenics. These findings indicate that neuronal VEGF protects against motor neuron degeneration, and may have therapeutic implications for ALS.

Oxidative stress is a hallmark of ischemic stroke pathogenesis causing neuronal malfunction and cell death. Up-regulation of anti-oxidative genes through activation of the NF-E2-related transcription factor 2 (Nrf2) is one of the key mechanisms in cellular defense against oxidative stress. Fumaric acid esters (FAEs) represent a class of anti-oxidative and anti-inflammatory molecules that are already in clinical use for multiple sclerosis therapy. Purpose of this study was to investigate whether FAEs promote neuronal survival upon ischemia, and analyze putative underlying molecular mechanisms in neurons. Murine organotypic hippocampal slice cultures, and two neuronal cell lines were treated with dimethyl fumarate (DMF) and monomethyl fumarate (MMF). Ischemic conditions were generated by exposing cells and slice cultures to oxygen-glucose deprivation (OGD), and cell death was determined through propidium iodide staining. Treatment with both DMF and MMF immediately after OGD during reoxygenation strongly reduced cell death in hippocampal cultures ex vivo. Both DMF and MMF promoted neuronal survival in HT-22 and SH-SY5Y cell lines exposed to ischemic stress. DMF but not MMF activated the anti-oxidative Nrf2 pathway in neurons. Accordingly, Nrf2 knockdown in murine neurons abrogated the protective effect of DMF but not MMF. Moreover, FAEs did not activate the hypoxia-inducible factor (HIF) pathway suggesting that this pathway may not significantly contribute to FAE mediated neuroprotection. Our results may provide the basis for a new therapeutic approach to treat ischemic pathologies such as stroke with a drug that already has a broad safety record in humans.

Background: Both normobaric (NBO) and hyperbaric (HBO) oxygen therapy are protective in transient cerebral ischemia. In contrast, in permanent ischemia models, which reflect the majority of clinical strokes, the effectiveness of NBO is unknown, and the effectiveness of HBO is controversial. The goals of the present study were to compare both oxygen therapies in 2 models of permanent ischemia, to study the effect of time window, and to evaluate the combination of both oxygen therapies. Methods: Distal or proximal permanent occlusion of middle cerebral artery (MCAO) was induced by coagulation or filament, respectively. Mice received air, NBO, a single or repeated HBO (3 ata) treatments. Infarct sizes were quantified at 7 days (coagulation) and 24 h (filament), respectively. Results: Following MCA coagulation, infarct volume was 12.9 ± 1.6 mm 3 in mice breathing air. When started 45 min or 120 min after MCAO, NBO (10.8 ± 2.2) and significantly more potently HBO (7.8 ± 0.9) reduced infarct size. Repeated HBO treatments had no additional effect (8.3 ± 2.3). HBO also significantly decreased TUNEL cell staining at 24 h. Combination of 60 min NBO plus 60 min HBO resulted in smaller cortical infarcts (8.7 ± 1.5) than 120 min NBO alone (11.1 ± 3.2). In contrast, infarct volumes in filament-induced permanent MCAO did not differ among rodents receiving air (50 ± 24 mm 3 ), NBO (48 ± 16), or HBO (46 ± 21). After filament-induced transient MCAO, however, HBO reduced infarct volume significantly. Conclusions: NBO and more effectively HBO protect the brain against permanent cortical ischemia. In extensive focal ischemia, however, oxygen therapy is only effective in case of early recanalization.

Local tissue oxygenation profoundly influences placental development. To elucidate the impact of hypoxia on cellular and molecular adaptation in vivo, pregnant mice at embryonic days 7.5–11.5 were exposed to reduced environmental oxygen (6–7% O2) for various periods of time. Hypoxia-inducible factor (HIF)-1α mRNA was highly expressed in the placenta, whereas HIF-2α was predominantly found in the decidua, indicating that HIF-1 is a relevant oxygen-dependent factor involved in placental development. During severe hypoxia, HIF-1α protein was strongly induced in the periphery but, however, not in the labyrinth layer of the placenta. Accordingly, no indication for tissue hypoxia in this central area was detected with 2-(2-nitro-1 H-imidazol-1-yl)- N-(2,2,3,3,3-pentafluoropropyl)acetamide staining and VEGF expression as hypoxic markers. The absence of significant tissue hypoxia was reflected by preserved placental architecture and trophoblast differentiation. In the search for mechanisms preventing local hypoxia, we found upregulation of endothelial nitric oxide synthase (NOS) expression in the labyrinth layer. Inhibition of NOS activity by Nω-nitro-l-arginine methyl ester application resulted in ubiquitous placental tissue hypoxia. Our results show that placental oxygenation is preserved even during severe systemic hypoxia and imply that NOS-mediated mechanisms are involved to protect the placenta from maternal hypoxia.

