MiRNA-132/212 regulates tight junction stabilization in blood–brain barrier after stroke

AbstractMicroRNA-132/212 has been supposed as a critical gene related to the blood–brain barrier (BBB) protection after stroke, but its regulation pathway including the upstream regulator and downstream targets is still unclear. Herein, we demonstrated the cAMP response element-binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) to be the upstream regulator of miRNA-132/212 using CRTC1 knockout and wild-type mice. CRTC1 deletion led to the reduction of miRNA-132/212 expression in mice brain after ischemic stroke, significantly increased infarct volume, and aggravated BBB permeability with worsening neurological deficits. Furthermore, we identified that miRNA-132 repressed Claudin-1, tight junction-associated protein-1 (TJAP-1), and RNA-binding Fox-1 (RBFox-1) by directly binding to their respective 3′-untranslated regions, which alleviated the ischemic damage by enhancing neuronal survival and BBB integrity. Moreover, the co-culture of endothelial cells with CRTC1-deficient neurons aggravated the cell vulnerability to hypoxia, also supporting the idea that miRNA-132/212 cluster is regulated by CRTC1 and acts as a crucial role in the mitigation of ischemic damage. This work is a step forward for understanding the role of miRNA-132/212 in neurovascular interaction and may be helpful for potential gene therapy of ischemic stroke.

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Network Pharmacology-Based Approach to Revealing Biological Mechanisms of Qingkailing Injection against IschemicStroke: Focusing on Blood-Brain Barrier

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/2914579 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Shuang Zhang ◽

Xueqian Wang ◽

Fafeng Cheng ◽

Chongyang Ma ◽

Shuning Fan ◽

...

Keyword(s):

Ischemic Stroke ◽

Tight Junction ◽

Blood Brain Barrier ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Brain Barrier ◽

Neurological Deficits ◽

Network Pharmacology ◽

Ppi Network ◽

Blood Brain

Ischemic stroke is the most common type of cerebrovascular accident worldwide. It causes long-term disability and death. Qingkailing (QKL) injection is a traditional Chinese patent medicine which has been clinically applied in the treatment of ischemic stroke for nearly thirty years. In the present study, network pharmacology combined with experimentation was used to elucidate the mechanisms of QKL. ADME screening and target prediction identified 62 active compounds and 275 targets for QKL. Topological screening of the protein-protein interaction (PPI) network was used to build a core PPI network consisting of 408 nodes and 17,830 edges. KEGG enrichment indicated that the main signaling pathway implicated in ischemic stroke involved hypoxia-inducible factor-1 (HIF-1). Experimentation showed that QKL alleviated neurological deficits, brain infraction, blood-brain barrier (BBB) leakage, and tight junction degeneration in a mouse ischemic stroke model. Two-photon laser scanning microscopy was used to evaluate BBB permeability and cerebral microvessel structure in living mice. HIF-1α, matrix metalloproteinase-9 (MMP-9), and tight junction proteins such as occludin, zonula occludins-1 (ZO-1), claudin-5, and VE-Cadherin were measured by western blotting. QKL upregulated ZO-1 and downregulated HIF-1α and MMP-9. QKL has a multiapproach, multitarget, and synergistic effect against ischemic stroke.

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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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Abstract TP80: Panaxatriol Saponins Attenuates Blood-brain Barrier Disruption and Promotes Angiogenesis After Ischemic Stroke in Rats

Stroke ◽

10.1161/str.48.suppl_1.tp80 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Hui Yang ◽

Zhen Hui ◽

Du Juan Sha ◽

Yun Xu

Keyword(s):

