ß1 integrins play a critical role maintaining vascular integrity in the hypoxic spinal cord, particularly in white matter.

Interactions between extracellular matrix (ECM) proteins and ß1 integrins play an essential role maintaining vascular integrity in the brain, particularly under vascular remodeling conditions. As blood vessels in the spinal cord are reported to have distinct properties from those in the brain, here we examined the impact of ß1 integrin inhibition on spinal cord vascular integrity, both under normoxic conditions, when blood vessels are stable, and during exposure to chronic mild hypoxia (CMH), when extensive vascular remodeling occurs. We found that a function-blocking ß1 integrin antibody triggered a small degree of vascular disruption in the spinal cord under normoxic conditions, but under hypoxic conditions, it greatly enhanced (20-fold) vascular disruption, preferentially in spinal cord white matter (WM). This resulted in elevated microglial activation as well as marked loss of myelin integrity and reduced density of oligodendroglial cells. To understand why vascular breakdown is localized to WM, we compared expression levels of major BBB components of WM and grey matter (GM) blood vessels, but this revealed no obvious differences. Interestingly however, hypoxyprobe staining demonstrated that the most severe levels of spinal cord hypoxia induced by CMH occurred in the WM. Analysis of brain tissue revealed a similar preferential vulnerability of WM tracts to show vascular disruption under these conditions. Taken together, these findings demonstrate an essential role for ß1 integrins in maintaining vascular integrity in the spinal cord, and unexpectedly, reveal a novel and fundamental difference between WM and GM blood vessels in their dependence on ß1 integrin function during hypoxic exposure. Our data support the concept that the preferential WM vulnerability described may be less a result of intrinsic differences in vascular barrier properties between WM and GM, and more a consequence of differences in vascular density and architecture.

Cerebral arterioles express the laminin subunits α4 and α5 in conjunction with α6ß4 integrin, but strongly downregulate laminin α4 during hypoxia-induced arteriogenic remodeling.

Previous studies have shown that expression of the endothelial laminin receptor α6ß4 integrin in the brain is uniquely restricted to arterioles. As exposure to chronic mild hypoxia (CMH, 8 % O2) stimulates robust angiogenic and arteriogenic remodeling responses in the brain, the goal of this study was to determine how CMH influences cerebrovascular expression of the ß4 integrin as well as its potential ligands, laminin 411 and 511, containing the α4 and α5 laminin subunits respectively, and then define how aging impacts this expression. We observed the following: (i) CMH launched a robust arteriogenic remodeling response both in the young (10 weeks) and aged (20 months) brain, correlating with an increased number of ß4 integrin+ vessels, (ii) while the laminin α4 subunit is expressed evenly across all cerebral blood vessels, laminin α5 was highly expressed preferentially on ß4 integrin+ arterioles, (iii) CMH-induced arteriolar remodeling was associated with strong downregulation of the laminin α4 subunit but no change in the laminin α5 subunit, (iv) in addition to its expression on arterioles, ß4 integrin was also expressed at lower levels on capillaries specifically in white matter (WM) tracts but not in the grey matter (GM), and (v), these observations were consistent in both the brain and spinal cord, and age had no obvious impact. Taken together, our findings suggest that laminin 511 may be a specific ligand for α6ß4 integrin and that dynamic switching of the laminin subunits α4 and α5 might play an instructive role in arteriogenic remodeling. Furthermore, ß4 integrin expression differentiates WM from GM capillaries, highlighting a novel and important difference.

Spinal Cord Blood Vessels in Aged Mice Show Greater Levels of Hypoxia-Induced Vascular Disruption and Microglial Activation.