Background and Purpose— Hyperbaric oxygen (HBO) has been shown to protect the brain parenchyma against transient focal cerebral ischemia, but its effects on the ischemic microcirculation are largely unknown. We examined the potential of HBO to reduce postischemic blood–brain barrier (BBB) damage and edema. Methods— Wistar rats and C57/BL6 mice underwent occlusion of the middle cerebral artery (MCAO) for 2 hours. Forty minutes after filament introduction, animals breathed either 100% O 2 at 3.0 atmospheres absolute (ata; HBO group) or at 1.0 ata (control) for 1 hour in an HBO chamber. In rats, MRI was performed 15 minutes after MCAO and after 15 minutes and 3, 6, 24, and 72 hours of reperfusion. In mice, BBB permeability for sodium fluorescein was measured after 24-hour reperfusion. Results— Increased BBB permeability on postcontrast T1-weighted (T1w) images had a biphasic pattern. HBO reduced volumes and intensity of enhancement. Mean abnormal enhancing volumes were 71±10 mm 3 (control) versus 47±10 mm 3 (HBO) at 15 minutes; 111±21 mm 3 versus 69±17 mm 3 3 hours; 147±44 mm 3 versus 83±21 mm 3 6 hours; 150±37 mm 3 versus 89±14 mm 3 24 hours; and 322±52 mm 3 versus 215±21 mm 3 72 hours (all P P Conclusions— Intraischemic HBO therapy reduces early and delayed postischemic BBB damage and edema after focal ischemia in rats and mice.

SUMMARYNormal tissue function in mammals depends on adequate supply of oxygen through blood vessels. A discrepancy between oxygen supply and consumption(hypoxia) induces a variety of specific adaptation mechanisms at the cellular,local and systemic level. These mechanisms are in part governed by the activation of hypoxia-inducible transcription factors (HIF-1, HIF-2), which in turn modulate expression of hypoxically regulated genes such as those encoding vascular endothelial growth factor (VEGF) and erythropoietin(EPO).EPO is a glycoprotein that is produced mainly by interstitial fibroblasts in the kidneys of the adult and in hepatocytes in the foetus. Released into the circulation, EPO makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, EPO has emerged as a multifunctional growth factor that plays a significant role in the nervous system. Both EPO and its receptor are expressed throughout the brain in glial cells, neurones and endothelial cells. Hypoxia and ischaemia have been recognised as important driving forces of EPO expression in the brain. EPO has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurones from ischaemic damage and by stimulating endothelial cells and thus supporting the angiogenic effect of VEGF in the nervous system. Thus,hypoxia-induced gene products such as VEGF and EPO might be part of a self-regulated physiological protection mechanism to prevent neuronal injury,especially under conditions of chronically reduced blood flow (chronic ischaemia).In this review, I will briefly summarize the recent findings on the molecular mechanisms of hypoxia-regulated EPO expression in general and give an overview of its expression in the central nervous system, its action as a growth factor with non-haematopoietic functions and its potential clinical relevance in various brain pathologies.

The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.

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.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homeostasis, including erythropoietin, vascular endothelial growth factor, and factors involved in glucose transport and metabolism. The mouse Hif1a gene is expressed from two distinct promoter/first exon combinations resulting in tissue-specific (mHIF-1alphaI.1) and ubiquitous (mHIF-1alphaI.2) mRNA isoforms. By in situ hybridization, we detected mHIF-1alphaI.1 mRNA exclusively in the elongated spermatids of the testis. In vitro studies indicated that the switch from mHIF-1alphaI.2 to mHIF-1alphaI.1 mRNA expression does not occur at the premeiotic stages of mouse spermatogenesis. Exposure of mice to hypoxic conditions induced mHIF-1alphaI.2 protein in spermatocytes and probably in Sertoli cells but not in spermatogonia. In contrast, expression of the putative mHIF-1alphaI.1 protein in spermatozoa of the testis and epididymis was oxygen independent and located to the midpiece of the spermatozoal flagellum. Both the switch in transcript expression during spermiogenesis and the unexpected protein localization in mature sperm cells suggest a so far unrecognized function of HIF-1alpha.

