Erythropoietin reduces experimental autoimmune encephalomyelitis severity via neuroprotective mechanisms.

BACKGROUND: Treatment with erythropoietin (Epo) in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis (MS), has consistently been shown to ameliorate disease progression and improve overall outcome. The effect has been attributed to modulation of the immune response and/or preservation of the central nervous system (CNS) tissue integrity. It remains unclear, however, if (a) Epo acts primarily in the CNS or the periphery and if (b) Epo's beneficial effect in EAE is mainly due to maintaining CNS tissue integrity or to modulation of the immune response. If Epo acts primarily by modulating the immune system, where is this modulation required? In the periphery, the CNS or both? METHODS: To address these questions, we used two well-characterized transgenic mouse strains that constitutively overexpress recombinant human Epo (rhEpo) either systemically (tg6) or in CNS only (tg21) in a MOG-induced EAE model. We assessed clinical severity, disease progression, immunomodulation, and CNS tissue integrity, including neuronal survival. RESULTS: Although disease onset remained unaffected, EAE progression was alleviated in transgenic animals compared to controls with both lines performing equally well showing that expression of Epo in the periphery is not required; Epo expression in the CNS is sufficient. Immunomodulation was observed in both strains but surprisingly the profile of modulation differed substantially between strains. Modulation in the tg21 strain was limited to a reduction in macrophages in the CNS, with no peripheral immunomodulatory effects observed. In contrast, in the tg6 strain, macrophages were upregulated in the CNS, and, in the periphery of this strain, T cells and macrophages were downregulated. The lack of a consistent immunomodulatory profile across both transgenic species suggests that immunomodulation by Epo is unlikely to be the primary mechanism driving amelioration of EAE. Finally, CNS tissue integrity was affected in all strains. Although myelin appeared equally damaged in all strains, neuronal survival was significantly improved in the spinal cord of tg21 mice, indicating that Epo may ameliorate EAE predominantly by protecting neurons. CONCLUSIONS: Our data suggests that moderate elevated brain Epo levels provide clinically significant neuroprotection in EAE without modulation of the immune response making a significant contribution.

HIF prolyl hydroxylase inhibition prior to transient focal cerebral ischaemia is neuroprotective in mice.

This study investigated the effects of 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8- to 12-week-old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood-brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up-regulated HIF-1α, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up-regulated HIF-1α and delocalized ZO-1. Pre-treating IOX3 on RBE4 cells 24 h before oxygen-glucose deprivation had a protective effect on endothelial barrier preservation with ZO-1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia. We show that IOX3, a selective small molecule (280.66 Da) HIF prolyl hydroxylase inhibitor, could up-regulate HIF-1α and increase erythropoietin expression in mice. We further demonstrate that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of blood-brain barrier (BBB) protection, while immediate application is detrimental both in vivo and in vitro. These findings provide new insights into the role of HIF stabilization in ischaemic stroke.

Cell-specific blood-brain barrier regulation in health and disease: a focus on hypoxia.

The blood-brain barrier (BBB) is a complex vascular structure consisting of microvascular endothelial cells that line the vessel wall, astrocyte end-feet, pericytes, as well as the basal lamina. BBB cells act in concert to maintain the characteristic impermeable and low paracellular flux of the brain vascular network, thus ensuring a homeostatic neuronal environment. Alterations in BBB stability that occur during injury have dire consequences on disease progression and it is clear that BBB cell-specific responses, positive or negative, must make a significant contribution to injury outcome. Reduced oxygenation, or hypoxia, is a characteristic of many brain diseases that significantly increases barrier permeability. Recent data suggest that hypoxia-inducible factor (HIF-1), the master regulator of the hypoxic response, probably mediates many hypoxic effects either directly or indirectly via its target genes. This review discusses current knowledge of physiological cell-specific regulation of barrier function, their responses to hypoxia as well as consequences of hypoxic- and HIF-1-mediated mechanisms on barrier integrity during select brain diseases. In the final sections, the potential of current advances in targeting HIF-1 as a therapeutic strategy will be overviewed.

Constitutive excessive erythrocytosis causes inflammation and increased vascular permeability in aged mouse brain.

Excessive erythrocytosis results in severely increased blood viscosity that may compromise the vascular endothelium. Using our transgenic mouse model of excessive erythrocytosis we previously showed that despite altered brain endothelial cell morphology and an activated vasculature, brain vascular integrity was largely maintained up to 4-5 months of age. We now present data showing that persistent long-term damage of the vascular wall during the later stages of adulthood (9-12 months) results in a chronic detrimental inflammatory phenotype and increased vessel permeability that likely contributes to the reduced life span of our erythropoietin overexpressing transgenic mouse. In aged transgenic animals inflammatory cells were detected in brain tissue and elevated RNA and protein levels of inflammatory markers such as IL-6 and TNFα were observed in both brain tissue and blood plasma. Additionally, increased expression of p53 and other pro-apoptotic markers, as well as decreased Bcl-xL expression in the brain vasculature, indicated ongoing cell death within the vascular compartment. Finally, abnormally elevated vascular permeability in all organs was detected in correlation with decreased expression of the tight junction protein occludin and the adherens junction protein ß-catenin in brain. Thus chronic erythrocytosis results in sustained activation of inflammatory pathways, vascular dysfunction and blood-brain barrier disruption.

Contribution of hypoxia to Alzheimer's disease: is HIF-1alpha a mediator of neurodegeneration?