In response to chronic mild hypoxia (CMH, 8% O2), spinal cord blood vessels launch a robust angiogenic response that is associated with transient disruption of the blood-spinal cord barrier (BSCB) which, in turn, triggers a microglial vasculo-protective response. Because hypoxia occurs in many age-related conditions, the goal of this study was to define how aging influences these responses by comparing events in young (8-10 weeks) and aged (20 months) mice. This revealed that aged mice had much greater (3-4-fold) levels of hypoxic-induced BSCB disruption than young mice and that, while the early stage of the angiogenic response in aged mice was no different to young mice, the maturation of newly formed vessels was significantly delayed. Interestingly, microglia in the spinal cords of aged mice were much more activated than young mice, even under normoxic conditions, and this was further enhanced by CMH, though, surprisingly, this resulted in reduced microglial clustering around leaky blood vessels and diminished vasculo-protection. Vascular disruption was associated with loss of myelin in spinal cord white matter (WM) in both young and aged mice. Furthermore, it was notable that the spinal cord of aged mice contained a lower density of Olig2+ oligodendroglial cells even under normoxic conditions and that CMH significantly reduced the density of Olig2+ cells in spinal cord WM of the aged, but not the young, mice. These results demonstrate that spinal cord blood vessels of aged mice are much more vulnerable to the damaging effects of hypoxia than young mice, in part due to the reduced vasculo-protection conferred by chronically activated microglial cells. These observations may have implications for the pathogenesis and/or treatment of spinal cord diseases such as amyotrophic lateral sclerosis (ALS) and suggest that an improvement in microglial function could offer therapeutic potential for treating these age-related conditions.

The importance of laminin at the blood-brain barrier.

The blood-brain barrier is a unique property of central nervous system blood vessels that protects sensitive central nervous system cells from potentially harmful blood components. The mechanistic basis of this barrier is found at multiple levels, including the adherens and tight junction proteins that tightly bind adjacent endothelial cells and the influence of neighboring pericytes, microglia, and astrocyte endfeet. In addition, extracellular matrix components of the vascular basement membrane play a critical role in establishing and maintaining blood-brain barrier integrity, not only by providing an adhesive substrate for blood-brain barrier cells to adhere to, but also by providing guidance cues that strongly influence vascular cell behavior. The extracellular matrix protein laminin is one of the most abundant components of the basement membrane, and several lines of evidence suggest that it plays a key role in directing blood-brain barrier behavior. In this review, we describe the basic structure of laminin and its receptors, the expression patterns of these molecules in central nervous system blood vessels and how they are altered in disease states, and most importantly, how genetic deletion of different laminin isoforms or their receptors reveals the contribution of these molecules to blood-brain barrier function and integrity. Finally, we discuss some of the important unanswered questions in the field and provide a "to-do" list of some of the critical outstanding experiments.

ß1 integrin is essential for blood-brain barrier integrity under stable and vascular remodelling conditions; effects differ with age.

BACKGROUND: Maintaining a tight blood-brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard. METHODS: Here, in young (8-10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking ß1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-hoc test. RESULTS: In both young and aged mice, ß1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, ß1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, ß1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, ß1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that ß1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins. CONCLUSIONS: These data demonstrate that ß1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As ß1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing ß1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

Reducing herbicide input and optimizing spray method can minimize nontarget impacts on native grassland plant species.

Invasive plants threaten biodiversity worldwide and effective management must control the target invader while conserving biodiversity. Herbicide is often used to control invasive plants, but potential negative impacts on biodiversity have led to spot spraying being recommended over boom spraying to minimize the exposure of nontarget species to chemicals. We examined the influence of herbicide application methods on off-target plant communities in threatened temperate grasslands of southeastern Australia, where spraying with the broadleaf herbicide fluroxypr is commonly used to control St. John's wort, Hypericum perforatum L. It is well established that fluroxypr effectively controls H. perforatum but few studies have examined its impact on native forbs. A spray drift experiment using water-sensitive cards indicated that ground surface coverage was higher for spot spraying (91%-99%) than for boom spraying (5%-31%). We established a replicated, 3-year, before-after-control-impact experiment across 48 1-m2 quadrats to determine how three herbicide application methods (spot spray, fine boom and coarse boom) affected nontarget native forbs, the group most likely to be affected by broadleaf herbicides. This experiment was conducted in grasslands where H. perforatum was almost absent, so responses would reflect the direct impacts of the chemical, rather than structural changes resulting from removal of the target invader. Spot spraying decreased the probability of occurrence of native leguminous forbs, while increasing the occurrence of exotic leguminous forbs and the richness of all exotic species and exotic annual forbs. Spot spraying reduced the occurrence of the native Desmodium varians and the abundance of the native Chrysocephalum apiculatum. During this 3-year study, native species appeared to be impacted either directly by fluroxypr or indirectly by increased competition with exotic species. Where herbicide application is deemed crucial in these grasslands, we recommend boom spraying when H. perforatum density is moderate to high. Spot spraying should only be used when the density of H. perforatum is very low. Given the regional variation in H. perforatum density, the spatial scale of invasion, soil depth, and conservation values, we present a decision tree to assist managers in evaluating the costs and benefits of chemical control, indicating situations where alternative or modified methods could be used.