In vivo, hypoxia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Primary cultures of porcine brain derived microvascular endothelial cells were used as an in vitro BBB model to evaluate the mechanisms by which hypoxia regulates paracellular permeability. Paracellular passage across endothelial cell monolayers is regulated by specialized intercellular structures like the tight junctions (TJ). Zonula occludens-1 (ZO-1), a protein of the TJ, lines the cytoplasmic face of intact TJ. The continuity of the ZO-1 expression was disrupted during 24 h of hypoxia which correlated with a decrease of the protein level to 32 +/- 8% and with a twofold increase in the phosphorylation of ZO-1 in comparison to values determined at the start of the experiment. The localization and expression level of ZO-1 were maintained during hypoxia in the presence of a polyclonal antibody to vascular endothelial growth factor (VEGF) demonstrating that hypoxia-induced changes of the ZO-1 expression are mediated by VEGF. The effect of hypoxia on the ZO-1 distribution probably is not tissue- or cell-specific because similar changes of ZO-1 distribution were observed when the rat brain endothelial cell line RBE4 or the murine epithelial cell line CSG was used. Furthermore, ZO-1 changes correlated with small changes in actin distribution. These results suggest that hypoxia increases the paracellular flux across the cell monolayer via the release of VEGF, which in turn leads to the dislocalization, decreased expression, and enhanced phosphorylation of ZO-1. Science.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset, dominantly inherited neurodegenerative disease caused by expansion of a glutamine repeat tract in ataxin-1 (ATXN1). Although the precise function of ATXN1 remains elusive, it seems to be involved in transcriptional repression. We find that mutant ATXN1 represses transcription of the neurotrophic and angiogenic factor vascular endothelial growth factor (VEGF). Genetic overexpression or pharmacologic infusion of recombinant VEGF mitigates SCA1 pathogenesis, suggesting a new therapeutic strategy for this disease.

Ischemic stroke results in disruption of the blood-brain barrier (BBB), edema formation and neuronal cell loss. Some neuroprotective factors such as vascular endothelial growth factor (VEGF) favor edema formation, while others such as erythropoietin (Epo) can mitigate it. Both factors are controlled by hypoxia inducible transcription factors (HIF) and the activity of prolyl hydroxylase domain proteins (PHD). We hypothesize that activation of the adaptive hypoxic response by inhibition of PHD results in neuroprotection and prevention of vascular leakage. Mice, subjected to cerebral ischemia, were pre- or post-treated with the novel PHD inhibitor FG-4497. Inhibition of PHD activity resulted in HIF-1α stabilization, increased expression of VEGF and Epo, improved outcome from ischemic stroke and reduced edema formation by maintaining BBB integrity. Additional in vitro studies using brain endothelial cells and primary astrocytes confirmed that FG-4497 induces the HIF signaling pathway, leading to increased VEGF and Epo expression. In an in vitro ischemia model, using combined oxygen and glucose deprivation, FG-4497 promoted the survival of neurons. Furthermore, FG-4497 prevented the ischemia-induced rearrangement and gap formation of the tight junction proteins zonula occludens 1 and occludin, both in cultured endothelial cells and in infarcted brain tissue in vivo. These results indicate that FG-4497 has the potential to prevent cerebral ischemic damage by neuroprotection and prevention of vascular leakage.

Sphingosine is a major storage compound in Niemann–Pick type C disease (NP–C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP–C patient fibroblasts and NP–C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome–lysosome fusion in NP–C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP–C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP–C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP–C neurons where defective SphK activity is due to impaired VEGF levels., Sphingosine is abnormally accumulated in Niemann–Pick type C disease (NP–C), but the causes of this accumulation have not been fully characterized. Here the authors show that sphingosine kinase activity is reduced in NP–C patient fibroblasts and NP–C mouse neurons due to defective vascular endothelial growth factor levels, suggesting therapeutic avenues.