The mammalian brain is extremely sensitive to alterations in cellular homeostasis as a result of environmental or physiological insults. In particular, hypoxic/ischemic challenges (i.e. reduced oxygen and/or glucose delivery) cause severe and detrimental alterations in brain function and can trigger neuronal cell death within minutes. Unfortunately, as we age, oxygen delivery to cells and tissues is impaired, thereby increasing the susceptibility of neurons to damage. Thus, hypoxic (neuronal) adaptation is significantly compromised during aging. Many neurological diseases, such as stroke, Alzheimer's disease (AD), Parkinson's disease and diabetes, are characterized by hypoxia, a state that is believed to only exacerbate disease progression. However, the contribution of hypoxia and hypoxia-mediated pathways to neurodegeneration remains unclear. This review discusses current evidence on the contribution of oxygen deprivation to AD, with an emphasis on hypoxia inducible transcription factor-1 (HIF-1)-mediated pathways and the association of AD with the cytoskeleton regulator cyclin-dependent kinase 5.

Maintaining blood-brain barrier integrity: pericytes perform better than astrocytes during prolonged oxygen deprivation.

The blood-brain barrier (BBB), consisting of specialized endothelial cells surrounded by astrocytes and pericytes, plays a crucial role in brain homeostasis. Many cerebrovascular diseases are associated with BBB breakdown and oxygen (O(2)) deprivation constitutes a critical factor that onsets its disruption. We investigated the impact of astrocytes and pericytes on brain endothelial cell permeability and survival during different degrees of O(2) deprivation. Prolonged exposure to 1% O(2) caused barrier breakdown and exposure to 0.1% O(2) dramatically accelerated disruption and induced cell death, mediated at least in part via caspase-3 activation. Reoxygenation allowed only cells exposed to 1% O(2) to re-establish barrier function. Notably co-culture with astrocytes and pericytes substantially enhanced barrier function under normoxic conditions, and produced differential responses during O(2) deprivation. At 1% O(2) astrocytes partially maintained barrier integrity whereas pericytes accelerated its disruption in the short-term, having positive effects only after prolonged exposure. Unexpectedly, at 0.1% O(2) pericytes were more effective than astrocytes in preserving barrier function although the protection afforded by both cells involved inhibition of caspase-3 pathways. Furthermore, cell-specific regulation of auto- and paracrine VEGF signaling pathways were also in part responsible for the differential modulation of barrier function. Our data suggests that cellular cross-talk within the neurovascular unit is crucial for preservation of barrier integrity and that pericytes, not astrocytes, play a significant role during severe and prolonged O(2) deprivation.

Chronic excessive erythrocytosis induces endothelial activation and damage in mouse brain.

Excessive erythrocytosis results in severely increased blood viscosity, which may have significant detrimental effects on endothelial cells and, ultimately, function of the vascular endothelium. Because blood-brain barrier stability is crucial for normal physiological function, we used our previously characterized erythropoietin-overexpressing transgenic (tg6) mouse line (which has a hematocrit of 0.8-0.9) to investigate the effect of excessive erythrocytosis on vessel number, structure, and integrity in vivo. These mice have abnormally high levels of nitric oxide (NO), a potent proinflammatory molecule, suggesting altered vascular permeability and function. In this study, we observed that brain vessel density of tg6 mice was significantly reduced (16%) and vessel diameter was significantly increased (15%) compared with wild-type mice. Although no significant increases in vascular permeability under normoxic or acute hypoxic conditions (8% O2 for 4 h) were detected, electron-microscopic analysis revealed altered morphological characteristics of the tg6 endothelium. Tg6 brain vascular endothelial cells appeared to be activated, with increased luminal protrusions reminiscent of ongoing inflammatory processes. Consistent with this observation, we detected increased levels of intercellular adhesion molecule-1 and von Willebrand factor, markers of endothelial activation and damage, in brain tissue. We propose that chronic excessive erythrocytosis and sustained high hematocrit cause endothelial damage, which may, ultimately, increase susceptibility to vascular disease.

Pivotal role of reduced glutathione in oxygen-induced regulation of the Na(+)/K(+) pump in mouse erythrocyte membranes.

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na(+)/K(+) pump, K(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na(+)/K(+) pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na(+)-loaded cells, revealing no effect of O(2) on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na(+)/K(+) pump activity.

Neuronal VEGF expression correlates with angiogenesis in postnatal developing rat brain.

When exposed to chronic sublethal hypoxia the developing brain responds with increases in permeability and angiogenesis. Vascular endothelial growth factor (VEGF) may mediate this response. Here, we present data on the localization of VEGF in the rat brain cortex during postnatal development and its correlation to vascularization. We reared newborn rats under normoxic conditions and in hypoxic chambers (FiO(2) 9.5%), removed them at postnatal days (P) 3, 8, 13, 24, and 33 and prepared the cortical brain tissue for immunohistochemistry, in situ hybridization (ISH), Western blot analyses and vessel density counting. When compared to age-matched controls, hypoxic-reared animals displayed a significant increase in platelet endothelial cell adhesion molecule 1 (PECAM-1) protein levels, cerebral microvascular lumen diameter and number and density of vessels (number of capillaries per area). In control animals, ISH and immunohistochemistry revealed that localization of VEGF is restricted almost exclusively to cortical neurons at early stages of development. As the vascular bed begins to stabilize, predominant VEGF expression switches to maturing glial cells which invest vessels while neuronal expression is reduced to a basal level. In hypoxic animals, early localization of VEGF is also restricted to cortical neurons, however, during later developmental stages, glial cells express elevated levels of VEGF protein and high neuronal expression also persists. Thus chronic sublethal hypoxia disrupts the temporal-spatial expression of VEGF, which correlates with continuing hypoxia-driven angiogenesis.