Blood-brain Barrier Tight Junction Research Articles

Endothelial-Leukocyte Interaction in Severe Malaria: Beyond the Brain.

Malaria is the most important parasitic disease worldwide, accounting for 1 million deaths each year. Severe malaria is a systemic illness characterized by dysfunction of brain tissue and of one or more peripheral organs as lungs and kidney. The most severe and most studied form of malaria is associated with cerebral complications due to capillary congestion and the adhesion of infected erythrocytes, platelets, and leukocytes to brain vasculature. Thus, leukocyte rolling and adhesion in the brain vascular bed during severe malaria is singular and distinct from other models of inflammation. The leukocyte/endothelium interaction and neutrophil accumulation are also observed in the lungs. However, lung interactions differ from brain interactions, likely due to differences in the blood-brain barrier and blood-air barrier tight junction composition of the brain and lung endothelium. Here, we review the importance of endothelial dysfunction and the mechanism of leukocyte/endothelium interaction during severe malaria. Furthermore, we hypothesize a possible use of adjunctive therapies to antimalarial drugs that target the interaction between the leukocytes and the endothelium.

BBB-endothelial tight junction response to mesenchymal stem cells in a model of MOG EAE

Experimental autoimmune encephalomyelitis (EAE), an induced autoimmune disease of the central nervous system, simulates the main histopathological and clinical aspects of multiple sclerosis including the impairment of the blood-brain barrier (BBB). In several experimental models of human neurodegenerative diseases, the intravenous (iv) injection of bone marrow-derived mesenchymal stem cells (MSCs) ameliorates clinical symptoms and histopathological features [1,2]. On the basis of these data, we have analyzed the status of BBB tight junctions (TJs) of cerebral cortex microvessels in a model of MOG-EAE with iv injection of MSCs (EAE-MSC). The observations were carried out on EAE-MSC mice sacrificed at 6-24 hrs and 10 days after MSCs iv injection. The expression of endothelial TJ proteins, claudin-5 and occludin, was analyzed in healthy, EAE, and EAE-MSC mice by immunofluorescence confocal microscopy, together with the evaluation of barrier function by FITC-Dextran, as an exogenous permeability tracer. The results demonstrate that unlike EAE animals, characterized by an interrupted junctional staining and a barrier leakage, EAE-MSC mice show together with attenuate disease symptoms, a continuous, control- like claudin-5 and occludin junctional pattern and a functionally recovered barrier efficiency. Overall, these findings suggest that during EAE, the neuroprotective effect of the injected MSCs includes a reparative BBB response that in turn may contribute to the reduction of the inflammatory infiltrates and to the significant amelioration of the disease.

Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

Blood-brain barrier permeability change and regulation mechanism after subarachnoid hemorrhage.

We aimed to investigate the blood brain barrier (BBB) change caused by subarachnoid hemorrhage (SAH) and to explore the molecular mechanisms of acute brain injury after SAH. The SD rat model of SAH was firstly established by endovascular filament perforation technique. The changes of regional cerebral blood flow (rCBF), BBB permeability and ultrastructure of brain tissue at different time points after SAH were respectively observed by Doppler flowmetry, evans blue extravasation and transmission electron microscopy. Meanwhile, the expression changes of Claudin-5, Occludin, Zo-1 and Caveolin-1 were detected by immunohistochemistry and Western blot. Furthermore, the expressions of Akt, P-Akt and Foxo1A were also measured by Western blot. The change of BBB permeability showed two peaks at 3 and 72 h after SAH, corresponding to the change of rCBF. The BBB tight junction opening can be observed after SAH, and the largest opening was occurred at 3 h and 72 h. There was no significant change in Caveolin-1, Claudin-5 and Akt expressions after SAH (P > 0.05), while Zo-1 and Occludin were significantly down-regulated (P < 0.05). The expression of P-Akt was obviously reduced at 30 min and then increased at 1 and 24 h, while Foxo1A was up-regulated at 1 and 24 h after SAH (P < 0.05). Down-regulated Zo-1 and Occludin, as well as Akt/FOXO signaling pathway may be involved in the regulation of tight junction opening and the BBB permeability in the early stage after SAH.

