FGF21 Protects against Aggravated Blood-Brain Barrier Disruption after Ischemic Focal Stroke in Diabetic db/db Male Mice via Cerebrovascular PPARγ Activation

Recombinant fibroblast growth factor 21 (rFGF21) has been shown to be potently beneficial for improving long-term neurological outcomes in type 2 diabetes mellitus (T2DM) stroke mice. Here, we tested the hypothesis that rFGF21 protects against poststroke blood–brain barrier (BBB) damage in T2DM mice via peroxisome proliferator-activated receptor gamma (PPARγ) activation in cerebral microvascular endothelium. We used the distal middle cerebral occlusion (dMCAO) model in T2DM mice as well as cultured human brain microvascular endothelial cells (HBMECs) subjected to hyperglycemic and inflammatory injury in the current study. We detected a significant reduction in PPARγ DNA-binding activity in the brain tissue and mRNA levels of BBB junctional proteins and PPARγ-targeting gene CD36 and FABP4 in cerebral microvasculature at 24 h after stroke. Ischemic stroke induced a massive BBB leakage two days after stroke in T2DM mice compared to in their lean controls. Importantly, all abnormal changes were significantly prevented by rFGF21 administration initiated at 6 h after stroke. Our in vitro experimental results also demonstrated that rFGF21 protects against hyperglycemia plus interleukin (IL)-1β-induced transendothelial permeability through upregulation of junction protein expression in an FGFR1 activation and PPARγ activity elevation-dependent manner. Our data suggested that rFGF21 has strong protective effects on acute BBB leakage after diabetic stroke, which is partially mediated by increasing PPARγ DNA-binding activity and mRNA expression of BBB junctional complex proteins. Together with our previous investigations, rFGF21 might be a promising candidate for treating diabetic stroke.

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Abstract WMP81: FGF21 Reduces Post-Stroke Blood Brain Barrier Damage in Diabetic db/db Male Mice

Stroke ◽

10.1161/str.51.suppl_1.wmp81 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Yinghua Jiang ◽

Ning Liu ◽

Xiaoying Wang

Keyword(s):

Dna Binding ◽

Binding Activity ◽

Junctional Complex ◽

Fibroblast Growth Factor 21 ◽

Nuclear Fraction ◽

Mrna Levels ◽

Protective Effects ◽

Post Stroke ◽

Dna Binding Activity ◽

Bbb Permeability

Background and Purpose: Our previous studies demonstrated that recombinant human Fibroblast growth factor 21 (rFGF21), an endocrine member of the FGF family is potently beneficial for improving long-term neurological outcomes of type 2 diabetes (T2D) stroke mice. Here we tested hypothesis that rFGF21 protects against post-stroke BBB damage by PPARγ activation of cerebral micovascular endothelium. Methods: T2D db/db mice and their non-diabetic counterparts db/+ mice were subjected to focal stroke of dMCAO. Four experimental groups: 1) db/+ stroke, 2) db/db stroke, 3) db/db stroke+rFGF21, and 4) db/db stroke+rFGF21+GW9662. rFGF21 (1.5mg/kg, i.p.) was injected at 6 hours after stroke and PPARγ inhibitor GW9662 (4mg/kg, i.p.) was injected 30 min prior to rFGF21 treatment. At 24 hours post-stroke, we collected peri-infarct nuclear fraction for PPARγ-DNA binding activity assay using EMSA, microvascular isolation for RT-PCR analyzing mRNA levels of proteins constituting BBB junctional complex (occluding, clauding-5, VE-cadherin and ZO-1) and PPARγ targeted downstream genes (CD36 and FABP4) as indicators of PPARγ activity in microvasculature. BBB permeability was assessed by measuring 3kDa FITC-dextran or Evans blue extravasations at 48 hour post-stroke. Results: Ischemic stroke induced a significant decrease of PPARγ-DNA binding activity and mRNA levels of BBB junctional proteins in peri-infarct area, and a significant increase of BBB permeability in diabetic db/db stroke mice compared to db/+ stroke mice. Changes of mRNA levels of CD36 and FABP4 in brain microvascular isolation were consistent with changes of PPARγ-DNA binding activity. rFGF21 administration significantly increased PPARγ-DNA binding activity, elevated mRNA levels of BBB junctional complex proteins and ameliorated BBB leakage. However, pre-treatment of GW9662 partially abolished the post-stroke BBB protective effects of rFGF21. Conclusions: rFGF21 has strong protective effects in acute BBB leakage after stroke, and the underlying mechanisms is partially via increase in PPARγ-DNA binding activity and mRNA expression of BBB junctional complex proteins. Together with our previous investigations, rFGF21 might be a promising candidate for treating diabetic stroke.