Hypoxia-induced vascular remodeling responses in the brain are much more robust than other organs.

Exposure to chronic mild hypoxia (CMH; 8-10% O2) promotes a robust vascular remodeling response in the brain resulting in 50% increased vessel density over a period of two weeks. It is currently unknown whether blood vessels in other organs show similar responses. To address this question, mice were exposed to CMH for 4 days and various markers of vascular remodeling were examined in the brain along with heart, skeletal muscle, kidney, and liver. In contrast to brain, where CMH strongly promoted endothelial proliferation, none of the peripheral organs showed this response and in heart and liver, CMH notably reduced endothelial proliferation. While the MECA-32 endothelial activation marker was strongly induced by CMH in brain, in peripheral organs it was constitutively expressed either on a sub-population of vessels (heart and skeletal muscle) or on all vessels (kidney and liver), and notably, CMH did not affect expression. Endothelial expression of the tight junction proteins claudin-5 and ZO-1 were markedly increased on cerebral vessels, but in the peripheral organs examined, CMH either had no effect or reduced ZO-1 expression (liver). Finally, while CMH had no impact on the number of Mac-1 positive macrophages in the brain, heart, or skeletal muscle, this number was markedly decreased in the kidney but increased in the liver. Our findings show that the vascular remodeling responses to CMH are organ-specific, with the brain showing a strong angiogenic response and enhanced tight junction protein expression, but heart, skeletal muscle, kidney, and liver failing to show these responses.

Age-associated changes in microglia activation and Sirtuin-1- chromatin binding patterns.

The aging process is associated with changes in mechanisms maintaining physiology, influenced by genetics and lifestyle, and impacting late life quality and longevity. Brain health is critical in healthy aging. Sirtuin 1 (Sirt1), a histone deacetylase with silencing properties, is one of the molecular determinants experimentally linked to health and longevity. We compared brain pathogenesis and Sirt1-chromatin binding dynamics in brain pre-frontal cortex from 2 groups of elder rhesus macaques, divided by age of necropsy: shorter-lived animals (18-20 years old (yo)), equivalent to 60-70 human yo; and longer-lived animals (23-29 yo), corresponding to 80-100 human yo and modeling successful aging. These were compared with young adult brains (4-7 yo). Our findings indicated drastic differences in the microglia marker Iba1, along with factors influencing Sirt1 levels and activity, such as CD38 (an enzyme limiting NAD that controls Sirt1 activity) and mir142 (a microRNA targeting Sirt1 transcription) between the elder groups. Iba1 was lower in shorter-lived animals than in the other groups, while CD38 was higher in both aging groups compared to young. mir142 and Sirt1 levels were inversely correlated in longer-lived brains (>23yo), but not in shorter-lived brains (18-20 yo). We also found that Sirt1 binding showed signs of better efficiency in longer-lived animals compared to shorter-lived ones, in genes associated with nuclear activity and senescence. Overall, differences in neuroinflammation and Sirt1 interactions with chromatin distinguished shorter- and longer-lived animals, suggesting the importance of preserving microglia and Sirt1 functional efficiency for longevity.

Exaggerated hypoxic vascular breakdown in aged brain due to reduced microglial vasculo-protection.