Rapid remodeling of tight junctions during paracellular diapedesis in a human model of the blood-brain barrier.

Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance and inflammation. Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that, at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed 10-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5 and claudin-3. We found that paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial lateral border recycling compartment is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.

Viral pathogen-associated molecular patterns regulate blood-brain barrier integrity via competing innate cytokine signals.

ABSTRACTPattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs), such as viral RNA, drives innate immune responses against West Nile virus (WNV), an emerging neurotropic pathogen. Here we demonstrate that WNV PAMPs orchestrate endothelial responses to WNV via competing innate immune cytokine signals at the blood-brain barrier (BBB), a multicellular interface with highly specialized brain endothelial cells that normally prevents pathogen entry. While Th1 cytokines increase the permeability of endothelial barriers, type I interferon (IFN) promoted and stabilized BBB function. Induction of innate cytokines by pattern recognition pathways directly regulated BBB permeability and tight junction formation via balanced activation of the small GTPases Rac1 and RhoA, which in turn regulated the transendothelial trafficking of WNV. In vivo, mice with attenuated type I IFN signaling or IFN induction (Ifnar−/− Irf7−/−) exhibited enhanced BBB permeability and tight junction dysregulation after WNV infection. Together, these data provide new insight into host-pathogen interactions at the BBB during neurotropic viral infection.

Idazoxan reduces blood–brain barrier damage during experimental autoimmune encephalomyelitis in mouse

We have previously shown that Idazoxan (IDA), an imidazoline 2 receptor ligand, is neuroprotective against spinal cord injury caused by experimental autoimmune encephalomyelitis (EAE) in mouse, an animal modal of multiple sclerosis (MS). However, the protective mechanism remains unclear. Here, we provided evidence to show that IDA confers neuroprotection through reduction in blood–brain barrier (BBB) damage. EAE was induced by immunizing C57 BL/6 mice with myelin oligodendrocyte glycoprotein35–55 amino acid peptide (MOG35–55). IDA was administrated for 14 days after MOG immunization at 2mg/kg (i.p., bid). Significant reduction in BBB damage occurred in the IDA-treated group of mice compared with the saline-treated group, as evidenced by the reduction in Evan׳s blue content in the brain tissue and the reduced BBB tight junction damage viewed under a transmission electron microscope. Moreover, EAE-induced reductions in tight junction proteins (JAM-1, Occludin, Claudin-5 and ZO-1) were also significantly ameliorated in IDA-treated mice, all of which supported the notion that IDA reduced BBB damage. Interestingly, the expression levels of extracellular matrix metalloproteinase-9 (MMP-9) and the ratio of MMP-9 against tissue inhibitor of metalloproteinase-1 (TIMP-1), which is known to be associated with MS-induced BBB damage, were significantly reduced in IDA-treated group, lending further support to the hypothesis that IDA confers brain protection through reducing BBB damage. This study raised a possibility that IDA is a promising pro-drug for development against MS.

Impact of altered glycaemia on blood-brain barrier endothelium: an in vitro study using the hCMEC/D3 cell line

