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 < 0.05, **P < 0.01 determined by one-way ANOVA with Tukey correction. For the raw data used for quantification, see Fig 1 in S1 Data. CCM, cerebral cavernous malformation; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; KLF2, kruppel-like factor 2; miR-27a, microRNA-27a; siRNA, small interfering RNA; VE-cadherin, vascular endothelial cadherin; WT, wild-type.
Fig 2.
CD5-2, a TSB to miR-27a/VE-cadherin, can be delivered to brain vasculature and regulates VE-cadherin.
(A) (i) CD5-2 increased VE-cadherin expression in CCM lesions from mouse samples given the control drug (Ctrl) or CD5-2. (ii) CD5-2 improves VE-cadherin localization in CCM lesions compared with mice given the control drug (Ctrl). In Ctrl, the white arrows point to disruptive VE-cadherin staining. In CD5-2, the red arrows point to junctional VE-cadherin. (B) In situ detection of CD5-2 in the blood vessels in the hindbrain of P6 Ccm2ECKO animal, 6 hours after IP injection of CD5-2. The image without CD5-2 probe was used as background control (top). CD5-2 was detected as the color of cyan (bottom) with ECs stained with CD31. Bar, 50 μm (left); 10 μm (right). Values are shown as mean ± SEM, **P < 0.01, determined by Student t test. For the raw data used for quantification, see Fig 2 in S1 Data. CCM, cerebral cavernous malformation; EC, endothelial cell; IP, intraperitoneal; miR-27a, microRNA-27a; TSB, target site blocker; VE-cadherin, vascular endothelial cadherin.
Fig 3.
CD5-2 rescues CCM lesions in the hindbrain of Ccm2ECKO mice.
(A) Visual appearance of CCM lesions in the hindbrain of representative three P12 Ccm2ECKO mice treated with control or CD5-2. Bar, 1 mm. (B) Composite microCT images of the same hindbrains shown in (A). Bar, 1 mm. CCM lesions are shown in red. (C) Quantitation of CCM lesion by (i) total lesion volume and (ii) the largest lesion volume, and (iii) the mean value of the largest 5 or (iv) 20 lesions. (D) Quantitation of lesion number. Ctrl, 186.6 ± 26.31; CD5-2, 127.3 ± 18.44. (E) Lesions were classified into three groups according to volume size (large: >10−2 mm3; medium: 10−3–10−2 mm3; small: <10−3 mm3). Large, Ctrl: 12.9 ± 1.6; CD5-2: 5.6 ± 1.3; medium, Ctrl: 39 ± 4.9; CD5-2: 26.3 ± 3.5; small, Ctrl: 134.7 ± 24.0; CD5-2: 95.1 ± 15.4, n = 7 mice for control-treated group (Ctrl) and n = 9 mice for CD5-2–treated group. Mice are from 4 different litters. Values are shown as mean ± SEM, N.S, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, determined by Student t test. For the raw data used for quantification, see Fig 3 in S1 Data. CCM, cerebral cavernous malformation; Ctrl, control; microCT, micro–computed tomography.
Fig 4.
CD5-2 decreases established CCM lesions in the hindbrain of Ccm2ECKO mice and lesions in the retina of Ccm2ECKO mice.
(A) Visual appearance of CCM lesions in the hindbrains of three representative P19 Ccm2ECKO animals treated with control or CD5-2. Bar, 1 mm. (B) microCT images of the hindbrains from two mice shown in (A). CCM lesions are shown in red. (C) Quantitation of CCM lesion volume (normalized to 1.0 for mice that received control) by total lesion volume and mean size of the largest 5 lesions. (D) Number of lesions per brain within different size ranges. In (C-D), n = 4 mice for control-treated group and n = 4 mice for CD5-2–treated group. Mice are from 4 different litters. (E) Representative images of whole mount retina isolated from control- and CD5-2–treated littermates stained with isolectin-B4. Mice were treated at P6 and P8. Retinas were dissected at P12. Images are representative of 6 retinas from 3 mice (3 litters) for each treatment. Bar, 200 μm. Quantification is given as the percentage of the marked condensed vascular plexus at the leading edge against the total area of the retinal vasculature. (F) Mice were treated at P8 and P12. Retinas were dissected at P19. Images are representative of 10 retinas from 5 mice (3 litters) for each treatment. Bar, 200 μm. Values are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, determined by Student t test. For the raw data used for quantification, see Fig 4 in S1 Data. CCM, cerebral cavernous malformation; microCT, micro–computed tomography.