Ischemic Stroke ◽

Blood Brain Barrier ◽

Cerebral Perfusion ◽

Infarct Volume ◽

Immunofluorescence Staining ◽

Brain Barrier ◽

Shh Pathway ◽

Blood Brain Barrier Disruption ◽

Blood Brain ◽

Brain Barrier Disruption

Background: The induction of angiogenesis and maintain the integrity of the blood brain barrier (BBB) after stroke may enhance neurorestorative processes. Panaxatriol Saponins (PTS), extracted from traditional Chinese herb Panaxnotoginseng, could noticeably prevent BBB disruption and promote angiogenesis in rodent stroke model. Methods: Middle cerebral artery occlusion (MCAO) model were applied to mimic acute stroke in vivo. Ischemic infarct volume and neurological functions were evaluated through 2,3,5-triphenyltetrazolium chloride (TTC) staining and Longa Scores (LS) respectively. The micro-PET scan was adopted to assess cerebral perfusion; evans blue extravasation assay was used to test BBB permeability; real time PCR and Western blot were used to evaluate the level of vascular growth factors, pro-inflammation factors, the components of Sonic hedgehog (Shh) pathway and NF-κB pathway. Enzyme Linked Immunosorbent Assay (ELISA) was used to detect the levels of pro-inflammation factors in the brain. The capillaries density in ischemic penumbra and tight junction in BBB were measured by immunofluorescence staining. Results: PTS treatment improved neurological function and reduced infarct volume in MCAO-rats. The result of micro-PET scan indicated that PTS could significantly enhance cerebral perfusion after MCAO operation. Treatment of PTS significantly attenuated BBB destruction. PTS could significantly increase the VEGF, Ang-1, VEGFR-2, Tie-2, CD31 and α-SMA mRNA expression at 3 d and 7 d after MCAO compared to vehicle group. Moreover, the expression levels of inflammation factors were decreased after PTS treatment. The co-immunofluorescence staining of α-SMA and Brdu with CD31 respectively showed that PTS promotes angiogenesis and endothelial cell proliferation after MCAO. Meanwhile, co-immunofluorescence staining of Claudin-5, Occludin and ZO-1 with CD31 respectively showed that PTS could protect tight junction from ischemia/reperfusion injury. PTS could also activate Shh pathway and inhibited NF-κB pathway. Conclusions: PTS alleviated ischemic stroke injury through attenuates blood-brain barrier disruption and promotes angiogenesis. PTS could be a potential medication for combating ischemic brain injury.

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MicroRNA-30a regulates acute cerebral ischemia-induced blood–brain barrier damage through ZnT4/zinc pathway

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x20926787 ◽

2020 ◽

pp. 0271678X2092678 ◽

Cited By ~ 1

Author(s):

Peng Wang ◽

Rong Pan ◽

John Weaver ◽

Mengjie Jia ◽

Xue Yang ◽

...

Keyword(s):

Ischemic Stroke ◽

Cerebral Ischemia ◽

Tight Junction ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Tight Junction Proteins ◽

Blood Brain ◽

Acute Cerebral Ischemia ◽

Junction Proteins

The mechanism of early blood–brain barrier (BBB) disruption after stroke has been intensively studied but still not fully understood. Here, we report that microRNA-30a (miR-30a) could mediate BBB damage using both cellular and animal models of ischemic stroke. In the experiments in vitro, inhibition of miR-30a decreased BBB permeability, prevented the degradation of tight junction proteins, and reduced intracellular free zinc in endothelial cells. We found that the zinc transporter ZnT4 was a direct target of negative regulation by miR-30a, and ZnT4/zinc signaling pathway contributed significantly to miR-30a-mediated BBB damage. Consistent with these in vitro findings, treatment with miR-30a inhibitor reduced zinc accumulation, increased the expression of ZnT4, and prevented the loss of tight junction proteins in microvessels of ischemic animals. Furthermore, inhibition of miR-30a, even at 90 min post onset of middle cerebral artery occlusion, prevented BBB damage, reduced infarct volume, and ameliorated neurological deficits. Together, our findings provide novel insights into the mechanisms of cerebral ischemia-induced BBB disruption and indicate miR-30a as a regulator of BBB function that can be an effective therapeutic target for ischemic stroke.

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TRA2A-induced upregulation of LINC00662 regulates blood-brain barrier permeability by affecting ELK4 mRNA stability in Alzheimer's microenvironment

10.21203/rs.2.17596/v2 ◽

2019 ◽

Author(s):

Qianshuo Liu ◽

Lu Zhu ◽

Xiaobai Liu ◽

Jian Zheng ◽

Yunhui Liu ◽

...

Keyword(s):

Tight Junction ◽

Blood Brain Barrier ◽

Rna Binding ◽

Rna Binding Proteins ◽

Brain Barrier ◽

Blood Brain ◽

Bbb Permeability ◽

Novel Target ◽

Related Proteins

Abstract The blood-brain barrier (BBB) plays a pivotal role in maintenance and regulation of the neural microenvironment. The occurrence of BBB disruption is the pathological change of early Alzheimer’s disease (AD). RNA-binding proteins and long non-coding RNAs are involved in the regulation of BBB permeability. Our study was performed to demonstrate TRA2A/LINC00662/ELK4 axis in regulating BBB permeability in AD microenvironment. In Aβ1-42-incubated microvascular endothelial cells (ECs) of BBB model in vitro, TRA2A and LINC00662 were enriched. TRA2A increased the stability of LINC00662 by binding with it. The knockdown of either TRA2A or LINC00662 decreased the BBB permeability via upregulating the expressions of tight junction-related proteins. ELK4 was lower expressed in BBB model in vitro in AD microenvironment. LINC00662 mediated the degradation of ELK4 mRNA by SMD pathway. The downregulated ELK4 increased the permeability of BTB by increasing the tight junction-related proteins expressions. TRA2A/LINC00662/ELK4 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment, which may provide a novel target for the therapy of AD.