BackgroundCerebrovascular complications involving endothelial dysfunction at the blood-brain barrier (BBB) are central to the pathogenesis of diabetes-related CNS disorders. However, clinical and experimental studies have reported contrasting evidence in relation to the effects of hyperglycemia on BBB permeability and function. Similarly the effect of hypoglycemia on BBB integrity is not well understood. Therefore, we assessed the differential impact of hypo and hyperglycemic conditions on BBB integrity and endothelial function in vitro using hCMEC/D3, a well characterized human brain microvascular endothelial cell line.MethodsParallel monolayers of hCMEC/D3 were exposed to normal, hypo- or hyperglycemic media, containing 5.5, 2.2 or 35 mM D-glucose, respectively. Following 3-24h exposure, the expression and distribution of BBB tight junction (ZO-1 and claudin-5) adherence junction (VE-cadherin) proteins, and glucose transporters as well as inflammatory (VCAM-1) and oxidative stress (Nrf-2) markers were analyzed by immunofluorescence and western blotting. Endothelial release of growth factors and pro-inflammatory cytokines were determined by ELISA. Further, the impact of altered glycemia on BBB permeability was assessed in hCMEC/D3 – astrocyte co-cultures on Transwell supports using fluorescent dextrans (4–70 kDa).ResultsCompared to controls, exposure to hypoglycemia (3 and 24h) down-regulated the expression of claudin-5 and disrupted the ZO-1 localization at cell-cell contacts, while hyperglycemia marginally reduced claudin-5 expression without affecting ZO-1 distribution. Permeability to dextrans (4-10 kDa) and VEGF release at 24h were significantly increased by hypo- and hyperglycemia, although 70 kDa dextran permeability was increased only under hypoglycemic conditions. The expression of SGLT-1 was up-regulated at 24h hypoglycemic exposure while only a modest increase of GLUT-1 expression was observed. In addition, the expression of Nrf-2 and release of interleukin-6 and PDGF-BB, were down-regulated by hypoglycemia (but not hyperglycemia), while both conditions induced a marginal and transient increase in VCAM-1 expression from 3 to 24h, including a significant increase in VE-cadherin expression at 3 h following hyperglycemia.ConclusionsIn summary, our findings demonstrate a potential impairment of BBB integrity and function by hypo or hyperglycemia, through altered expression/distribution of TJ proteins and nutrient transporters. In addition, hypoglycemic exposure severely affects the expression of oxidative and inflammatory stress markers of BBB endothelium.

Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by increasing cerebrovascular oxidative stress

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood–brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.

Abstract W MP40: Pyruvate Protects Post-Hypoxic Neuronal Cells and a Blood Brain Barrier Model From rtPA Toxicity

Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved treatment for ischemic stroke. However, rtPA’s therapeutic window is limited to 4.5 h after stroke onset due to hemorrhagic transformation and neurotoxicity. Here, we demonstrated that the intermediary metabolite pyruvate protects neuronal cells and a blood brain barrier (BBB) model from delayed rtPA toxicity in an in vitro oxygen-glucose deprivation (OGD, 0.5% O 2 )-reoxygenation model of ischemic stroke. After 3 or 6 h OGD, neuronal cells were reoxygenated with 11 mM glucose ± 8 mM pyruvate and/or 10 μg/ml rtPA. Cellular viability, reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP2) activity, and cellular contents of NADPH, NADP + , ATP, MMP2, tissue inhibitor of metalloproteinase-2 (TIMP2), and phosphor-activation of anti-apoptotic p70s6 kinase, Akt and Erk were measured. Pyruvate treatment after 3 h OGD decreased cell death by 80% in the absence (P &lt; 0.01) and 64% in the presence (P &lt; 0.01) of rtPA. After 6 h OGD, rtPA exacerbated cell death; pyruvate dampened this effect. Three hours OGD and 4 h reoxygenation + rtPA increased ROS formation by 50%. Pyruvate prevented this ROS formation and doubled antioxidant NADPH/NADP + ratio and ATP content. In the BBB model, 3 h OGD and 24 h reoxygenation increased FITC-dextran leakage, indicating disruption of intercellular junctions. Although rtPA exacerbated this effect, pyruvate prevented it while sharply lowering MMP2/TIMP2 ratio and increasing phosphorylation of p70s6 kinase, Akt and Erk. Pyruvate protects neuronal cells and BBB tight junctions from OGD-reoxygenation and rtPA toxicity while reducing ROS and activating anti-apoptotic signaling. These results support the use of pyruvate as an adjuvant to dampen rtPA’s side effects, thereby extending rtPA’s therapeutic window.

Blood-Brain Barrier Permeability in Aging and Alzheimer's Disease.

