Heike Beck

Essential Role for Mitochondrial Thioredoxin Reductase in Hematopoiesis, Heart Development, and Heart Function

ABSTRACT Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2 TrxR2−/− minus;/TrxR2−/− minus; embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2 TrxR2−/− minus;/TrxR2−/− minus; embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreichs ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.

Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4−/− mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4−/− mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

Cell Type Specific Upregulation of Vascular Endothelial Growth Factor in an MCA-occlusion Model of Cerebral Infarct

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.

Angiogenesis after cerebral ischemia.

Though the vascular system of the adult brain is extremely stable under normal baseline conditions, endothelial cells start to proliferate in response to brain ischemia. The induction of angiogenesis, primarily in the ischemic boundary zone, enhances oxygen and nutrient supply to the affected tissue. Additionally, the generation of new blood vessels facilitates highly coupled neurorestorative processes including neurogenesis and synaptogenesis which in turn lead to improved functional recovery. To take advantage of angiogenesis as a therapeutic concept for stroke treatment, the knowledge of the precise molecular mechanisms is mandatory. Especially, since a couple of growth factors involved in post-ischemic angiogenesis may have detrimental adverse effects in the brain by increasing vascular permeability. This article summarizes the knowledge of molecular mechanisms of angiogenesis following cerebral ischemia. Finally, experimental pharmacological and cellular approaches to stimulate and enhance post-ischemic angiogenesis are discussed.

Impaired brain angiogenesis and neuronal apoptosis induced by conditional homozygous inactivation of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is essential for the differentiation of the primitive embryonic vascular system and has been implicated in the vascularization of organs. Recently, VEGF has also been proposed to play a role in neural development, neuroprotection, and adult neurogenesis. Here we have investigated the function of VEGF in the developing brain by cre-lox technology. We show that VEGF produced by the embryonic neuroectoderm is required for the vascularization and the development of the brain. Both the invasion and the directed growth of capillaries were severely impaired in the fore-, mid- and hindbrain of VEGF(lox/lox)/nestin-cre mouse embryos homozygous for a VEGF mutation in the neural tube. These observations demonstrate that VEGF, via local secretion by neural progenitors, induces brain angiogenesis and guides the growth of capillaries toward the ventricular zone. VEGF deficiency led to developmental retardation and progressive destruction of neural tissue in all brain regions. The defect was most pronounced in telencephalic structures, such as the hippocampus, and caused microcephaly. Taken together, the findings establish the critical importance of neuroectoderm-derived VEGF in the morphogenesis of the brain. VEGF acts as a key regulator of brain angiogenesis and provides instructive cues for the correct spatial organization of the vasculature.

Embryonic endothelial progenitor cells expressing a broad range of proangiogenic and remodeling factors enhance vascularization and tissue recovery in acute and chronic ischemia

Clonal embryonic endothelial progenitor cells (eEPCs) isolated from embryonic day 7.5 mice home specifically to hypoxic areas in mouse tumor metastases but spare normal organs and do not form carcinomas. Based on these results, we assessed the potential of eEPCs to enhance vascularization and limit organ dysfunction after ischemia in syngenic and xenotypic organisms. The angiogenic potential of eEPCs was evaluated in chronic ischemic rabbit hindlimbs after regional application by retroinfusion. eEPC treatment improved limb perfusion, paralleled by an increase in capillary density and collateral blood vessel number. Systemic eEPC infusion into mice after ischemic cardiac insult increased postischemic heart output measured by a marked improvement in left ventricle developed pressure and both systolic and diastolic functions. In vitro, eEPCs strongly induced vascular outgrowths from aortic rings. To address the molecular basis of this intrinsic angiogenic potential, we investigated the eEPC transcriptome. Genomewide Affymetrix GeneChip analysis revealed that the eEPCs express a wealth of secreted factors known to induce angiogenesis, tissue remodeling, and organogenesis that may contribute to the eEPC‐mediated beneficial effects. Our findings show that eEPCs induce blood vessel growth and cardioprotection in severe ischemic conditions providing a readily available source to study the mechanisms of neovascularization and tissue recovery.

Cell type specific expression of vascular endothelial growth factor and angiopoietin-1 and -2 suggests an important role of astrocytes in cerebellar vascularization.

The vascularization of the central nervous system occurs by angiogenic sprouting, a process in which different factors like vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1/2 must act in a coordinated fashion. We investigated how these factors participate in capillarization of the cerebellum, an area experiencing marked reorganization processes during its postnatal development. VEGF and Ang-1 mRNA were predominantly expressed by astrocytes, while Ang-2 mRNA was specifically induced at the tip of invading endothelial cell cords. Similar to the cerebral cortex, vascularization of the cerebellum occurred in an inside-out pattern, following closely the generation and differentiation of each cerebellar layer. VEGF and Ang-1/2 expression patterns shifted in a similar inside-out fashion, supporting their proposed function in vessel growth and maturation.

Participation of Bone Marrow-Derived Cells in Long-Term Repair Processes after Experimental Stroke

Bone marrow-derived cells participate in remodeling processes of many ischemia-associated diseases, which has raised hopes for the use of bone marrow as a source for cell-based therapeutic approaches. To study the participation of bone marrow-derived cells in a stroke model, bone marrow from C57BL/6-TgN(ACTbEGFP)1Osb mice that express green fluorescent protein (GFP) in all cells was transplanted into C57BL/6J mice. The recipient mice underwent permanent occlusion of the middle cerebral artery, and bone marrow-derived cells were tracked by fluorescence. The authors investigated the involvement of bone marrow-derived cells in repair processes 6 weeks and 6 months after infarction. Six weeks after occlusion of the artery, more than 90% of the GFP-positive cells in the infarct border zone were microglial cells. Very few GFP-positive cells expressed endothelial markers in the infarct/infarct border zone, and no bone marrow-derived cells transdifferentiated into astrocytes, neurons, or oligodendroglial cells at all time points investigated. The results indicate the need for additional experimental studies to determine whether therapeutic application of nonselected bone marrow will replenish brain cells beyond an increase in microglial engraftment.

The Role of Thioredoxin Reductases in Brain Development

The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells.

Loss of Thioredoxin Reductase 1 Renders Tumors Highly Susceptible to Pharmacologic Glutathione Deprivation

Tumor cells generate substantial amounts of reactive oxygen species (ROS), engendering the need to maintain high levels of antioxidants such as thioredoxin (Trx)- and glutathione (GSH)-dependent enzymes. Exacerbating oxidative stress by specifically inhibiting these types of ROS-scavenging enzymes has emerged as a promising chemotherapeutic strategy to kill tumor cells. However, potential redundancies among the various antioxidant systems may constrain this simple approach. Trx1 and thioredoxin reductase 1 (Txnrd1) are upregulated in numerous cancers, and Txnrd1 has been reported to be indispensable for tumorigenesis. However, we report here that genetic ablation of Txnrd1 has no apparent effect on tumor cell behavior based on similar proliferative, clonogenic, and tumorigenic potential. This finding reflects widespread redundancies between the Trx- and GSH-dependent systems based on evidence of a bypass to Txnrd1 deficiency by compensatory upregulation of GSH-metabolizing enzymes. Because the survival and growth of Txnrd1-deficient tumors were strictly dependent on a functional GSH system, Txnrd1-/- tumors were highly susceptible to experimental GSH depletion in vitro and in vivo. Thus, our findings establish for the first time that a concomitant inhibition of the two major antioxidant systems is highly effective in killing tumor, highlighting a promising strategy to combat cancer.