In a recent study of young mice, we showed that chronic mild hypoxia (CMH, 8% O2 ) triggers transient blood-brain barrier (BBB) disruption, and that microglia play an important vasculo-protective function in maintaining BBB integrity. As hypoxia is a common component of many age-related diseases, here we extended these studies to aged mice and found that hypoxia-induced vascular leak was greatly amplified (5-fold to 10-fold) in aged mice, being particularly high in the olfactory bulb and midbrain. While aged mice showed no obvious difference in the early stages of hypoxic angiogenic remodeling, the compensatory increase in vascularity and vessel maturation was significantly delayed. Compared with young brain, microglia in the normoxic aged brain were markedly activated, and this was further increased under hypoxic conditions, but paradoxically, this correlated with reduced vasculo-protection. Microglial depletion studies showed that microglial still play an important vasculo-protective role in aged brain, but interestingly, partial attenuation of microglial activation with minocycline resulted in fewer vascular leaks and reduced loss of endothelial tight junction proteins. Taken together, these findings suggest that increased BBB disruption in hypoxic aged mice can be explained both by a delayed vascular remodeling response and reduced microglial vasculo-protection. Importantly, they show that overly activated microglia in the aged brain are less effective at maintaining vascular integrity, though this can be improved by reducing microglial activation with minocycline, suggesting therapeutic potential for enhancing BBB integrity in the hypoxia-predisposed elderly population.

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

The impact of genetic manipulation of laminin and integrins at the blood-brain barrier.

Blood vessels in the central nervous system (CNS) are unique in having high electrical resistance and low permeability, which creates a selective barrier protecting sensitive neural cells within the CNS from potentially harmful components in the blood. The molecular basis of this blood-brain barrier (BBB) is found at the level of endothelial adherens and tight junction protein complexes, extracellular matrix (ECM) components of the vascular basement membrane (BM), and the influence of adjacent pericytes and astrocyte endfeet. Current evidence supports the concept that instructive cues from the BBB ECM are not only important for the development and maturation of CNS blood vessels, but they are also essential for the maintenance of vascular stability and BBB integrity. In this review, we examine the contributions of one of the most abundant ECM proteins, laminin to BBB integrity, and summarize how genetic deletions of different laminin isoforms or their integrin receptors impact BBB development, maturation, and stability.

Polygenic networks in peripheral leukocytes indicate patterns associated with HIV infection and context-dependent effects of cannabis use.

In spite of suppressive antiretroviral therapies (ART), Human Immunodeficiency Virus (HIV)-infected subjects still experience the consequences of viral persistence and chronic inflammation. In the brain, where most HIV-1 targets are of innate immune origin, neurological and cognitive impairments are detectable and enhanced by highly prevalent substance use disorders. Cannabis is one of the most prevalent substances among HIV+ â€‹subjects, compared to non-infected populations, either prescribed for improving various symptoms or used recreationally, as well as a component of polysubstance use. The mechanisms by which addictive substances and HIV interact are multifactorial and poorly understood. Importantly, the HIV brain target cells, macrophages and microglia, express receptors to neurotransmitters elevated by such drugs, and express receptors to cannabinoids, particularly CB2R. We have tested a panel of 784 transcripts associated with neurological disorders, digitally multiplexed and detectable in peripheral blood cells from a small cohort (n â€‹= â€‹102) of HIV-positive (HIV+) and HIV-negative (HIV-) specimens, stratified based on criteria of lifetime (LT) dependence of cannabis (CAN+) or not (CAN-). Demographic homogeneity and low incidence of co-morbidities helped increase power and allowed the identification of key differences consistent with HIV infection, cannabis exposure, or their interactions. A small percentage of these subjects used cannabis as well as other drugs. The data was analyzed using robust systems and visualization strategies to detect orchestrated patterns in gene networks connected based on molecular interfaces with higher power than in single genes. We found that the effects of cannabis differed drastically between HIV- and HIV+ â€‹groups, particularly in gene networks playing a role in inflammation, neurodegeneration, apoptosis and leukocyte adhesion and transmigration. At the level of individual genes, we identified detrimental effects that were associated with polysubstance use as a covariate, particularly methamphetamine. Transcription factor usage predictions suggest that the effects of cannabis are associated with transcriptional co-regulation at the gene promoters by multiple factors that vary by context. Overall, we have found that the effects of cannabis may be context-dependent, with potential benefits in the context of HIV reflected by improvements in cognition, but in the absence of the polysubstance use component.