Blood-Brain Barrier Permeability in Aging and Alzheimer's Disease.

Effects of fumaric acid esters on blood–brain barrier tight junction proteins

The blood–brain barrier (BBB) is composed of a network of tight junctions (TJ) which interconnect cerebral endothelial cells (EC). Alterations in the TJ proteins are common in inflammatory diseases of the central nervous system (CNS) like multiple sclerosis (MS). Modulation of the BBB could thus represent a therapeutic mechanism. One pathway to modulate BBB integrity could be the induction of nuclear-factor (erythroid derived 2) related factor-2 (Nrf2) mediated oxidative stress responses which are targeted by fumaric acid esters (FAE). Here we analyze effects of FAE on the expression of TJ proteins in the human cerebral endothelial cell line hCMEC/D3 and experimental autoimmune encephalomyelitis (EAE). We show that dimethylfumarate (DMF) and its primary metabolite monomethylfumarate (MMF) induce the expression of the Nrf2/NQO1 pathway in endothelial cells. Neither MMF nor DMF had a consistent modulatory effect on the expression of TJ molecules in hCMEC/D3 cells. Tumor necrosis factor (TNFα)-induced downregulation of TJ proteins was at least partially reversed by treatment with FAE. However, DMF had no effect on claudin-5 expression in EAE, despite its effect on the clinical score and infiltration of immune cells. These data suggest that the modulation of the BBB is not a major mechanism of action of FAE in inflammatory demyelinating diseases of the CNS.

Quantitative Atlas of Blood–Brain Barrier Transporters, Receptors, and Tight Junction Proteins in Rats and Common Marmoset

The purpose of this study was to determine the protein amounts of blood-brain barrier (BBB) permeability-related transporters, receptors, and tight junction proteins in Sprague Dawley and Wistar rats and common marmoset, and also to investigate inter-species and inter-strain differences across rodents and primates. Quantification of target proteins in isolated brain capillaries was conducted by liquid chromatography-tandem mass spectrometry-based quantitative targeted absolute proteomics, with in silico peptide selection. Most target proteins showed inter-rodent, inter-primate species, and inter-rat strain differences of less than 2-fold. Comparison of rat and human BBB showed that P-glycoprotein, multidrug resistance-associated protein 4, monocarboxylate transporter 1, l-type amino acid transporter, and organic anion transporter 3 exhibited differences of more than two-fold in protein abundance, whereas the amounts of breast cancer resistance protein, glucose transporter 1, and insulin receptor were similar in rat and human. In contrast, the differences between marmoset and human BBB were less than 2-fold for almost all measured proteins. Thus, the molecular basis of BBB functions may be similar in marmoset and human, whereas that of rats shows significant differences. The marmoset may be a good model to access in vivo human BBB permeability characteristics, as an alternative to rat and macaque monkey.

Effects of selective hypothermia on blood-brain barrier integrity and tight junction protein expression levels after intracerebral hemorrhage in rats

Hypothermia has neuroprotective effects on global cerebral ischemic injuries. However, its efficacy after intracerebral hemorrhage (ICH) is inconclusive. In this study, bacterial collagenase was used to induce ICH stroke in male Wistar rats. We assessed the effects of normothermia and 4 h of local hypothermia (~33.2°C) initiated 1, 6, or 12 h after collagenase infusion on hemorrhage volume and neurological outcomes. Following early cooling initiated after 1 h, blood-brain barrier (BBB) disruption and brain water content were tested. Furthermore, the expression levels of tight junction (TJ) proteins (claudin 5 and occludin) and the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were determined using Western blotting, real-time quantitative PCR, and immunohistochemical staining at 1 and 3 d after ICH. Early local hypothermia tends to reduce hemorrhagic volume and neurological deficits, but the difference is not statistically significant compared with other groups. However, early hypothermia significantly reduces BBB disruption, edema formation, the expression levels of IL-1β and TNF-α, and the loss of TJ proteins. Together, these data suggest that local hypothermia is an effective treatment for edema formation and BBB disruption via the upregulation of TJ proteins and the suppression of TNF-α and IL-1β.