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Mycoplasma genitaliumLipoproteins Induce Human Monocytic Cell Expression of Proinflammatory Cytokines and Apoptosis by Activating Nuclear FactorκB

Mediators of Inflammation ◽

10.1155/2008/195427 ◽

2008 ◽

Vol 2008 ◽

pp. 1-8 ◽

Cited By ~ 20

Author(s):

Yimou Wu ◽

Hong Qiu ◽

Yanhua Zeng ◽

Xiaoxing You ◽

Zhongliang Deng ◽

...

Keyword(s):

Dna Binding ◽

Proinflammatory Cytokines ◽

Cell Apoptosis ◽

Molecular Mechanisms ◽

Binding Activity ◽

Mrna Levels ◽

Dependent Manner ◽

Monocytic Cell ◽

Dna Binding Activity ◽

Human Monocytic Cell

This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared fromMycoplasma genitalium. Cultured cells were stimulated withM. genitaliumLP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-κB (NF-κB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-κB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-κB. Our results indicate thatM. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-κB.

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Abstract TP108: Blood Occludin Level Indicates the Extent of Early Blood Brain Barrier Damage in Ischemic Stroke

Stroke ◽

10.1161/str.47.suppl_1.tp108 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Rong Pan ◽

Kewei Yu ◽

Theodore Weatherwax1 ◽

Handong Zheng ◽

Yirong Yang ◽

...

Keyword(s):

Ischemic Stroke ◽

Blood Brain Barrier ◽

Evans Blue ◽

Brain Barrier ◽

Blue Dye ◽

Dependent Manner ◽

Cerebral Microvessels ◽

Evans Blue Dye ◽

Blood Brain ◽

Junction Protein

Background and Purpose: Fear of symptomatic intracerebral hemorrhage (ICH) has been the primary reason for withholding tPA thrombolysis from acute ischemic stroke patients. Early blood brain barrier (BBB) damage is appreciated to be closely associated with post-thrombolysis ICH, while it remains a technical challenge for rapid assessment of BBB damage before tPA administration. Our recent data showed that cerebral ischemia induced rapid degradation of tight junction protein occludin in ischemic cerebromicrovessels. This study further investigates whether the cleaved occludin is released into the blood stream and how blood occludin levels correlate to the extent of ischemic BBB damage. Methods: Male Sprague Dawley rats were subjected to 1.5, 3, 4.5, 12 and 24 hours of middle cerebral artery occlusion (MCAO), followed by 5-min reperfusion. Blood samples were taken before and after MCAO. Blood occludin was assessed by ELISA. BBB permeability was measured by Evans blue dye leakage. Occludin cleavage was identified on immunoblots. Results: MCAO induced Evans blue dye leakage and blood occludin increase in a duration-dependent manner. Blood occludin increase concurrently occurred with the loss of occludin from ischemic cerebral microvessels. Western blot analysis identified two cleaved occludin fragments (31- and 55- kDa) in the blood. Lastly, blood occludin levels remained significantly higher than its basal level within the first 24 hours after MCAO onset. Conclusions: Our results indicate that blood occludin levels correlate well with the extents of BBB damage and thus may serve as a potential biomarker for evaluating the risk of hemorrhagic transformation before tPA administration.

