Hugo H. Marti

Erythropoietin Gene Expression in Human, Monkey and Murine Brain

The haematopoietic growth factor erythropoietin is the primary regulator of mammalian erythropoiesis and is produced by the kidney and the liver in an oxygen‐dependent manner. We and others have recently demonstrated erythropoietin gene expression in the rodent brain. In this work, we show that cerebral erythropoietin gene expression is not restricted to rodents but occurs also in the primate brain. Erythropoietin mRNA was detected in biopsies from the human hippocampus, amygdala and temporal cortex and in various brain areas of the monkey Macaca mulatta. Exposure to a low level of oxygen led to elevated erythropoietin mRNA levels in the monkey brain, as did anaemia in the mouse brain. In addition, erythropoietin receptor mRNA was detected in all brain biopsies tested from man, monkey and mouse. Analysis of primary cerebral cells isolated from newborn mice revealed that astrocytes, but not microglia cells, expressed erythropoietin. When incubated at 1% oxygen, astrocytes showed >l OO‐fold time‐dependent erythropoietin mRNA accumulation, as measured with the quantitative reverse transcription‐polymerase chain reaction. The specificity of hypoxic gene induction in these cells was confirmed by quantitative Northern blot analysis showing hypoxic up‐regulation of mRNA encoding the vascular endothelial growth factor, but not of other genes. These findings demonstrate that erythropoietin and its receptor are expressed in the brain of primates as they are in rodents, and that, at least in mice, primary astrocytes are a source of cerebral erythropoietin expression which can be up‐regulated by reduced oxygenation.

Brain Protection by Erythropoietin: A Manifold Task

Many hematopoietic growth factors are produced locally in the brain. Among these, erythropoietin (Epo), has a dominant role for neuroprotection, neurogenesis, and acting as a neurotrophic factor in the central nervous system. These functions make erythropoietin a good candidate for treating diseases associated with neuronal cell death.

Neuron-Specific Prolyl-4-Hydroxylase Domain 2 Knockout Reduces Brain Injury After Transient Cerebral Ischemia

Background and Purpose— Numerous factors involved in the adaptive response to hypoxia, including erythropoietin and vascular endothelial growth factor are transcriptionally regulated by hypoxia-inducible factors (HIFs). During normoxia, prolyl-4-hydroxylase domain (PHD) proteins hydroxylate HIF-&agr; subunits, resulting in their degradation. We investigated the effect of neuronal deletion of PHD2, the most abundant isoform in brain, for stroke outcome. Methods— We generated neuron-specific Phd2 knockout mice and subjected animals to systemic hypoxia or transient middle cerebral artery occlusion. Infarct volume and cell death were determined by histology. HIF-1&agr;, HIF-2&agr;, and HIF target genes were analyzed by immunoblotting and real-time polymerase chain reaction, respectively. Results— Neuron-specific ablation of Phd2 significantly increased protein stability of HIF-1&agr; and HIF-2&agr; in the forebrain and enhanced expression of the neuroprotective HIF target genes erythropoietin and vascular endothelial growth factor as well as glucose transporter and glycolysis-related enzymes under hypoxic and ischemic conditions. Mice with Phd2-deficient neurons subjected to transient cerebral ischemia exhibited a strong reduction in infarct size, and cell death of hippocampal CA1 neurons located in the peri-infarct region was dramatically reduced in these mice. Vessel density in forebrain subregions, except for caudate–putamen, was not altered in Phd2-deficient animals. Conclusions— Our findings denote that the endogenous adaptive response on hypoxic–ischemic insults in the brain is at least partly dependent on the activity of HIFs and identify PHD2 as the key regulator for the protective hypoxia response. The results suggest that specific inhibition of PHD2 may provide a useful therapeutic strategy to protect brain tissue from ischemic injury.

Influenza Virus Infection Aggravates Stroke Outcome

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 &agr;7 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 &agr;7 nicotinic acetylcholine receptor agonists is a novel therapeutic option to treat stroke in a proinflammatory context.

Fetuses from preeclamptic mothers show reduced hepatic erythropoiesis.

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-1β 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.

Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations

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 Alzheimers 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.

Early Blood–Brain Barrier Disruption in Ischemic Stroke Initiates Multifocally Around Capillaries/Venules

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 &mgr;m. 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.

Oxygen sensors and neuronal adaptation to ischemia

Hypoxia research is in the focus, as three of its pioneers receive the Lasker Prize 2016 for their discovery of hypoxia-inducible transcription factors (HIFs) and prolyl-4-hydroxlase domain proteins (PHDs) as cellular oxygen sensors [1]. HIFs are heterodimers consisting of a labile α and a stable β unit. In the presence of molecular oxygen the α subunit is hydroxylated by PHDs leading to immediate proteasomal degradation. In cerebral ischemia, brain hypoperfusion results in tissue hypoxia, combined with nutrient depletion. Given that neuronal energy generation primarily relies on oxidative glucose metabolism, and their striking susceptibility to excitotoxicity, neurons exhibit the highest vulnerability to ischemic stress among all cells of the central nervous system. PHDs control endogenous mechanisms in nerve cells that promote adaptation to hypoxia/ischemia. As PHDs have many additional targets beside HIFα subunits, it remains to be established whether PHD-mediated effects are HIF-dependent or rather HIF-independent. Our major finding is that neuronal inactivation of PHD2 in mice improves recovery from cerebral ischemia [2, 3]. One could hypothesize that this protective effect is either a direct one or alternatively occurs in an indirect manner, whereby PHD2 deficient neurons produce factors that act in a paracrine fashion on neighboring endothelial cells, astrocytes or microglia. The resulting activation/ modulation of these cells could then in turn support survival of neurons. We and others have demonstrated experimental evidence for both-direct and indirect-protective mechanisms. PHD2 inactivation in neurons resulted in HIF stabilization and subsequent transcriptional activation of erythropoietin and vascular endothelial growth factor (VEGF) [2, 3]. Both mediators are well known to be potent anti-apoptotic factors, which directly support neuronal survival. Accordingly, application of the PHD inhibitor FG-4497 strongly increased cell survival in pure neuronal cultures exposed to ischemic conditions [4]. In addition, neuronal PHD2 deletion resulted in increased expression of HIF-dependent glucose transporters and glycolytic enzymes [2]. A switch from oxidative to glycolytic metabolism might improve ischemic tolerance of PHD2 deficient neuronal cells by enabling oxygen-independent energy generation, and reduction of mitochondrial reactive oxygen species (ROS) production. Recently, Quaegebeur et al. showed that genetic loss or inhibition of PHD1 in mice improves brain tissue damage and sensorimotor deficit upon focal cerebral ischemia by reprogramming neuronal glucose metabolism [5]. PHD1 deficient neurons maintained energy production via oxidative phosphorylation in mitochondria, but preferentially metabolized glutamine instead of glucose. PHD1 inactivation in neurons further redirected glucose away from glycolysis into the oxidative pentose phosphate pathway (oxPPP), which preserved …