Differences and similarities in the transcriptional profile of peripheral whole blood in early and late-onset preeclampsia: insights into the molecular basis of the phenotype of preeclampsiaa

Preeclampsia (PE) can be sub-divided into early- and late-onset phenotypes. The pathogenesis of these two phenotypes has not been elucidated. To gain insight into the mechanisms of disease, the transcriptional profiles of whole blood from women with early- and late-onset PE were examined. A cross-sectional study was conducted to include women with: i) early-onset PE (diagnosed prior to 34 weeks, n=25); ii) late-onset PE (after 34 weeks, n=47); and iii) uncomplicated pregnancy (n=61). Microarray analysis of mRNA expression in peripheral whole blood was undertaken using Affymetrix microarrays. Differential gene expression was evaluated using a moderated t-test (false discovery rate <0.1 and fold change >1.5), adjusting for maternal white blood cell count and gestational age. Validation by real-time qRT-PCR was performed in a larger sample size [early PE (n=31), late PE (n=72) and controls (n=99)] in all differentially expressed genes. Gene ontology analysis and pathway analysis were performed. i) 43 and 28 genes were differentially expressed in early- and late-onset PE compared to the control group, respectively; ii) qRT-PCR confirmed the microarray results for early and late-onset PE in 77% (33/43) and 71% (20/28) of genes, respectively; iii) 20 genes that are involved in coagulation (SERPINI2), immune regulation (VSIG4, CD24), developmental process (H19) and inflammation (S100A10) were differentially expressed in early-onset PE alone. In contrast, only seven genes that encoded proteins involved in innate immunity (LTF, ELANE) and cell-to-cell recognition in the nervous system (CNTNAP3) were differentially expressed in late-onset PE alone. Thirteen genes that encode proteins involved in host defense (DEFA4, BPI, CTSG, LCN2), tight junctions in blood-brain barrier (EMP1) and liver regeneration (ECT2) were differentially expressed in both early- and late-onset PE. Early- and late-onset PE are characterized by a common signature in the transcriptional profile of whole blood. A small set of genes were differentially regulated in early- and late-onset PE. Future studies of the biological function, expression timetable and protein expression of these genes may provide insight into the pathophysiology of PE.

Perforin expression is required for fatal blood-brain barrier disruption in the Plasmodium berghei ANKA model of cerebral malaria (P3061)

Abstract The most severe clinical complication of Plasmodium falciparum infection is cerebral malaria (CM). Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice is an established model of CM. Previous work has implied a role for CD8 T cells and perforin expression in fatal stages of PbA infection. We therefore infected C57BL/6 and C57BL/6 Perforin-/- mice with PbA and assessed the CNS for cerebral endothelial cell blood-brain barrier (BBB) disruption using FITC albumin leakage into tissue, confocal microscopy and 3D volumetric T2 and T1 gadolinium enhanced MRI. PbA infected C57BL/6 mice: 1.) had extensive disruption of BBB tight junction proteins Occludin and Claudin 5, 2.) developed severe CNS vascular permeability of FITC albumin and gadolinium leakage, and 3.) became moribund. We also observed increased VEGF cytokine expression coinciding with CNS vascular permeability in these animals. Despite having similar CD8 T cell infiltration into the brain, PbA infected C57BL/6 Perforin-/- mice had intact BBB tight junction, were devoid of CNS vascular permeability, presented with normal brain MRI, and had normal behavioral scores on the rotarod assay. This study is therefore the first to demonstrate the critical importance of perforin expression in promoting BBB tight junction alteration during PbA infection. Therefore, development of therapies to block factors related perforin mediated delivery could have significant benefit to the treatment of CM.

