Endothelial Myosin IIA Is Required for the Maintenance of Blood–Brain Barrier Integrity
<p>Brain endothelial deletion of Myosin IIA impairs the integrity of the BBB in mice. (<b>A</b>) Representative PCR gel image for genotyping mice using tail genomic DNA, showing floxed allele band at 480 bp, WT allele band at 349 bp, and Cre-positive band at 421 bp. (<b>B</b>) Representative Western blot image, showing Myosin IIA protein levels in primary brain ECs from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">n</span> = 3). (<b>C</b>) Statistical analysis of Myosin IIA protein levels in primary brain ECs from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">n</span> = 3). *** <span class="html-italic">p</span> < 0.001; Student’s <span class="html-italic">t</span>-test. (<b>D</b>) Representative immunofluorescence staining of brain tissue sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice, showing CD31 (green), Myosin IIA (red), and DAPI (blue). Scale bar: 50 μm. (<b>E</b>) Representative image of brain tissues from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice following intravenous injection of Evans blue. Scale bar: 5 mm. (<b>F</b>) Statistical analysis of total Evans blue tracer leakage into the brain parenchyma of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">Myh9</span><sup>fl/fl</sup>, <span class="html-italic">n</span> = 4; <span class="html-italic">Myh9</span><sup>ECKO</sup>, <span class="html-italic">n</span> = 5). * <span class="html-italic">p</span> < 0.05; Student’s <span class="html-italic">t</span>-test. (<b>G</b>) Representative immunofluorescence staining of brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice following intravenous injection of Sulfo-NHS-Biotin, showing Sulfo-NHS-Biotin (green) and DAPI (blue). Scale bar: 1 mm.</p> "> Figure 2
<p>Brain endothelial deletion of Myosin IIA increases seizure susceptibility and seizure-induced mortality. (<b>A</b>) Experimental design for a mouse epilepsy model, involving intraperitoneal injection of scopolamine followed by pilocarpine 30 min later, with mouse behavior monitored over the next 120 min. (<b>B</b>) Representative images of Evans blue tracer leakage in brain tissues of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice after pilocarpine-induced epilepsy. Scale bar: 5 mm. (<b>C</b>) Statistical analysis of total Evans blue tracer leakage into the brain tissues of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice post-pilocarpine administration (<span class="html-italic">n</span> = 6). * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001; one-way ANOVA test. (<b>D</b>) Representative immunofluorescence co-staining images of brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice post-pilocarpine administration, showing CD31 (red) and Sulfo-NHS-Biotin (green). Scale bar: 100 μm. (<b>E</b>) Statistical analysis of Sulfo-NHS-Biotin leakage index in brain tissues of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice after pilocarpine-induced epilepsy (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> < 0.05; Student’s <span class="html-italic">t</span>-test. (<b>F</b>) Statistical analysis of the incidence of seizure occurrence in <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice after pilocarpine administration (NS, no seizure group; Seizure, seizure group; <span class="html-italic">n</span> = 14). *** <span class="html-italic">p</span> <0.001; Fisher’s exact test. (<b>G</b>) Time to first seizure onset in <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice following pilocarpine administration (<span class="html-italic">n</span> = 6). * <span class="html-italic">p</span> < 0.05; Student’s <span class="html-italic">t</span>-test. (<b>H</b>) Survival time of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice after pilocarpine administration, with a total monitoring duration of 120 min (<span class="html-italic">n</span> = 14). * <span class="html-italic">p</span> < 0.05; Student’s <span class="html-italic">t</span>-test.</p> "> Figure 3
