Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice

Luca Bravi, Noemi Rudini, Roberto Cuttano, Costanza Giampietro, Luigi Maddaluno, Luca Ferrarini, Ralf H Adams, Monica Corada, Gwenola Boulday, Elizabeth Tournier-Lasserve, Elisabetta Dejana, Maria Grazia Lampugnani, Luca Bravi, Noemi Rudini, Roberto Cuttano, Costanza Giampietro, Luigi Maddaluno, Luca Ferrarini, Ralf H Adams, Monica Corada, Gwenola Boulday, Elizabeth Tournier-Lasserve, Elisabetta Dejana, Maria Grazia Lampugnani

Abstract

Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of β-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a β-catenin-driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

Keywords: cerebral cavernous malformation; endothelial cells; sulindac metabolites; vascular pathology; β-catenin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Brain endothelial cells in CCM3-ECKO mice show enhanced β-catenin transcription activity earlier than activation of TGF-β/BMP signaling. (A) Brain sections from wild-type (WT) and CCM3-ECKO mice stained for β-gal (red, Upper), and p-Smad1 (red, Lower) in endothelial cells (Podocalyxin-positive, green) at early (3 dpn) and late (9 dpn) time points after CCM3 recombination (1 dpn). Blue, DAPI-stained nuclei. (B) Costaining for β-gal (red), p-Smad1 (green), and Podocalyxin (blue). (A and B) Arrows, β-gal– and p-Smad1–positive nuclei; empty arrows, p-Smad1–negative nuclei in endothelial cells of a vascular malformation (caverna in A) and telangiectasia in B) in 3-dpn pups. (Insets) Magnification of boxed areas. (Scale bars, 50 μm; Insets in A, 10 μm.) (C) Quantification of β-gal–positive and p-Smad–positive endothelial cells in brain sections as in A and B. At least 450 nuclei were counted in 40 random fields at 63× magnification for each condition in samples from matched littermate pups in three independent experiments. *P < 0.01 versus indicated controls (t test).
Fig. 2.
Fig. 2.
Brain endothelial cells in CCM3-ECKO mice express stem-cell/EndMT markers in association with enhanced β-catenin transcription activity. (A–C) Brain sections from wild-type (WT) and CCM3-ECKO mice stained for β-gal (red) in combination with Podocalyxin (blue) and different stem-cell/EndMT markers (Klf4, S100a4, Id1, all green), at 3 and 9 dpn after CCM3 recombination at 1 dpn. Arrows point to endothelial cells (Podocalyxin-positive) expressing both β-gal and stem-cell/EndMT markers (see Merge, yellow). (Scale bar, 40 μm.) (D) Quantification of endothelial nuclei positive for β-gal, Klf4, S100a4, and Id1 (single positive) and of their colocalization in brain sections as in A–C. Colocalization was calculated in two populations of endothelial cells: the β-gal–positive one with EndMT-positive nuclei and the EndMT-positive one with β-gal–positive nuclei. At least 600 nuclei were counted in 50 random fields at 63× magnification for each condition in samples from matched littermate pups in three independent experiments. *P < 0.05 versus respective WT values (t test); ^P < 0.05 versus value in 3-dpn CCM3-ECKO pups.
Fig. 3.
Fig. 3.
β-Catenin controls the expression of stem-cell/EndMT markers in CCM3-knockout endothelial cells in culture. (A and B) Quantification of typical β-catenin transcription targets (A) and of stem-cell/EndMT markers (B) without (−) and with (+) expression of a dominant-negative Tcf4 in lung wild-type (WT) and CCM3-knockout (KO) endothelial cells. Data are means (±SD) of triplicate RT-PCR assays from three independent experiments. Tubulin transcripts (α, β), which are not targets of the CCM3 knockout, were not modified by dominant-negative Tcf4. *P < 0.05 for CCM3-knockout versus control (WT). ^P < 0.05 for CCM3 knockout plus dominant-negative Tcf4 versus CCM3 knockout plus GFP (t test).
Fig. 4.
Fig. 4.