Myelin basic protein induces inflammatory mediators from primary human endothelial cells and blood–brain barrier disruption: implications for the pathogenesis of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by demyelination of white matter, loss of myelin forming oligodendrocytes, changes in the blood-brain barrier (BBB) and leucocyte infiltration. Myelin basic protein (MBP) is a component of the myelin sheath. Degradation of myelin is believed to be an important step that leads to MS pathology. Transmigration of leucocytes across the vasculature, and a compromised BBB participate in the neuroinflammation of MS. We examined the expression and regulation of the chemokine (C-C motif) ligand 2 (CCL2) and the cytokine interleukin-6 (IL-6) in human endothelial cells (EC), a component of the BBB, after treatment with MBP. EC were treated with full-length MBP. CCL2 and IL-6 protein were determined by ELISA. Western blot analysis was used to determine signalling pathways. A BBB model was treated with MBP and permeability was assayed using albumin conjugated to Evan's blue dye. The levels of the tight junction proteins occludin and claudin-1, and matrix metalloprotease (MMP)-2 were assayed by Western blot. MBP significantly induced CCL2 and IL-6 protein from EC. This induction was partially mediated by the p38 MAPK pathway as there was phosphorylation after MBP treatment. MBP treatment of a BBB model caused an increase in permeability that correlated with a decrease in occludin and claudin-1, and an induction of MMP2. These data demonstrate that MBP induces chemotactic and inflammatory mediators. MBP also alters BBB permeability and tight junction expression, indicating additional factors that may contribute to the BBB breakdown characteristic of MS.

Abstract WP443: KLF2 Protects Against Ischemic Stroke by Regulating Endothelial Blood Brain Barrier Function

Background During an ischemic stroke normal brain endothelial function is abrogated resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells, and serves as a key molecular switch regulating endothelial function and promoting vascular health. Purpose To determine the role of KLF2 in ischemic stroke. Methods and Results —To examine the specific role of KLF2 in the brain and cerebrovascular disease, we performed ischemic strokes and conducted BBB assays in KLF2 deficient (K2-/-), KLF2 overexpressing (K2tg), and control mice. K2 -/- mice exhibited larger strokes in the transient middle cerebral artery occlusion model (95% greater stroke volume vs. control; n=10-20/group; P=0.001) and significant impairment in BBB function as assessed by in vivo real time quantitative PET analysis and evans blue dye (EBD) assays (86% greater EBD permeability vs. control; n=3/group; P=0.002). In contrast, K2tg mice were protected against ischemic stroke (49% smaller stroke volume vs. control; n=8-9/group, P=0.004) and demonstrated less impairment in BBB function (30% less EBD permeability vs control; n=3/group; P=0.003). In concordance, gain and loss of function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective (32% less permeability in adenoviral KLF2 overexpression vs. control; P=0.005 and 107% greater permeability in siRNA mediated KLF2 knockdown vs. control; P&lt;0.0005). Mechanistically, KLF2 was demonstrated to regulate the critical BBB tight junction factor occludin, both in vitro (7.0±0.3 fold expression in adenoviral KLF2 overexpression vs. control; P&lt;0.0001 and 0.43±0.03 fold expression in siRNA mediated KLF2 knockdown vs. control; P&lt;0.0005) and in vivo (0.56±0.11 fold expression in K2 -/- vs. control; n=4-7/group; P=0.01 and 2.5±0.5 fold expression in K2tg vs. control; n=3-4/group; P&lt;0.05). Conclusions These data are first to identify KLF2 as a key regulator of brain endothelial function and cerebrovascular homeostasis, thereby serving as a novel neuroprotective factor in ischemic stroke.

Kruppel-like factor 2 protects against ischemic stroke by regulating endothelial blood brain barrier function

During an ischemic stroke normal brain endothelial function is perturbed, resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells (ECs), and serves as a key molecular switch regulating endothelial function and promoting vascular health. In this study we sought to determine the role of KLF2 in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion was performed in KLF2-deficient (KLF2(-/-)), KLF2 overexpressing (KLF2(tg)), and control mice, and stroke volume was analyzed. BBB function was assessed in vivo by real-time neuroimaging using positron emission tomography and Evan's blue dye assay. KLF2(-/-) mice exhibited significantly larger strokes and impairment in BBB function. In contrast, KLF2(tg) mice were protected against ischemic stroke and demonstrated preserved BBB function. In concordance, gain- and loss-of-function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective. Mechanistically, KLF2 was demonstrated, both in vitro and in vivo, to regulate the critical BBB tight junction factor occludin. These data are first to identify endothelial KLF2 as a key regulator of the BBB and a novel neuroprotective factor in ischemic stroke.