The GFAP Monoclonal Antibody GA-5 Identifies Astrocyte Remodeling and Glio-Vascular Uncoupling During the Evolution of EAE.

To examine how astrocyte activation is regulated at different phases of relapsing-remitting EAE, we performed an immunofluorescent analysis of the spinal cord using the anti-glial fibrillary acidic protein (GFAP) monoclonal antibody GA-5. In keeping with previous studies, gray matter astrocytes showed strongly increased GFAP expression during the peak phase of disease (14 days post-immunization), which remained elevated during the remission phase (21-28 days post-immunization). In sharp contrast, during the peak phase of disease, the GA-5 signal in sub-meningeal white matter transiently disappeared in areas containing high levels of infiltrating leukocytes, but during the remission phase, the GFAP signal was fully restored. Parallel staining of the same sections with a polyclonal GFAP antibody confirmed elevated GFAP expression in the gray matter but no loss of signal in white matter. Interestingly, loss of GA-5 signal in sub-meningeal white matter was strongly associated with vascular disruption as defined by extravascular fibrinogen leak and by glio-vascular uncoupling, as defined by dissociation of AQP4-positive astrocyte endfeet and CD31-positive blood vessels. GA-5-negative areas were also associated with demyelination. These findings demonstrate a novel staining pattern of a GFAP antibody during EAE progression and suggest that the GFAP epitope recognized by the GA-5 monoclonal antibody transiently disappears as white matter astrocytes undergo remodeling during the peak phase of EAE. They also suggest that the GA-5 antibody provides a novel tool to identify astrocyte remodeling in other neurological conditions.

The impact of chronic mild hypoxia on cerebrovascular remodelling; uncoupling of angiogenesis and vascular breakdown.

BACKGROUND: Chronic mild hypoxia (CMH, 8% O2) stimulates robust vascular remodelling in the brain, but it also triggers transient vascular disruption. This raises the fundamental question: is the vascular leak an unwanted side-effect of angiogenic remodelling or is it a pathological response, unrelated to endothelial proliferation, in which declining oxygen levels trigger endothelial dysfunction? METHODS: To answer this question, mice were exposed to CMH (8% O2) for periods up to 14 days, after which, brain tissue was examined by immunofluorescence (IF) to determine which type of blood vessel (arteriole, capillary or venule) was most commonly associated with endothelial proliferation and vascular leak and how this correlated with tight junction protein expression. Vascular perfusion was examined using DiI. Data were analysed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-hoc test. RESULTS: The following was observed: (1) most endothelial proliferation and extravascular fibrinogen leak occurred in capillaries and to a lesser degree in venules, (2) much to our surprise, endothelial proliferation and extravascular fibrinogen leak never colocalized, (3) interestingly however, endothelial proliferation was strongly associated with an intravascular fibrinogen staining pattern not seen in stable blood vessels, (4) DiI perfusion studies revealed that angiogenic vessels were adequately perfused, suggesting that fibrinogen retention in angiogenic vessels is not due to temporary closure of the vessel, but more likely because fibrinogen is retained within the vessel wall, (5) bromodeoxyuridine (BrdU) labelling as a means to more permanently label proliferating endothelial cells, confirmed lack of any connection between endothelial proliferation and extravascular fibrinogen leak, while (6) in contrast, proliferating microglia were detected within extravascular leaks. CONCLUSIONS: Taken together, our findings support the concept that in the short-term, hypoxia-induced endothelial proliferation triggers transient fibrinogen deposition within the walls of angiogenic blood vessels, but no overt vascular leak occurs in these vessels. Importantly, endothelial proliferation and extravascular fibrinogen leaks never co-localize, demonstrating that extravascular leak is not an unwanted side-effect of angiogenic endothelial proliferation, but rather a dysfunctional vascular response to hypoxia that occurs in a distinct group of non-angiogenic blood vessels.