Fig 5.
CD5-2 normalizes CCM lesion structure in Ccm2ECKO mice.
(A) Sample scanning electron micrographs of the blood vessels in hindbrain from mice that received Ctrl (top) and CD5-2 (bottom). Images were taken from multiple vessels. Two magnifications are shown: magnification 1, approximately 2,000×, bar: 10 μm; magnification 2, approximately 5,000×, top bar: 5 μm, bottom bar: 2 μm (EC junctions are indicated by red arrow). (B) Hindbrains photographed after fluorescent cadaverine injection. Bright-field (top) and fluorescence image with 555/60-nm excitation and 610/75-nm emission (bottom). An animal without cadaverin Alexa 555 injection was used as the background (i,ii); fluorescence from control (Ctrl) and CD5-2 treated animals was measured by stereomicroscope (iii-vi) and quantified by Fiji imageJ software (vii). Ctrl n = 5; CD5-2 n = 4 from 2 litters. Bar, 2 mm. (C) CCM1 or CCM2 in hCMEC/D3 was depleted by siRNAs. Cells were then transfected with control (Ctrl) or CD5-2. Endothelial monolayer permeability to FITC-dextran (40 KDa) was determined by measurement of absorbance at 490 nm (A490) (n = 2–3). (D) Miles assay of dermal permeability in heterozygous Ccm1 (Tie2-Cre, Ccm1 fl/+) mice in response to PBS or VEGF after pretreatment with control or CD5-2 (n = 6). Values are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, determined by Student t test or one-way ANOVA with Tukey correction. For the raw data used for quantification, see Fig 5 in S1 Data. CCM, cerebral cavernous malformation; EC, endothelial cell; FITC, fluorescein isothiocyanate; hCMEC/D3, human cerebral microvascular endothelial cell line; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.
Fig 6.
CD5-2 inhibits inflammatory response in CCM lesion via VE-cadherin.
(A) CD5-2 effects on ICAM-1 expression in CCM lesion. Sections were triple-stained for DAPI (blue) or CD31 (green), ICAM-1 (red); 3 mice were utilized for each condition. Representative sections are shown. Bar, 8 μm. (B-C) Western blot analysis of ICAM-1 (B) and phospho-p65 (C) in brain microvascular ECs (hCMEC/D3) treated with 10 nM siRNAs for scramble control (Ctrl), CCM1, or CCM2 for 4 hours, followed by transfection of 15 nM CD5-2 or controls then cultured overnight. Molecular weights in kilodaltons are shown. ICAM-1 antibody (Cell signalling #4915) gave two bands (89 and 92 kDa). This antibody recognizes the ICAM-1 C-terminal portion, which is dense with ubiquitination and phosphorylation sites and, as such, #4915 can detect multiple sized ICAM moieties. Representative blots are shown (n = 3) with α-tubulin or β-actin rabbit used as loading control. (D) Representative images of dynamic adhesion of neutrophils to control- or CD5-2–treated ECs following the stimulation with TNF-α for 4 hours. Quantification of number of adherent neutrophils with control or CD5-2–treated ECs is given (n = 3). (E) Representative images of paracellular transmigration of neutrophils (green) through TNF-α–stimulated endothelium (VE-cadherin, red) in the presence of control and CD5-2 in vitro. Transmigrated neutrophils appear GFPdim. (F) (i) Heatmap of all genes between control (Ctrl) and CD5-2 (CD5-2) in VE-cadherin–expressing ECs (VE-cadherin+/+). (ii) GSEA analysis of differentially expressed genes (DEGs) between Ctrl- and CD5-2–treated ECs. (iii) Bar plot of selected inflammation-related DEGs inhibited by CD5-2. Values are shown as mean ± SEM. *P < 0.05, **P < 0.01, N.S, not significant, determined by Student t test or one-way ANOVA with Tukey correction. For the raw data used for quantification, see Fig 6 in S1 Data; Fig 6B and 6C in S1 Raw images. CCM, cerebral cavernous malformation; CD31, cluster of differentiation 31; EC, endothelial cell; GFP, green fluorescent protein; GSEA, Gene Set Enrichment Analysis; hCMEC/D3, human cerebral microvascular endothelial cell line; ICAM-1, intercellular adhesion molecule 1; NF-κB, nuclear factor kappa B; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; TSB, target site blocker; VE-cadherin, vascular endothelial cadherin.