scholarly journals Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xing-Yong Chen ◽  
Shao-Fen Wan ◽  
Nan-Nan Yao ◽  
Ze-Jing Lin ◽  
Yan-Guang Mao ◽  
...  
Keyword(s):  
Cell Migration ◽  
Tight Junction ◽  
Blood Brain Barrier ◽  
Western Blotting ◽  
Tight Junction Proteins ◽  
Control Shrna ◽  
And Migration ◽  
Junction Proteins ◽  
Proliferation And Migration

Abstract Background Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear. Methods Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test. Results Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R. Conclusion This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

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

2020 ◽  
pp. 0271678X2092678 ◽  
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.

scholarly journals The blood-brain barrier studied in vitro across species

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0236770
Author(s):  
Maj Schneider Thomsen ◽  
Nanna Humle ◽  
Eva Hede ◽  
Torben Moos ◽  
Annette Burkhart ◽  
...  
Keyword(s):  
Tight Junction ◽  
Cell Size ◽  
Blood Brain Barrier ◽  
Transferrin Receptor ◽  
Brain Barrier ◽  
Tight Junction Proteins ◽  
Apparent Permeability ◽  
Blood Brain ◽  
Junction Proteins

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, meaning that only a few drugs can pass the BBB. Thus, for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify species-dependent differences that could explain the variations in TEER and aid to the selection of models for future BBB studies. The BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Additionally, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared. Expression and organization of tight junction proteins were in all three species influenced by co-culturing, supporting the findings, that TEER increases after co-culturing with astrocytes. All models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. The most interesting discovery was that even though the pBECs had higher TEER than rBECs and mBECs, the Papp did not show the same variation between species, which could be explained by a significantly larger cell size of pBECs. In conclusion, our results imply that the choice of species for a given BBB study should be defined from its purpose, instead of aiming to reach the highest TEER, as the models studied here revealed similar BBB properties.

scholarly journals The blood-brain barrier studied in vitro across species

2020 ◽  
Author(s):  
Maj Schneider Thomsen ◽  
Nanna Humle ◽  
Eva Hede ◽  
Torben Moos ◽  
Annette Burkhart ◽  
...  
Keyword(s):  
Tight Junction ◽  
Cell Size ◽  
Blood Brain Barrier ◽  
Transferrin Receptor ◽  
Brain Barrier ◽  
Tight Junction Proteins ◽  
Apparent Permeability ◽  
Blood Brain ◽  
Junction Proteins

AbstractThe blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis and protects the brain against toxic substances circulating in the blood, which consequently means that only a few drugs can pass the BBB. Thus for drug screening, understanding cell interactions, and pathology, in vitro BBB models have been developed using BECs from various animal sources. When comparing models of different species, differences exist especially in regards to the transendothelial electrical resistance (TEER). Thus, in the present study, we compared primary mice, rat, and porcine BECs (mBECs, rBECs, and pBECs) cultured in mono- and co-culture with astrocytes, to identify potential species-dependent differences that could explain the variations in TEER and aid to the selection of models for future in vitro BBB studies.The in vitro BBB models based on primary mBECs, rBECs, and pBECs were evaluated and compared in regards to major BBB characteristics. The barrier integrity was evaluated by the expression of tight junction proteins and measurements of TEER and apparent permeability (Papp). Furthermore, the cell size, the functionality of the P-glycoprotein (P-gp) efflux transporter, and the expression of the transferrin receptor were evaluated and compared.pBECs exhibit the highest TEER followed by rBECs and mBECs, but surprisingly the Papp was not that different, which might be explained by a significantly larger cell size of pBECs than rBECs and mBECs. The expression and organization of many tight junction proteins were in all three species influenced by co-culturing, which supports the findings that the TEER increases after co-culturing with astrocytes. Furthermore, all models had functional polarised P-gp efflux transporters and expressed the transferrin receptor. In conclusion, the choice of species for in vitro BBB modeling should be based on the purpose of the study.

Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle

2017 ◽  
Vol 95 (3) ◽  
pp. 1313 ◽  
Author(s):  
L. Zhang ◽  
L. F. Schütz ◽  
C. L. Robinson ◽  
M. L. Totty ◽  
L. J. Spicer
Keyword(s):  
Gene Expression ◽  
Tight Junction ◽  
Tight Junction Proteins ◽  
Junction Proteins

scholarly journals Cyclooxygenase Inhibition Alters Proliferative, Migratory, and Invasive Properties of Human Glioblastoma Cells In Vitro

2021 ◽  
Vol 22 (9) ◽  
pp. 4297
Author(s):  
Matthew Thomas Ferreira ◽  
Juliano Andreoli Miyake ◽  
Renata Nascimento Gomes ◽  
Fábio Feitoza ◽  
Pollyana Bulgarelli Stevannato ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Biology ◽  
Gelatin Zymography ◽  
Viable Cell ◽  
Cell Counting ◽  
Gelatinolytic Activity ◽  
Ep4 Receptor ◽  
And Migration ◽  
Proliferation And Migration

Prostaglandin E2 (PGE2) is known to increase glioblastoma (GBM) cell proliferation and migration while cyclooxygenase (COX) inhibition decreases proliferation and migration. The present study investigated the effects of COX inhibitors and PGE2 receptor antagonists on GBM cell biology. Cells were grown with inhibitors and dose response, viable cell counting, flow cytometry, cell migration, gene expression, Western blotting, and gelatin zymography studies were performed. The stimulatory effects of PGE2 and the inhibitory effects of ibuprofen (IBP) were confirmed in GBM cells. The EP2 and EP4 receptors were identified as important mediators of the actions of PGE2 in GBM cells. The concomitant inhibition of EP2 and EP4 caused a significant decrease in cell migration which was not reverted by exogenous PGE2. In T98G cells exogenous PGE2 increased latent MMP2 gelatinolytic activity. The inhibition of COX1 or COX2 caused significant alterations in MMP2 expression and gelatinolytic activity in GBM cells. These findings provide further evidence for the importance of PGE2 signalling through the EP2 and the EP4 receptor in the control of GBM cell biology. They also support the hypothesis that a relationship exists between COX1 and MMP2 in GBM cells which merits further investigation as a novel therapeutic target for drug development.

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