Sarcomas in the groin and inguinal canal: A 16-year single-centre experience.

INTRODUCTION: Soft tissue sarcomas arising in the groin and inguinal canal can be difficult to diagnose and manage. This is in part explained by the complex anatomy of the region. Early referral to specialist centres has been advocated, as inadvertent excision of these tumours can jeopardise definitive treatment. We present our 16-year experience at a regional sarcoma service. MATERIALS AND METHODS: A retrospective review of patients treated for a sarcoma in the groin and inguinal canal within the North of England Bone and Soft Tissue Tumour Service was performed. Demographic information, along with therapeutic approach and outcomes, was recorded and analysed. RESULTS: A total of 67 patients were identified, out of which 18 presented with new lesions, 32 presented after having a previous inadvertent sarcoma excision, 10 had a planned resection and 7 presented with recurrent disease. Liposarcomas were the most common histological subtype (55%), and the spermatic cord the most common origin (45%). Fifty-seven patients had surgery for this condition, with seven incomplete excision. Regional flaps were used in 60% of the cases, to allow an adequate oncological resection and soft tissue cover. Patients who had undergone a previous inadvertent sarcoma excision did not have worse rates of local recurrence, metastases and disease-specific mortality. Kaplan-Meier disease-specific survival at 5 years was 82%. DISCUSSION: Inadvertent and inadequate groin sarcoma excision outside of specialist centres remains a problem despite clear guidance. Despite this, an aggressive oncological approach to inadequately managed tumours shows similar outcomes as tumour managed exclusively by our specialist centre.

Hypoxia in multiple sclerosis; is it the chicken or the egg?

Over the past 50 years, intense research effort has taught us a great deal about multiple sclerosis. We know that it is the most common neurological disease affecting the young-middle aged, that it affects two to three times more females than males, and that it is characterized as an autoimmune disease, in which autoreactive T lymphocytes cross the blood-brain barrier, resulting in demyelinating lesions. But despite all the knowledge gained, a key question still remains; what is the initial event that triggers the inflammatory demyelinating process? While most research effort to date has focused on the immune system, more recently, another potential candidate has emerged: hypoxia. Specifically, a growing number of studies have described the presence of hypoxia (both 'virtual' and real) at an early stage of demyelinating lesions, and several groups, including our own, have begun to investigate how manipulation of inspired oxygen levels impacts disease progression. In this review we summarize the findings of these hypoxia studies, and in particular, address three main questions: (i) is the hypoxia found in demyelinating lesions 'virtual' or real; (ii) what causes this hypoxia; and (iii) how does manipulation of inspired oxygen impact disease progression?

Mild hypoxia triggers transient blood-brain barrier disruption: a fundamental protective role for microglia.

We recently demonstrated that when mice are exposed to chronic mild hypoxia (CMH, 8% O2), blood vessels in the spinal cord show transient vascular leak that is associated with clustering and activation of microglia around disrupted vessels. Importantly, microglial depletion profoundly increased hypoxia-induced vascular leak, implying that microglia play a critical role maintaining vascular integrity in the hypoxic spinal cord. The goal of the current study was to examine if microglia play a similar vasculo-protective function in the brain. Employing extravascular fibrinogen leak as an index of blood-brain barrier (BBB) disruption, we found that CMH provoked transient vascular leak in cerebral blood vessels that was associated with activation and aggregation of Mac-1-positive microglia around leaky vessels. Interestingly, CMH-induced vascular leak showed regional selectivity, being much more prevalent in the brainstem and olfactory bulb than the cerebral cortex and cerebellum. Pharmacological depletion of microglia with the colony stimulating factor-1 receptor inhibitor PLX5622, had no effect under normoxic conditions, but markedly increased hypoxia-induced cerebrovascular leak in all regions examined. As in the spinal cord, this was associated with endothelial induction of MECA-32, a marker of leaky CNS endothelium, and greater loss of endothelial tight junction proteins. Brain regions displaying the highest levels of hypoxic-induced vascular leak also showed the greatest levels of angiogenic remodeling, suggesting that transient BBB disruption may be an unwanted side-effect of hypoxic-induced angiogenic remodeling. As hypoxia is common to a multitude of human diseases including obstructive sleep apnea, lung disease, and age-related pulmonary, cardiac and cerebrovascular dysfunction, our findings have important translational implications. First, they point to a potential pathogenic role of chronic hypoxia in triggering BBB disruption and subsequent neurological dysfunction, and second, they demonstrate an important protective role for microglia in maintaining vascular integrity in the hypoxic brain.

