Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice

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Open Access
Peer-reviewed

Research Article

Jia Li ,

Yang Zhao ,

Jaesung Choi,

Ka Ka Ting,

Paul Coleman,

Jinbiao Chen,

Victoria C. Cogger,

Li Wan,

Zhongsong Shi,

Thorleif Moller,

Xiangjian Zheng ,

Mathew A. Vadas ,

Jennifer R. Gamble

Jia Li, 

Yang Zhao, 

Jaesung Choi, 

Ka Ka Ting, 

Paul Coleman, 

Jinbiao Chen, 

Victoria C. Cogger, 

Li Wan, 

Zhongsong Shi, 

Thorleif Moller

x

Published: June 5, 2020

https://doi.org/10.1371/journal.pbio.3000734

?This is an uncorrected proof.

AbstractCerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

Citation: Li J, Zhao Y, Choi J, Ting KK, Coleman P, Chen J, et al. (2020) Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice. PLoS Biol 18(6):
e3000734.

https://doi.org/10.1371/journal.pbio.3000734Academic Editor: Richard Daneman, UCSD, UNITED STATESReceived: May 1, 2019; Accepted: May 20, 2020; Published: June 5, 2020Copyright: © 2020 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the paper and its Supporting Information files, S1 Data and S1 and S2 Movies. All the gel images were included in S1 Raw images; the RNA-seq data have been deposited on GEO (GSE149948).Funding: This work was supported by the National Health and Medical Research Council (NHMRC) of Australia, #571408 (JRG), #1074664 (JL), and #161558 (XZ), https://nhmrc.gov.au/, and the National Natural Science Foundation of China, #81873752 (ZS), http://www.nsfc.gov.cn/english/site_1/index.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.Abbreviations:
ADAMTS7,
a disintegrin and metalloproteinase with thrombospondin motifs 7; Akt,
protein kinase B; AVM,
arteriovenous malformation; CCM,
cerebral cavernous malformation; CCM1/2/3,
cerebral cavernous malformations 1/2/3; CNS,
central nervous system; DEG,
differentially expressed gene; EC,
endothelial cell; ECM,
extracellular matrix; ENG,
endoglin; eNOS,
endothelial nitric oxide synthase; FDR,
false discovery rate; FITC,
fluorescein isothiocyanate; FPKM,
fragments per kilo bases per million reads; GFP,
green fluorescent protein; Gr,
granulocyte; GSEA,
Gene Set Enrichment Analysis; HE,
hematoxylin–eosin; HHT,
hereditary hemorrhagic telangiectasia; HUVEC,
human umbilical vein endothelial cell; ICAM-1,
intercellular adhesion molecule 1; IP,
intraperitoneal; JAM-A,
junctional adhesion molecule-A; KLF2/4,
kruppel-like factor 2/4; KRIT1,
krev interaction trapped 1; LENG8,
leukocyte receptor cluster member 8; microCT,
micro–computed tomography; miR-27a,
microRNA-27a; Notch1,
Notch homolog 1; P,
day(s) post birth; PDCD10,
programmed cell death 10; PDGF,
platelet-derived growth factor; PD1,
programmed cell death protein 1; pMLC,
phospho myosin light chain; PMN,
polymorphonuclear neutrophil; PPARγ,
peroxisome proliferator-activated receptor gamma; RNA-seq,
RNA sequencing; SEM,
scanning electron microscope; SEMA6A,
semaphoring 6A; siRNA,
small interfering RNA; TGF-β,
transforming growth factor-β; TLR4,
toll-like receptor 4; TNF-α,
tumor necrosis factor-α; TSB,
target site blocker; TSP1,
thrombospondin 1; VE-cadherin,
vascular endothelial cadherin; VEGF,
vascular endothelial growth factor; WT,
wild-type; ZO-1,
zonula occludens-1