Neurodegeneration, Mitochondrial Dysfunction, and Oxidative Stress

Mitochondria are intracellular organelles that play a crucial role in energy metabolism. Most cell energy is obtained through mitochondrial metabolic pathways, especially the Krebs cycle and electron transport chain which is the main site for production of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals. Brain tissue is highly sensitive to oxidative stress due to its high oxygen consumption, iron and lipid contents, and low activity of antioxidant defenses. Thus, energy metabolism impairment and oxidative stress are important events that have been related to the pathogenesis of diseases affecting the central nervous system. In the present issue, the pathogenesis of common Alzheimer's (AD) and Parkinson's (PD) diseases is addressed in five papers. Mondragon-Rodriguez et al. proposed that phosphorylated tau protein could play the role of potential connector and that a combined therapy involving antioxidants and check points for synaptic plasticity during early stages of the disease could become a viable therapeutic option for AD treatment. This paper is accompanied by a study by T. Rohn that explores the potential role that the triggering receptor expressed on myeloid cells 2 (TREM2) normally plays and how loss of function may contribute to AD pathogenesis by enhancing oxidative stress and inflammation within the central nervous system. Additionally, O. Myhre et al. review the possible impact of environmental exposures in metal dyshomeostasis and inflammation in AD and PD. Furthermore, T. Omura et al. explore recent studies on the mechanism of endoplasmic reticulum stress-induced neuronal death related to PD, focusing on the involvement of human ubiquitin ligase HRD1 in the prevention of neuronal death as well as a potential therapeutic approach for PD based on the upregulation of HRD1. Lastly, S. Matsuda et al. showed a concise overview on the cellular functions of the mitochondrial kinase PINK1 and the relationship between Parkinsonism and mitochondrial dynamics, with particular emphasis on a mitochondrial damage response pathway and mitochondrial quality control. Three of the papers deal with aspects of oxidative stress implicated in the pathogenesis of neurodegenerative diseases. W. Liu et al. reviewed the current literature on the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2) and Bcl-2/Bax in rat skeletal muscles. A.-M. Enciu et al. explore the possibility of oxidative-induced molecular mechanisms of blood-brain barrier disruption and tight junction protein expression alteration, in relation to aging and neurodegeneration. Moreover, M. Tajes et al. suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia. A. Hosseini and M. Abdollahi review the pathogenesis of diabetic neuropathy with a focus on oxidative stress and introduced therapies dependent or independent of oxidative stress. A. Sekigawa et al. review the currently available evidence that neither mitochondria nor leucine-rich repeat kinase 2 (LRRK2) was present in the swellings of mice expressing P123H β-synuclein, suggesting that α- and β-synucleins might play differential roles in the mitochondrial pathology of α-synucleinopathies. The paper by P. F. Schuck et al. showed that trans-glutaconic acid is toxic to brain cells in vitro, by causing alterations in cell ion balance and probably neurotransmission, as well as oxidative stress in rat cerebral cortex. To complete the issue, M. J. Rodriguez et al. discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome. Fortes et al. showed that 5TIO1 can protect the brain against neuronal damage regularly observed during neuropathologies. These papers are accompanied by a review by Z. Yu et al. on how the neuroglobin's neuroprotection is related to mitochondria function and regulation. By compiling these papers, we hope to enrich our readers and researchers with respect to mitochondrial dysfunction, energy metabolism impairment, and oxidative stress in the pathophysiology of neurodegenerative diseases. Emilio L. Streck Grzegorz A. Czapski Cleide Goncalves da Silva