Activated Protein C Attenuates Experimental Autoimmune Encephalomyelitis Progression by Enhancing Vascular Integrity and Suppressing Microglial Activation.

BACKGROUND: Activated protein C (APC), a serine protease with antithrombotic effects, protects in animal models of ischemic stroke by suppressing inflammation and enhancing vascular integrity, angiogenesis, neurogenesis and neuroprotection. A small number of animal studies suggest it might also have therapeutic potential in multiple sclerosis (MS), though results have been mixed. Based on these conflicting data, the goals of this study were to clarify the therapeutic potential of APC in the experimental autoimmune encephalomyelitis (EAE) model of MS and to determine mechanistically how APC mediates this protective effect. METHODS: The protective potential of APC was examined in a chronic progressive model of EAE. Vascular breakdown, tight junction protein expression and vascular expression of fibronectin and α5ß1 integrin as well as vascularity and glial activation were analyzed using immunofluorescence (IF) of spinal cord sections taken from mice with established EAE. The direct influence of APC on microglial activation was evaluated in vitro by a combination of morphology and MMP-9 expression. RESULTS: APC attenuated the progression of EAE, and this was strongly associated at the histopathological level with reduced levels of leukocyte infiltration and concomitant demyelination. Further analysis revealed that APC reduced vascular breakdown which was associated with maintained endothelial expression of the tight junction protein zonula occludens-1 (ZO-1). In addition, APC suppressed microglial activation in this EAE model and in vitro studies revealed that APC strongly inhibited microglial activation at both the morphological level and by the expression of the pro-inflammatory protease MMP-9. CONCLUSION: These findings build on the work of others in demonstrating strong therapeutic potential for APC in the treatment of inflammatory demyelinating disease and suggest that enhancement of vascular integrity and suppression of microglial activation may be important mediators of this protection.

Chronic mild hypoxia accelerates recovery from preexisting EAE by enhancing vascular integrity and apoptosis of infiltrated monocytes.

While several studies have shown that hypoxic preconditioning suppresses development of the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), no one has yet examined the important clinically relevant question of whether mild hypoxia can impact the progression of preexisting disease. Using a relapsing-remitting model of EAE, here we demonstrate that when applied to preexisting disease, chronic mild hypoxia (CMH, 10% O2) markedly accelerates clinical recovery, leading to long-term stable reductions in clinical score. At the histological level, CMH led to significant reductions in vascular disruption, leukocyte accumulation, and demyelination. Spinal cord blood vessels of CMH-treated mice showed reduced expression of the endothelial activation molecule VCAM-1 but increased expression of the endothelial tight junction proteins ZO-1 and occludin, key mechanisms underlying vascular integrity. Interestingly, while equal numbers of inflammatory leukocytes were present in the spinal cord at peak disease (day 14 postimmunization; i.e., 3 d after CMH started), apoptotic removal of infiltrated leukocytes during the remission phase was markedly accelerated in CMH-treated mice, as determined by increased numbers of monocytes positive for TUNEL and cleaved caspase-3. The enhanced monocyte apoptosis in CMH-treated mice was paralleled by increased numbers of HIF-1α+ monocytes, suggesting that CMH enhances monocyte removal by amplifying the hypoxic stress manifest within monocytes in acute inflammatory lesions. These data demonstrate that mild hypoxia promotes recovery from preexisting inflammatory demyelinating disease and suggest that this protection is primarily the result of enhanced vascular integrity and accelerated apoptosis of infiltrated monocytes.