Erythropoietin (Epo), the major hormone controlling the hypoxia-induced increase in the number of erythrocytes, has also a functional role in the brain. However, few data exist as to the cellular source of brain-derived Epo as well as to the molecular mechanisms that control Epo expression in the central nervous system. Using patch-clamp and RT-PCR methods, we provide direct evidence that, besides astrocytes, neurons are a source of Epo in the brain. Both the astrocytic and neuronal expression of Epo mRNA are induced not only by hypoxia, but also by desferrioxamine (DFX) and cobalt chloride (CoCl(2)), two agents known to mimic the hypoxic induction of Epo in hepatoma cells. This induction is blocked by cycloheximide suggesting that de novo protein synthesis is required. Furthermore, the addition of H(2)O(2) decreases the hypoxia-induced Epo mRNA levels. These data indicate that, following hypoxia, a common oxygen sensing and signaling pathway leads to increased Epo gene expression in both nervous and hepatoma cells; this pathway would be dependent on the redox-state of the brain. Furthermore, we show that the in vivo administration of CoCl(2) and DFX to mice induces an increased Epo mRNA level in the neocortex. As Epo protects the brain against ischemia, our in vivo experiments suggest that the use of molecules such as CoCl(2) or DFX, that provoke an increased Epo gene expression in the brain, could be useful in the development of potential therapeutic strategies for the treatment of hypoxic or ischemic brain injury.

The tissue type plasminogen activator (t-PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t-PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI-1), the protease nexin-1 (PN-1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI-1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor-β1 (TGF-β1) in NMDA-induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT-PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI-1 without any effect on PN-1, NSP, or t-PA. Then we showed that although the expression of PAI-1 is restricted to astrocytes, PN-1, NSP, and t-PA are expressed in both neurons and astrocyt...

The present study describes, for the first time, a temporal and spatial cellular expression of erythropoietin (Epo) and Epo receptor (Epo-R) with the evolution of a cerebral infarct after focal permanent ischemia in mice. In addition to a basal expression of Epo in neurons and astrocytes, a postischemic Epo expression has been localized specifically to endothelial cells (1 day), microglia/macrophage-like cells (3 days), and reactive astrocytes (7 days after occlusion). Under these conditions, the Epo-R expression always precedes that of Epo for each cell type. These results support the hypothesis that there is a continuous formation of Epo, with its corresponding receptor, during the active evolution of a focal cerebral infarct and that the Epo/Epo-R system might be implicated in the processes of neuroprotection and restructuring (such as angiogenesis and gliosis) after ischemia. To support this hypothesis, a significant reduction in infarct volume (47%; P < 0.0002) was found in mice treated with recombinant Epo 24 hours before induction of cerebral ischemia. Based on the above, we propose that the Epo/Epo-R system is an endogenous mechanism that protects the brain against damages consequent to a reduction in blood flow, a mechanism that can be amplified by the intracerebroventricular application of exogenous recombinant Epo.

SummaryAngiogenic growth factors and their endothelial receptors function as major regulators of blood vessel formation. The VEGF/VEGFR and the Angiopoietin/Tie2 receptor systems represent key signal transduction pathways involved in the regulation of embryonic vascular development. Inactivation of any of the genes encoding these molecules results in defective vascular development and lethality between embryonic day 8.5 and 12.5. In addition, VEGF and its receptors are also critically involved in the regulation of pathological blood vessel growth in the adult during various angiogenesis-dependent diseases that are associated with tissue hypoxia, such as solid tumor growth and ischemic diseases. It is now well established that therapeutic angiogenesis can be achieved in animal models of hind limb and myocardial ischemia by exogenously adding VEGF and/or other angiogenic growth factors. Available clinical data from human trials also suggests that patients with severe cardiovascular diseases could potentially benefit from such therapies. However, much more work needs to be done to compare the potency of different angiogenic factors or the combination thereof, as well as the best way of delivery, either as recombinant proteins, as naked DNA or via adenoviral vectors. Nevertheless, the therapeutic efficacy of simply injecting naked plasmid DNA or proteins into ischemic tissue to deliver secreted angiogenic factors is an encouraging finding. Time will show whether the adverse side effects of therapeutic angiogenesis, mainly vascular permeability and edema formation, can be minimized and angiogenic factors can be used as an effective therapy in patients for the treatment of ischemic diseases such as arterial occlusive disease, myocardial infarction, and, eventually, also stroke.