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Abstract T P250: Role of Autophagy in Blood-Brain Barrier Disruption and Tight Junction Degradation Under Ischemic Condition

Stroke ◽

10.1161/str.46.suppl_1.tp250 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Kyeong-A Kim ◽

Young-Jun Shin ◽

Eun-Sun Kim ◽

Muhammad Akram ◽

Dabi Noh ◽

...

Keyword(s):

Ischemic Stroke ◽

Tight Junction ◽

Cell Viability ◽

Blood Brain Barrier ◽

Neuronal Damage ◽

Cell Damage ◽

Brain Barrier ◽

Blood Brain ◽

Junction Protein

During ischemic stroke, the integrity of blood-brain barrier (BBB), which shows selective permeability for substances to brain, is significantly damaged amplifying ischemic neuronal damage. There have been attempts to identify the exact mechanism ischemic BBB disruption to minimize brain damage under ischemic stroke. Autophagy is catabolic process which involves degradation and recycling of damaged or unnecessary organelles. However, excessive autophagy can induce cell damage and death under pathological conditions such as ischemia. In this study, we evaluated if autophagy is a key mechanism of BBB dysfunction under ischemic stroke. In vitro BBB model of bEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), an ischemic mimic condition. After exposure to OGD for 18 hours, cell viability was significantly decreased and cellular permeability was impaired. The conversion of LC3-I to LC3-II and puncta of LC3 in bEnd.3 were increased, demonstrating that autophagy is induced under ischemic condition. Modulation of autophagy by 3-methyladenine, an autophagy inhibitor, reversed the conversion of LC3 as well as decreased cell viability, suggesting that autophagy involves in ischemic BBB damage. The level of occludin, a tight junction protein in BBB, was decreased after OGD, and this was reversed by inhibition of autophagy. Our findings showed that induction of autophagy might contribute to increased permeability through occludin degradation in brain endothelial cells under ischemia, providing a new mechanism of BBB disruption in ischemic stroke.

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Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection

AJP Cell Physiology ◽

10.1152/ajpcell.00095.2018 ◽

2018 ◽

Vol 315 (3) ◽

pp. C343-C356 ◽

Cited By ~ 74

Author(s):

Wazir Abdullahi ◽

Dinesh Tripathi ◽

Patrick T. Ronaldson

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Tight Junction ◽

Tight Junctions ◽

Blood Brain Barrier ◽

Functional Expression ◽

Brain Barrier ◽

Vascular Protection ◽

Blood Brain ◽

Junction Protein

The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely controls cerebral homeostasis. It also plays a central role in the regulation of blood-to-brain flux of endogenous and exogenous xenobiotics and associated metabolites. This is accomplished by molecular characteristics of brain microvessel endothelial cells such as tight junction protein complexes and functional expression of influx and efflux transporters. One of the pathophysiological features of ischemic stroke is disruption of the BBB, which significantly contributes to development of brain injury and subsequent neurological impairment. Biochemical characteristics of BBB damage include decreased expression and altered organization of tight junction constituent proteins as well as modulation of functional expression of endogenous BBB transporters. Therefore, there is a critical need for development of novel therapeutic strategies that can protect against BBB dysfunction (i.e., vascular protection) in the setting of ischemic stroke. Such strategies include targeting tight junctions to ensure that they maintain their correct structure or targeting transporters to control flux of physiological substrates for protection of endothelial homeostasis. In this review, we will describe the pathophysiological mechanisms in cerebral microvascular endothelial cells that lead to BBB dysfunction following onset of stroke. Additionally, we will utilize this state-of-the-art knowledge to provide insights on novel pharmacological strategies that can be developed to confer BBB protection in the setting of ischemic stroke.

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TRA2A-induced upregulation of LINC00662 regulates blood-brain barrier permeability by affecting ELK4 mRNA stability in Alzheimer's microenvironment

10.21203/rs.2.17596/v1 ◽

2019 ◽

Author(s):

Qianshuo Liu ◽

Lu Zhu ◽

Xiaobai Liu ◽

Jian Zheng ◽

Yunhui Liu ◽

...

Keyword(s):

Tight Junction ◽

Blood Brain Barrier ◽

Rna Binding ◽

Rna Binding Proteins ◽

Brain Barrier ◽

Blood Brain ◽

Bbb Permeability ◽

Novel Target ◽

Related Proteins

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Weisheng-Tang Ameliorates Acute Ischemic Brain Damage in Mice by Maintaining Blood-Brain Barrier Integrity

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/4379732 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Min Jae Kim ◽

Ki Hyun Park ◽

Ji Yun Lee ◽

Ki-Tae Ha ◽

Byung Tae Choi ◽

...