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Hyperhomocysteinemia increases permeability of the blood-brain barrier by NMDA receptor-dependent regulation of adherens and tight junctions

Blood ◽

10.1182/blood-2011-02-338269 ◽

2011 ◽

Vol 118 (7) ◽

pp. 2007-2014 ◽

Cited By ~ 68

Author(s):

Richard S. Beard ◽

Jason J. Reynolds ◽

Shawn E. Bearden

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Barrier Permeability ◽

Blood Brain Barrier Permeability ◽

Blood Brain ◽

Junction Protein ◽

Brain Barrier Permeability

Abstract Hyperhomocysteinemia (HHcy) increases permeability of the blood-brain barrier, but the mechanisms are undetermined. Homocysteine (Hcy) is an agonist of the neuronal N-methyl-D-aspartate receptor (NMDAr). We tested the hypothesis that HHcy disrupts the blood-brain barrier by an NMDAr-dependent mechanism in endothelium. In brain microvascular endothelial cells, there was no change in expression of the adherens junction protein VE-cadherin with Hcy treatment, but there was a significant decrease in the amount of β-catenin at the membrane. Moreover, Hcy caused nuclear translocation of β-catenin and attachment to the promoter for the tight junction protein claudin-5, with concomitant reduction in claudin-5 expression. Using a murine model of HHcy (cbs+/−), treatment for 2 weeks with an NMDAr antagonist (memantine) rescued cerebrovascular expression of claudin-5 and blood-brain barrier permeability to both exogenous sodium fluorescein and endogenous IgG. Memantine had no effect on these parameters in wild-type littermates. The same results were obtained using an in vitro model with brain microvascular endothelial cells. These data provide the first evidence that the NMDAr is required for Hcy-mediated increases in blood-brain barrier permeability. Modulating cerebral microvascular NMDAr activity may present a novel therapeutic target in diseases associated with opening of the blood-brain barrier in HHcy, such as stroke and dementia.

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Abstract TP256: Mouse Model of Uremic Cerebral Microbleeds

Stroke ◽

10.1161/str.47.suppl_1.tp256 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Wei Ling Lau ◽

Mary Tarbiat-Boldaji ◽

Hayley Smalls ◽

Ane Nunes ◽

Javad Savoj ◽

...

Keyword(s):

Mouse Model ◽

Tight Junction ◽

Blood Brain Barrier ◽

Cerebral Microbleeds ◽

Brain Barrier ◽

Barrier Integrity ◽

Blood Brain ◽

Increased Risk ◽

Junction Protein ◽

Blood Brain Barrier Integrity

Introduction: Cerebral microbleeds are more common in chronic kidney disease (CKD) and dialysis patients compared to the general population. Diminished kidney function alone appears to be a risk factor for microbleeds, independent of age and hypertension. Microbleed burden in CKD patients is associated with increased risk of future hemorrhagic stroke and with cognitive dysfunction. The mechanisms that drive uremic microbleed formation are unclear. Hypothesis: We hypothesized that CKD mice are predisposed to develop cerebral microhemorrhages (the pathologic substrate of microbleeds), and that a standardized inflammatory stimulus (lipopolysaccharide, LPS) will amplify microhemorrhage burden in CKD mice compared to non-CKD controls (CTL). We also hypothesized that uremia induces depletion of tight junction proteins, altering blood-brain barrier integrity and representing a potential mechanism of microbleed formation. Methods: Animal groups included CTL (n=3), CKD (n=3), CTL+LPS (n=5) and CKD+LPS (n=5). CKD induction in male C57BL/6 mice was achieved via nephrotoxic adenine diet x18 days. Two weeks following CKD induction, CKD and control mice were treated with LPS 1 mg/kg i.p. dosed at 0, 6 and 24 hours. Brains were harvested one week after LPS injections and 40-micron sections were stained using Prussian blue to identify microhemorrhages. Immunohistochemistry was performed for the blood-brain barrier tight junction protein claudin-5. Results: CKD mice had significantly elevated blood urea nitrogen, and tubulointerstitial fibrosis was present on kidney histology. Total number of microhemorrhages per brain was 2.3±1.5 (mean ± standard error of the mean) for CTL mice, 8.3±1.5 for CKD mice, 23.2±4.2 for CTL+LPS mice, and 27.6±6.2 for CKD+LPS mice (p<0.05 for CKD+LPS vs. CTL). Immunostaining showed decreased claudin-5 expression in CKD mice compared to CTL. Conclusions: We have generated a mouse model that will facilitate future mechanistic studies in the field of uremic microbleeds. Our initial findings suggest that CKD alters blood-brain barrier integrity and that inflammation amplifies development of microbleeds in CKD.