Brain endothelial cells in CCM3-ECKO mice show sulindac sulfide reduction of β-catenin transcription activity and induction of relocalization of VE-cadherin from diffused distribution to adherens junctions. (A) Brain sections without (Vehicle) and with sulindac sulfide treatment of the CCM3-ECKO mice at different time points after CCM3 recombination. Arrowheads, β-gal reactivity (red) in the nucleus of endothelial cells (Podocalyxin-positive, green). (Scale bar, 50 μm.) (B) Quantification of immunofluorescence microscopy data as in A. At least 500 nuclei were counted in 40 random fields at 63× magnification for each condition in samples from matched littermate pups in three independent experiments. *P < 0.05 versus respective Vehicle (t test). (C) Brain sections (9-dpn pups) stained for VE-cadherin (green), diffused (Vehicle), and junctional (sulindac sulfide, arrowheads) in blood-vessel endothelial cells of CCM3-ECKO and of wild-type (WT) mice (junctional, arrowheads). (Scale bar, 25 μm.)
Fig. 5.
Fig. 5.
CCM3-ECKO mice show sulindac-sulfide–induced constraint of brain and retinal vascular lesions and prolonged survival. (A) Macroscopic appearance of CCM3-ECKO mice brains following dissection without (Vehicle) and with sulindac sulfide treatment in pups at different time points after CCM3 recombination at 1 dpn. (B) Endothelial cells (Pecam-positive, red) of different types of vascular lesions [mulberry (multiple cavernae), single caverna, or telangiectases (Telang.: tortuous small vessels with abnormally dilated lumen) (33)] in brain sections without (Vehicle) and with sulindac sulfide treatment of the CCM3-ECKO mice (9 dpn). (C, Left and Middle) Quantification of mean number of brain lesions as illustrated in B. Matched littermates from five independent litters were Vehicle-treated (n = 8) or sulindac sulfide-treated (n = 7). *P < 0.005, Wilcoxon signed-rank test. (Right) Quantification of mean size of brain lesions (μm; see SI Appendix, Methods for details). *P < 0.05, t test. (D) Endothelial cells (Pecam-positive, red) of vessels in the retina of wild-type (WT) and CCM3-ECKO mice without (Vehicle) and with sulindac sulfide treatment. Multiple-lumen vascular lesions (arrowheads) develop from veins (arrow). (E) Percentages of the retinal perimeter affected by vascular lesions illustrated in D (n = 7 for both Vehicle and sulindac sulfide treatments). *P < 0.05, t test. (F) Detail of the retina vascular lesions illustrated in D. As well as the peripheral vascular malformations, sulindac sulfide induced reductions in the diameters of the veins (green, isolectin B4-labeled endothelial cells). Arteries of these CCM3-ECKO mice do not show this aberrant phenotype. [Scale bars, 0.3 cm (A); 100 μm (B); 700 μm (D); 60 μm (F).] (G) CCM3-flox/flox–Cdh5(PAC)-CreERT2–BAT-gal pups were treated with tamoxifen at 1 dpn to induce endothelial-specific recombination of CCM3. Treatment with sulindac sulfide was started the following day. Kaplan–Meier curves of Vehicle- and sulindac- sulfide-treated pups were significantly different (P = 0.0046, log-rank test). Matched littermate pups were the following: Vehicle-treated, n = 13; sulindac sulfide-treated, n = 15 (in three independent experiments). (H) Pups were recombined for CCM3 at 6 dpn to retard the development of CCM lesions and prolong life span. Sulindac sulfide was started the following day. Kaplan–Meier curves of Vehicle- and sulindac sulfide-treated pups were significantly different (P = 0.0032, log-rank test). Matched littermate pups were the following: Vehicle-treated, n = 8; sulindac sulfide-treated, n = 9 (in two independent experiments).

Source: PubMed

Tulevat kliiniset tutkimukset

Sponsorit ja yhteistyökumppanit

Sairaudet

Huumeiden interventiot

Evästeasetukset

Käytämme evästeitä varmistaaksemme, että annamme sinulle parhaan kokemuksen verkkosivustollamme. Lisätietoja on tietosuoja- ja evästekäytännössämme. ...>>>Voit muuttaa asetuksiasi tai peruuttaa suostumuksesi milloin tahansa poistamalla evästeet verkkosivustoltasi tai tietokoneeltasi käytännössä kuvatulla tavalla. Hyväksy se ja valitse mieltymyksesi merkitsemällä vastaavat ruudut alla olevaan "Asetusten hallinta" -osioon. Lue huolellisesti käyttöehdot ennen kuin jatkat minkä tahansa tämän verkkosivuston osan käyttöä.
«Hallitse asetuksia