<p>Deletion of brain endothelial Myosin IIA downregulates junctional proteins of the BBB. (<b>A</b>) Representative electron microscopy image of vascular endothelial tight junction structures in brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice, with yellow arrows indicating disrupted tight junctions. Scale bar: 200 nm. (<b>B</b>) Statistical analysis of the proportion of abnormal tight junction structures in brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">n</span> = 3). *** <span class="html-italic">p</span> < 0.001; Student’s <span class="html-italic">t</span>-test. (<b>C</b>) Western blot analysis of ZO-1, Ve-cadherin, Occludin, and Claudin-5 protein levels in primary brain ECs from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice. (<b>D</b>) Statistical analysis of protein expression levels of ZO-1, Ve-cadherin, Occludin, and Claudin-5 in primary brain ECs from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01; Student’s <span class="html-italic">t</span>-test. (<b>E</b>) Representative immunofluorescence co-staining image of brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice, showing CD31 (green) and ZO-1 (red). Scale bar: 50 μm. (<b>F</b>) Representative immunofluorescence co-staining image of brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice, showing CD31 (green) and Occludin (red). Scale bar: 50 μm. (<b>G</b>) Representative immunofluorescence co-staining image of brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice, showing CD31 (green) and Claudin-5 (red). Scale bar: 50 μm. (<b>H</b>) Statistical analysis of colocalization of ZO-1, Occludin, Claudin-5, and CD31 in brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">n</span> = 3). *** <span class="html-italic">p</span> < 0.001; Student’s <span class="html-italic">t</span>-test.</p> "> Figure 4
<p>Myosin IIA mediates the transcription of <span class="html-italic">Ctnnb1</span> gene and interacts with its protein β-catenin. (<b>A</b>) RNA was extracted from primary brain ECs of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice, and RT-PCR was conducted to assess mRNA expression levels of key molecules regulating the blood–brain barrier (<span class="html-italic">n</span> = 3–4). ** <span class="html-italic">p</span> < 0.01; Student’s <span class="html-italic">t</span>-test. (<b>B</b>) Western blot analysis of β-catenin protein levels in primary brain ECs from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice. (<b>C</b>) Statistical analysis of β-catenin protein levels in primary brain ECs from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> < 0.05; Student’s <span class="html-italic">t</span>-test. (<b>D</b>) RT-PCR analysis of <span class="html-italic">Ctnnb1</span> mRNA levels in SCR-shRNA and <span class="html-italic">Myh9</span>-shRNA group cells (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> < 0.01; Student’s <span class="html-italic">t</span>-test. (<b>E</b>) Representative immunofluorescence staining of Myosin IIA in HCMEC/D3 brain ECs, with Myosin IIA (red) and DAPI (blue). Scale bar: 20 μm. (<b>F</b>) Construction strategy for a luciferase reporter plasmid containing the Ctnnb1 gene promoter. (<b>G</b>) Brain ECs overexpressing either control or Myosin IIA were co-transfected with the <span class="html-italic">Ctnnb1</span> gene promoter luciferase reporter plasmid and Renilla luciferase reporter plasmid. Cells were collected 24 h post-transfection to measure their activity (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> < 0.01; Student’s <span class="html-italic">t</span>-test. (<b>H</b>) Interaction between Myosin IIA and β-catenin. Once bEnd.3 brain ECs reached a confluent monolayer, cells were lysed, proteins were harvested, and co-immunoprecipitation was performed to detect interactions between Myosin IIA and β-catenin proteins.</p> "> Figure 5
<p>Overexpression of β-catenin ameliorates BBB leakage in <span class="html-italic">Myh9</span><sup>ECKO</sup> mice. (<b>A</b>) Construction strategy for AAV-GFP and AAV-HA-β-catenin overexpression plasmids. (<b>B</b>) Transfection of AAV-GFP and AAV-HA-β-catenin plasmids into 293T cells; 48 h later, cell proteins were harvested and analyzed by Western blot to assess β-catenin protein levels. (<b>C</b>) Representative immunofluorescence co-staining images of brain sections from <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice 30 days post-injection with AAV-GFP virus showing CD31 (red) and GFP (green); and 30 days post-injection with AAV-HA-β-catenin virus showing CD31 (red) and HA-tag (green). Scale bar: 100 μm. (<b>D</b>) Intravenous injection of AAV-GFP or AAV-HA-β-catenin virus in <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice; 30 days later, Evans blue was injected intravenously, and the total amount of Evans blue in the