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Time-dependent dual effect of NLRP3 inflammasome in brain ischemia

10.1101/2020.10.08.332007 ◽

2020 ◽

Author(s):

Alejandra Palomino-Antolin ◽

Paloma Narros-Fernández ◽

Víctor Farré-Alins ◽

Javier Sevilla-Montero ◽

Celine Decouty-Pérez ◽

...

Keyword(s):

Brain Injury ◽

Blood Brain Barrier ◽

Inflammatory Cytokines ◽

Nlrp3 Inflammasome ◽

Neurovascular Unit ◽

Time Dependent ◽

Brain Barrier ◽

Dependent Manner ◽

Dual Effect ◽

Blood Brain

AbstractBackground and purposePost-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit.MethodsWT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining.ResultsWe identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration.ConclusionsThese findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

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Rufinamide (RUF) suppresses inflammation and maintains the integrity of the blood–brain barrier during kainic acid-induced brain damage

Open Life Sciences ◽

10.1515/biol-2021-0090 ◽

2021 ◽

Vol 16 (1) ◽

pp. 845-855

Author(s):

Huaxu Yu ◽

Bin He ◽

Xu Han ◽

Ting Yan

Keyword(s):

Kainic Acid ◽

Blood Brain Barrier ◽

Neuronal Damage ◽

Brain Barrier ◽

Protective Mechanism ◽

Dependent Manner ◽

Nissl Staining ◽

Microglia Cell ◽

Protein Levels ◽

Blood Brain

Abstract Rufinamide (RUF) is a structurally unique anti-epileptic drug, but its protective mechanism against brain injury remains unclear. In the present study, we validated how the RUF protected mice with kainic acid (KA)-induced neuronal damage. To achieve that, a mouse epilepsy model was established by KA intraperitoneal injection. After Nissl staining, although there was a significant reduction in Nissl bodies in mice treated with KA, 40, 80, and 120 mg/kg, RUF significantly reduced KA-induced neuronal damage, in a dose-dependent manner. Among them, 120 mg/kg RUF was most pronounced. Immunohistochemistry (IHC) and western blot analysis showed that RUF inhibited the IBA-1 overexpression caused by KA to block microglia cell overactivation. Further, RUF treatment partially reversed neuroinflammatory cytokine (IL-1β, TNFα, HMGB1, and NLRP3) overexpression in mRNA and protein levels in KA mice. Moreover, although KA stimulation inhibited the expression of tight junctions, RUF treatment significantly upregulated expression of tight junction proteins (occludin and claudin 5) in both mRNA and protein levels in the brain tissues of KA mice. RUF inhibited the overactivation of microglia, suppressed the neuroinflammatory response, and reduced the destruction of blood–brain barrier, thereby alleviating the excitatory nerve damage of the KA-mice.

