Endothelial Conditional Knockdown of NMMHC IIA (Nonmuscle Myosin Heavy Chain IIA) Attenuates Blood-Brain Barrier Damage During Ischemia-Reperfusion Injury

Shuaishuai Gong; Guosheng Cao; Fang Li; Zhuo Chen; Xuewei Pan; Huifen Ma; Yuanyuan Zhang; Boyang Yu; Junping Kou

doi:10.1161/STROKEAHA.120.031410

Endothelial Conditional Knockdown of NMMHC IIA (Nonmuscle Myosin Heavy Chain IIA) Attenuates Blood-Brain Barrier Damage During Ischemia-Reperfusion Injury

Stroke. 2021 Mar;52(3):1053-1064. doi: 10.1161/STROKEAHA.120.031410. Epub 2021 Feb 16.

Authors

Shuaishuai Gong^#¹, Guosheng Cao^#^{1

2}, Fang Li¹, Zhuo Chen¹, Xuewei Pan¹, Huifen Ma¹, Yuanyuan Zhang¹, Boyang Yu¹, Junping Kou¹

Affiliations

¹ State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Material Medical, School of Traditional Pharmacy, China Pharmaceutical University, Nanjing, PR China (S.G., G.C., F.L., Z.C., X.P., H.M., Y.Z., B.Y., J.K.).
² College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, PR China (G.C.).

^# Contributed equally.

PMID: 33588591
DOI: 10.1161/STROKEAHA.120.031410

Abstract

Background and purpose: In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Endothelial detachment contributes to BBB disruption and neurovascular dysfunction, but its regulation in stroke has yet to be clarified. We investigated the function of NMMHC IIA (nonmuscle myosin heavy chain IIA) in the endothelium on BBB breakdown and its potential mechanisms.

Methods: Endothelial conditional knockdown NMMHC IIA (Myh9^ECKD) was constructed in vivo and in vitro, and its role was explored in middle cerebral artery occlusion/reperfusion-injured mice and oxygen-glucose deprivation/reoxygenation-injured brain microvascular endothelial cells. The degree of brain injury was analyzed using staining (2,3,5-triphenyltetrazolium chloride, hematoxylin, and eosin) and electron microscopy. BBB breakdown was investigated with leakage of Evans Blue dye and expression of TJs (tight junctions) and MMP (matrix metallopeptidase)-2/9. Transcriptomics for enrichment analysis was adopted to explore the potential downstream signaling pathways of NMMHC IIA involved in middle cerebral artery occlusion/reperfusion-induced BBB dysfunction.

Results: NMMHC IIA expression was upregulated in endothelial cells after cerebral ischemia/reperfusion injury. Myh9^ECKD mice exhibited improvement in endothelial barrier hyperpermeability and TJs integrity stimulated by cerebral ischemia/reperfusion. Blebbistatin (NMMHC II inhibitor) treatment exerted the same effect. Transcriptomics showed that NMMHC IIA was involved in regulating various BBB-related genomic changes in the middle cerebral artery occlusion/reperfusion model, and NMMHC IIA was confirmed to significantly modulate Hippo and peroxisome proliferator-activated receptor gamma/nuclear factor-kappa B signaling pathways, which are closely related to BBB damage.

Conclusions: Our findings provide some new insights into how NMMHC IIA contributes to maintaining the integrity of the cerebral endothelial barrier. NMMHC IIA could be a potential therapeutic target for ischemic stroke.

Keywords: blood-brain barrier; endothelial cells; ischemic stroke; myosin heavy chain; reperfusion.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Blood-Brain Barrier / metabolism*
Brain / metabolism
Brain Ischemia / drug therapy*
Cell Survival
Endothelial Cells / metabolism*
Infarction, Middle Cerebral Artery / metabolism*
Male
Mice
Mice, Inbred C57BL
Myosin Heavy Chains / metabolism*
Oxygen / metabolism
Permeability
Reperfusion Injury / metabolism*
Signal Transduction
Tight Junctions / metabolism

Substances

Myosin Heavy Chains
Oxygen