Keyword(s):

Tight Junction ◽

Brain Damage ◽

Blood Brain Barrier ◽

Infarct Volume ◽

Brain Barrier ◽

Tight Junction Proteins ◽

Ischemic Brain Damage ◽

Ischemic Brain ◽

Blood Brain ◽

Junction Proteins

Stroke is one of the major causes of death and long-term disability worldwide; the associated breakdown of the blood-brain barrier (BBB) aggravates ischemic brain damage. Accordingly, many medicinal herbs and formulas have been used to treat stroke-related symptoms. In this study, we selected two Korean herbal medicine formulas, Weisheng-tang and Tongxuewan, through Dongeuibogam text-mining analysis, and evaluated their protective effect on BBB disruption and brain damage in stroke. Ischemic brain damage was induced in mice by photothrombotic cortical ischemia. The infarct volume, brain edema, neurological deficits, and motor function 24 h after ischemic injury were analyzed. We investigated BBB breakdown by measuring Evans blue extravasation in addition to endothelial cells, tight junction proteins, protease-activated receptor-1 (PAR-1), and matrix metalloproteinase-9 (MMP-9) using immunofluorescence staining and confocal microscopy. Pretreatment with Weisheng-tang significantly reduced infarct volume and edema and improved neurological and motor functions; however, Tongxuewan did not. In addition, Weisheng-tang decreased brain infarction and edema and recovered neurological and motor deficit in a dose-dependent manner (30, 100, and 300 mg/kg). Weisheng-tang pretreatment resulted in significantly less BBB damage and higher brain microvasculature after focal cerebral ischemia. Tight junction proteins, such as zonula occludens-1 (ZO-1) and claudin-5, were preserved in Weisheng-tang-pretreated mice. Moreover, the ischemic brain in these mice showed suppressed PAR-1 and MMP-9 expression. In conclusion, our findings show that Weisheng-tang, which was selected through literature analysis but has not previously been used as a stroke remedy, exerts protective effects against ischemic brain damage and suggest its possible application for potential stroke patients, especially in the elderly.

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DL-3n-Butylphthalide Improves Blood–Brain Barrier Integrity in Rat After Middle Cerebral Artery Occlusion

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.610714 ◽

2021 ◽

Vol 14 ◽

Author(s):

Muyassar Mamtilahun ◽

Zhenyu Wei ◽

Chuan Qin ◽

Yongting Wang ◽

Yaohui Tang ◽

...

Keyword(s):

Enzyme Activity ◽

Ischemic Stroke ◽

Blood Brain Barrier ◽

Infarct Volume ◽

Artery Occlusion ◽

Brain Barrier ◽

Barrier Integrity ◽

Aqp4 Expression ◽

Blood Brain ◽

Blood Brain Barrier Integrity

Objective: DL-3n-butylphthalide (NBP) has beneficial effects in different stages of ischemic stroke. Our previous studies have demonstrated that NBP promoted angiogenesis in the perifocal region of the ischemic brain. However, the molecular mechanism of NBP for blood–brain barrier protection in acute ischemic stroke was unclear. Here, we explored the neuroprotective effects of NBP on blood–brain barrier integrity in the acute phase of ischemic stroke in a rat model.Methods: Adult male Sprague–Dawley rats (n = 82) underwent 2 h of transient middle cerebral artery occlusion and received 90 mg/kg of NBP for 3 days. Brain edema, infarct volume, surface blood flow, and neurological severity score were evaluated. Blood–brain barrier integrity was evaluated by Evans blue leakage and changes in tight junction proteins. We further examined AQP4 and eNOS expression, MMP-9 enzyme activity, and possible signaling pathways for the role of NBP after ischemic stroke.Results: NBP treatment significantly increased eNOS expression and surface blood flow in the brain, reduced brain edema and infarct volume, and improved neurological severity score compared to the control group (p < 0.05). Furthermore, NBP attenuated Evans blue and IgG leakage and increased tight junction protein expression compared to the control after 1 and 3 days of ischemic stroke (p < 0.05). Finally, NBP decreased AQP4 expression, MMP-9 enzyme activity, and increased MAPK expression during acute ischemic stroke.Conclusion: NBP protected blood–brain barrier integrity and attenuated brain injury in the acute phase of ischemic stroke by decreasing AQP4 expression and MMP-9 enzyme activity. The MAPK signaling pathway may be associated in this process.

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