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Effects of fasudil on blood-brain barrier integrity

10.21203/rs.3.rs-1116440/v1 ◽

2021 ◽

Author(s):

Kei Sato ◽

Shinsuke Nakagawa ◽

Yoichi Morofuji ◽

Yuki Matsunaga ◽

Takashi Fujimoto ◽

...

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Tight Junction ◽

Acute Ischemic Stroke ◽

Blood Brain Barrier ◽

Tight Junction Protein ◽

Brain Barrier ◽

Blood Brain ◽

Junction Protein ◽

Culture Models

Abstract Background Cerebral infarction accounts for 85% of all stroke cases. Even in an era of rapid and effective recanalization using an intravascular approach, the majority of patients have poor functional outcomes. Thus, there is an urgent need for the development of therapeutic agents to treat acute ischemic stroke. We evaluated the effect of fasudil, a Rho kinase inhibitor, on blood brain barrier (BBB) functions under normoxia or oxygen-glucose deprivation (OGD) conditions using a primary cell-based in vitro BBB model. Medhods: BBB models from rat primary cultures (brain capillary endothelial cells, astrocytes, and pericytes) were subjected to either normoxia or 6-hour OGD/24-hour reoxygenation. To assess the effects of fasudil on BBB functions, we evaluated real time impedance, transendothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction protein expression using immunohistochemistry and western blotting. Lastly, to understand the observed protective mechanism on BBB functions by fasudil we examined the role of cyclooxygenase-2 and thromboxane A2 receptor agonist U-46619 in BBB-forming cells. Results We found that treatment with 0.3–30 µM of fasudil increased cellular impedance. Fasudil enhanced barrier properties in a concentration-dependent manner, as measured by an increased (TEER) and decreased permeability. Fasudil also increased the expression of tight junction protein claudin-5. Reductions in TEER and increased permeability were observed after OGD/reoxygenation exposure in mono- and co-culture models. The improvement in BBB integrity by fasudil was confirmed in both of the models, but was significantly higher in the co-culture than in the monoculture model. Treatment with U-46619 did not show significant changes in TEER in the monoculture model, whereas it showed a significant reduction in TEER in the co-culture model. Fasudil significantly improved the U-46619-induced TEER reduction in the co-culture models. Pericytes and astrocytes have opposite effects on endothelial cells and may contribute to endothelial injury in hyperacute ischemic stroke. Overall, fasudil protects the integrity of BBB both by a direct protective effect on endothelial cells and by a pathway mediated via pericytes and astrocytes. Conclusions Our findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

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Flavonoid transport across RBE4 cells: A blood-brain barrier model

Cellular & Molecular Biology Letters ◽

10.2478/s11658-010-0006-4 ◽

2010 ◽

Vol 15 (2) ◽

Cited By ~ 61

Author(s):

Ana Faria ◽

Diogo Pestana ◽

Diana Teixeira ◽

Joana Azevedo ◽

Victor Freitas ◽

...

Keyword(s):

Blood Brain Barrier ◽

Cerebrovascular Diseases ◽

Brain Barrier ◽

Array Detector ◽

Dependent Manner ◽

Blood Brain ◽

Parkinson’S Diseases ◽

Blood Brain Barrier Model ◽

Immortalized Cell ◽

Barrier Model

AbstractThere is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer’s and Parkinson’s diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.

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Culture-Induced Changes in Blood—Brain Barrier Transcriptome: Implications for Amino-Acid Transporters in vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.72 ◽

2009 ◽

Vol 29 (9) ◽

pp. 1491-1502 ◽

Cited By ~ 94

Author(s):

Ruth Lyck ◽

Nadine Ruderisch ◽

Anton G Moll ◽

Oliver Steiner ◽

Clemens D Cohen ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Amino Acid Transporters ◽

Mrna Levels ◽

Blood Brain ◽

Barrier Formation ◽

Platelet Endothelial Cell Adhesion ◽

Induced Changes

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood—brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1+) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven ( 4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two ( Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y+ Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

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