Hemangioblastomas are highly vascular tumors of the central nervous system that overexpress the hypoxia-inducible gene, vascular endothelial growth factor (VEGF), as a consequence of mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL). Previous reports showed that hemangioblastomas can also express erythropoietin (Epo), which is also hypoxia-inducible. However, Epo expression in hemangioblastomas was observed only in individual cases, and the analyses were mainly based on indirect determination of erythropoiesis-stimulating activity. Therefore, we analyzed a series of 11 hemangioblastomas for Epo, VEGF, and VHL expression by Northern blot analysis and compared the results with normal brain and glioblastomas. Surprisingly, we observed Epo mRNA expression in all hemangioblastoma specimens analyzed, but in none of four glioblastomas. In contrast, VEGF mRNA was expressed in all hemangioblastomas and all glioblastomas. In situ hybridization revealed neoplastic stromal cells as Epo- and VEGF-producing cells in hemangioblastomas. These results suggest that in the nonhypoxic microenvironment of hemangioblastoma, Epo, similar to VEGF, might be negatively regulated by the VHL gene product.© 1998 by The American Society of Hematology.

The fetal liver is the main hematopoietic organ during intrauterine life. Morphometrical studies were performed on liver sections to detect changes occurring with intrauterine growth retardation and preeclampsia. Compared with the controls (n = 10), fetuses from preeclamptic mothers showed a severe reduction of erythroid cells by 60% on average (n = 18). Closer examination revealed that the erythroid cells at early stages of differentiation were more affected (80% reduction) than at later stages (55%). Seven out of 18 fetuses from preeclamptic mothers did not show growth retardation but exhibited severely reduced hepatic erythropoiesis. We suggest that the prime factor for impaired red blood cell production is preeclampsia itself rather than intrauterine growth retardation. Regulation of erythropoiesis in utero might depend on the interaction of many hematopoietic growth factors, and preeclampsia might alter the balance. To test this notion, we quantitated erythropoietin in fetal blood and various cytokines in the amniotic fluid. An elevation of erythropoietin and interleukin (IL)-3 levels was seen in babies born under the conditions of preeclampsia, whereas the concentrations of granulocyte/macrophage-colony-stimulating factor (CSF), granulocyte-CSF, and IL-1 beta were reduced, and the levels of IL-6 and IL-8 remained constant. With preeclampsia, a discrepancy between elevation of erythrocyte numbers in peripheral blood and depression of hematopoiesis at the main production site, the fetal liver, is seen. Concomitantly, there is elevation of some but reduction of other hematopoietic cytokines. We envision that during the course of preeclampsia quantitation of hematopoietic growth factors might allow to predict the deterioration of in utero life conditions.

Hypoxia-inducible factors (HIFs) are key transcriptional regulators that play a major role in oxygen homeostasis. HIF activity is tightly regulated by oxygen-dependent hydroxylases, which additionally require iron and 2-oxoglutarate as cofactors. Inhibition of these enzymes has become a novel target to modulate the hypoxic response for therapeutic benefit. Inhibition of prolyl-4-hydroxylase domains (PHDs) have been shown to delay neuronal cell death and protect against ischemic injury in the hippocampus. In this study we have examined the effects of prolyl hydroxylase inhibition on synaptic transmission and plasticity in the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) and excitatory postsynaptic currents (EPSCs) were elicited by stimulation of the Schaffer collateral pathway in the CA1 region of the hippocampus. Treatment of rat hippocampal slices with low concentrations (10 µM) of the iron chelator deferosoxamine (DFO) or the 2-oxoglutarate analogue dimethyloxalyl glycine (DMOG) had no effect on fEPSP. In contrast, application of 1 mM DMOG resulted in a significant decrease in fEPSP slope. Antagonism of the NMDA receptor attenuated the effects of DMOG on baseline synaptic signalling. In rat hippocampal slices pretreated with DMOG and DFO the induction of long-term potentiation (LTP) by tetanic stimulation was strongly impaired. Similarly, neuronal knockout of the single PHD family member PHD2 prevented murine hippocampal LTP. Preconditioning of PHD2 deficient hippocampi with either DMOG, DFO, or the PHD specific inhibitor JNJ-42041935, did not further decrease LTP suggesting that DMOG and DFO influences synaptic plasticity primarily by inhibiting PHDs rather than unspecific effects. These findings provide striking evidence for a modulatory role of PHD proteins on synaptic plasticity in the hippocampus.