NOGOB receptor deficiency increases cerebrovascular permeability and hemorrhage via impairing histone acetylation–mediated CCM1/2 expression
Zhi Fang, … , Wenquan Hu, Qing Robert Miao
Zhi Fang, … , Wenquan Hu, Qing Robert Miao
Published March 22, 2022
Citation Information: J Clin Invest. 2022;132(9):e151382. https://doi.org/10.1172/JCI151382.
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Research Article Angiogenesis Vascular biology

NOGOB receptor deficiency increases cerebrovascular permeability and hemorrhage via impairing histone acetylation–mediated CCM1/2 expression

Abstract

The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain-barrier hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-sequencing analysis to examine changes in the transcriptome. Surprisingly, we found that the acetyltransferase HBO1 and histone acetylation were downregulated in NGBR-deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of HBO1 and acetylated histone H4K5 and H4K12 on the promotor of the CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that is modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.

Authors

Zhi Fang, Xiaoran Sun, Xiang Wang, Ji Ma, Thomas Palaia, Ujala Rana, Benjamin Miao, Louis Ragolia, Wenquan Hu, Qing Robert Miao

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Figure 5

The overexpression of CCM1 and CCM2 in brain ECs diminishes Ngbr deficiency–promoted microvessel leakage and hemorrhage in vivo.

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The overexpression of CCM1 and CCM2 in brain ECs diminishes Ngbr deficie...
CCM1 and CCM2 overexpression in brain ECs was achieved by AAV-CCM1 and AAV-CCM2 administration. AAV-BR1-GFP was used as control (AAV-ctrl). (A) Tamoxifen was injected 1 week after AAV injection, and mice were euthanized 3 weeks after tamoxifen injection as shown in the diagram protocol. (B) The efficacy of AAV-BR1–mediated overexpression of CCM1 and CCM2 was determined by Western blotting of the lysates of MBMVECs extracted from the brain of mice injected with AAV-CCM1 and -2 or AAV-ctrl. The results demonstrated sufficient overexpression of CCM1 and CCM2 in MBMVECs in vivo. (C) Representative images of hemorrhage in fresh brain tissues and H&E staining. Hemorrhage sites were observed in the brain of AAV-ctrl–injected NgbrECKO mice. In contrast, scarce hemorrhage sites were observed in the brain of the AAV-CCM1/2–injected NgbrECKO group. Scale bars: 100 μm. (D and E) CCM1 and CCM2 overexpression significantly diminished the Ngbr deficiency–promoted hyperpermeability, determined by calculation of Evans blue extravasation and brain water content. Data are presented as mean ± SD, n = 5 mice per group. Significance was tested by 1-way ANOVA with Dunnett’s post hoc test. ***P < 0.001 versus Ngbrfl/fl mice treated with AAV-ctrl; ###P < 0.001 versus NgbrECKO mice treated with AAV-ctrl. (F) IgG staining showing increased IgG-positive staining in AAV-ctrl–injected NgbrECKO mice compared with AAV-ctrl–injected Ngbrfl/fl mice, while significantly decreased IgG-positive staining was observed in AAV-CCM1/2–injected NgbrECKO mice. Scale bars: 200 μm. (G) Western blotting was used to determine the protein levels in MBMVECs extracted from 5 mice in each group. Results showed that overexpression of CCM1 and CCM2 genes mitigated the hyperactivation of RhoA/phos-MLC signaling and the impairment of AJs (VE-cadherin) and TJs (claudin-5) in MBMVECs of NgbrECKO mice.