brain tissues of different treatment groups was quantitatively analyzed <span class="html-italic">(n</span> = 5). *** <span class="html-italic">p</span> < 0.001; one-way ANOVA test. (<b>E</b>) Intravenous injection of AAV-GFP or AAV-HA-β-catenin virus in <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice; 30 days later, Sulfo-NHS-Biotin was injected, and brain sections were co-stained for CD31 (red) and Sulfo-NHS-Biotin (green). Scale bar: 100 μm. (<b>F</b>) Statistical analysis of Sulfo-NHS-Biotin leakage index in brain tissues of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice 30 days post-injection with AAV-GFP or AAV-HA-β-catenin virus (<span class="html-italic">n</span> = 3). Scale bar: 200 μm. * <span class="html-italic">p</span> < 0.05; one-way ANOVA test.</p> "> Figure 6
<p>Overexpression of β-catenin in cerebral endothelium of <span class="html-italic">Myh9</span><sup>ECKO</sup> mice ameliorates epilepsy-induced injury. (<b>A</b>) Statistical analysis of the total amount of Evans blue tracer in the brain tissues of <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice injected intravenously with AAV-GFP or AAV-HA-β-catenin viruses, followed by pilocarpine-induced epilepsy 30 days later (<span class="html-italic">n</span> = 5). *** <span class="html-italic">p</span> < 0.001; one-way ANOVA test. (<b>B</b>) Statistical analysis of the incidence of epilepsy in <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice after intravenous injection of AAV-GFP or AAV-HA-β-catenin viruses and subsequent pilocarpine administration 30 days later (NS, no seizure group; Seizure, seizure group; <span class="html-italic">n</span> = 10). ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001; Fisher’s exact test. (<b>C</b>) Statistical analysis of the time to first seizure in <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice treated with AAV-GFP or AAV-HA-β-catenin viruses and subsequently induced with pilocarpine (<span class="html-italic">Myh9</span><sup>fl/fl</sup>+Vector and <span class="html-italic">Myh9</span><sup>fl/fl</sup>+HA-β-catenin, <span class="html-italic">n</span> = 5; <span class="html-italic">Myh9</span><sup>ECKO</sup>, <span class="html-italic">n</span> = 8; <span class="html-italic">Myh9</span><sup>ECKO</sup> +HA-β-catenin, <span class="html-italic">n</span> = 6). ** <span class="html-italic">p</span> < 0.01; one-way ANOVA test. (<b>D</b>) Statistical analysis of survival time for <span class="html-italic">Myh9</span><sup>fl/fl</sup> and <span class="html-italic">Myh9</span><sup>ECKO</sup> mice injected with AAV-GFP or AAV-HA-β-catenin viruses and then induced with pilocarpine 30 days later, with a total monitoring duration of 120 min (<span class="html-italic">n</span> = 10). * <span class="html-italic">p</span> < 0.05; one-way ANOVA test.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Cell Lines
2.3. FACS Sorting and Analysis of Brain ECs
2.4. Evans Blue Leakage
2.5. BBB Permeability Assay
2.6. Immunohistochemistry
2.7. Transmission Electron Microscopy (TEM)
2.8. Western Blot
2.9. Real-Time PCR (RT-PCR) Analysis
2.10. Seizure Model
2.11. Statistics
3. Results
3.1. Cerebral Endothelial Deletion of Myosin IIA Impairs the Integrity of the BBB in Mice
3.2. Cerebral Endothelial Deletion of Myosin IIA Increases Seizure Susceptibility and Seizure-Induced Mortality
3.3. Cerebral Endothelial Deletion of Myosin IIA Disrupts Brain Endothelial Tight Junctions
3.4. Myosin IIA Mediates the Transcription of the Ctnnb1 Gene and Interacts with Its Protein β-Catenin
3.5. Overexpression of β-Catenin Ameliorates BBB Leakage in Myh9ECKO Mice
3.6. Overexpression of β-Catenin Ameliorates Epileptic Damage in Myh9ECKO Mice
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Deng, Y.; Qiao, Z.; Zhou, C.; Pei, Y.; Xu, H.; Kang, X.; Luo, J. Endothelial Myosin IIA Is Required for the Maintenance of Blood–Brain Barrier Integrity. Cells 2024, 13, 1635. https://doi.org/10.3390/cells13191635
Deng Y, Qiao Z, Zhou C, Pei Y, Xu H, Kang X, Luo J. Endothelial Myosin IIA Is Required for the Maintenance of Blood–Brain Barrier Integrity. Cells. 2024; 13(19):1635. https://doi.org/10.3390/cells13191635
Chicago/Turabian StyleDeng, Yanan, Ziqi Qiao, Changping Zhou, Yujun Pei, Han Xu, Xuya Kang, and Jincai Luo. 2024. "Endothelial Myosin IIA Is Required for the Maintenance of Blood–Brain Barrier Integrity" Cells 13, no. 19: 1635. https://doi.org/10.3390/cells13191635