IntroductionCerebral cavernous malformations (CCMs) are vascular malformations mostly occurring in the central nervous system (CNS) [1]. CCM lesions are formed by abnormal dilated blood capillaries. One of the key features of CCM is the existence of gaps between endothelial cells (ECs) lining the lesions [2], which are leaky and can cause hemorrhagic stroke and seizure [3–5]. Currently, there is no treatment for CCMs other than surgery, which is limited by size and depth of the lesions.
Familial CCMs in humans are a result of loss-of-function mutation in either one of three genes, CCM1 (krev interaction trapped 1 [KRIT1]), CCM2, and CCM3 (programmed cell death 10 [PDCD10]) [6]. Mice with postnatal loss of Ccm genes in ECs develop similar brain vascular lesions as seen in human CCM patients. In both humans and mice, loss of CCM1 or CCM2 leads to similar defects in vascular integrity, morphology, and burden of CCM lesions, with CCM3 deletion giving a more severe phenotype [7–10].
The molecular pathways involved in CCM development include the kruppel-like factor (KLF)2/4 pathway, endothelial-to-mesenchymal transition, RhoA/ROCK/phospho myosin light chain (pMLC), and, more recently, toll-like receptor 4 (TLR4) signaling through gut microbiome composition [3,9,11–14]. Although the clinical course of CCM disease is highly variable, a common feature is the disruptive EC–EC junctions and enhanced permeability [2,4,11,15], which likely explains the hemorrhage and inflammatory response in CCMs. Consistent with this, key endothelial junction molecules, including vascular endothelial cadherin (VE-cadherin), zonula occludens-1 (ZO-1), and claudin-5, are decreased or disorganized in lesions [10,16]. VE-cadherin, the ‘hub’ of EC junctional molecules, regulates the actin cytoskeleton through RhoA [17]. It also functions upstream of claudin-5, a key protein in tight junctions in the CNS [18,19]. In addition, VE-cadherin is a critical endothelial regulator of transforming growth factor-β (TGF-β) signaling [20]. Thus, VE-cadherin serves as an appealing target for CCM, since RhoA, claudin-5, and TGF-β are all deregulated in this disease [9,11,12,21] and are linked to disruption of vascular integrity. Therapies that increase the expression of VE-cadherin could restore vascular integrity and have a profound impact on CCM disease [22]. Indeed, the RhoA kinase inhibitors fasudil and, to a lesser extent, simvastatin decreased CCM lesions through restoring vascular integrity in mice [9,23].
MicroRNAs are key regulators of gene expression and play important roles in regulation of vascular integrity [24]. We previously showed microRNA-27a (miR-27a) targets VE-cadherin to disrupt vascular integrity [25,26]. Herein, we show miR-27a is overexpressed in brain ECs isolated from mice with CCMs. CD5-2 is a 15-nt-long target site blocker (TSB), which binds to the miR-27a binding site in the VE-cadherin 3′ UTR [25,26]. We previously showed CD5-2 increases endogenous VE-cadherin expression and restores vascular integrity in retinopathy, in peripheral ischemic vessels, and cancer vessels [25–27]. Based on this knowledge, we propose a strategy to treat CCMs through restoring VE-cadherin expression. We demonstrate here that the miR-27a/VE-cadherin interaction is CCM relevant and can be targeted by CD5-2 to normalize the vasculature of CCM lesions, resulting in decreased inflammation and inhibition of CCM pathologies. These data highlight VE-cadherin as a druggable target in diseases with abnormal vascular integrity.

Results
Up-regulation of microRNA-27a in the context of abnormal VE-cadherin expression in CCM pathology
Disruption of EC junctions and abnormal vascular integrity are key features in CCM pathology [9,11]. In the lesions of human patients with CCM, VE-cadherin expression is disrupted compared with its expression in normal vessels (Fig 1A). In a neonatal mouse model of CCM disease (the EC specific Ccm2 deleted model, Ccm2ECKO), numerous CCM lesions appeared in the eye (S1A Fig) and hindbrains (S1B Fig) [3,21]. Furthermore, depletion of CCM1 or CCM2 in both primary human umbilical vein endothelial cells (HUVECs) and brain microvascular EC line, hCMEC/D3, using small interfering RNA (siRNA), resulted in decreased mRNA expression of key junctional molecules such as VE-cadherin and claudin-5 (S2A and S2B Fig) [28], and disruption of EC junctions, as shown by VE-cadherin staining (S2C Fig).
Fig 1. VE-cadherin and miR-27a are down-regulated and up-regulated in CCMs, respectively.(A) Representative immunohistochemistry staining of VE-cadherin of human lesion-free and CCM brain tissue. Arrowheads, VE-cadherin; dashed line, vascular lumen of CCM lesions. Bar, 100 μm (n=6). (B) Real-time PCR measurement of microRNA expression in HUVECs treated with siRNA negative control (si-Ctrl) or siRNA to CCM2 (n=3–4). (C) miR-27a was up-regulated and miR-125 was down-regulated in ECs isolated at P8 mice. RNA were from two separate preparations. The first includes the following: WT (n=5), Ccm1ECKO (n=3), and Ccm2ECKO (n=2) mice. The second includes the following: WT (n=4), Ccm1ECKO (n=3), and Ccm2ECKO (n=2) mice. (D) siRNA knockdown of KLF2 or KLF4 or both in HUVECs blocks loss-of-CCM2–induced expression of miR-27a (n=3–4). (E) Measurement of KLF2 and KLF4 in VE-cadherin null ECs with CCM2 depleted by siRNAs (n=3). Data represent mean ± SEM; *P 10−2 mm3) and medium (10−3–10−2 mm3) lesions but not the small (10−2 mm3; medium: 10−3–10−2 mm3; small:
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