Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice

Significance

Cerebral cavernous malformation (CCM) disease can lead to brain hemorrhages, seizures, and paralysis. No pharmacological therapy is currently available. Here we define, to our knowledge for the first time in vivo, the sequence of molecular events that lead to CCM vascular cavernomas. We found that β-catenin activation is the first trigger followed by TGF-β signaling, which, in turn, mediates the progression of the disease. We also show that β-catenin signaling is cell-autonomous and independent of Wnt-receptor activation. Most importantly, these studies prompted us to identify pharmacological agents that, by targeting the altered β-catenin signaling, limit the formation of brain vascular cavernomas in mice with CCM3 ablation in endothelial cells. These drugs are currently used in clinics for different pathologies and may be repurposed for CCM therapy.

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.

The vascular malformations that characterize the disease known as cerebral cavernous malformation (CCM) are concentrated in the central nervous system, and they typically show multiple lumens and vascular leakage (1). These abnormalities can result in severe neurological symptoms, including hemorrhagic stroke (2), and, to date, the only possible therapy is surgery (3). In humans, loss-of-function mutations in any one of three independent genes known as cerebral cavernous malformation 1, 2, and 3 (CCM1, CCM2, and CCM3) are the cause of the genetic form of CCM (4). Similarly, in murine models, the vascular phenotype can be reproduced by endothelium-specific loss-of-function mutations of any one of these three CCM-linked genes (5–7).

We have recently reported (7) that TGF-β/BMP signaling is activated after ablation of CCM1, CCM2, or CCM3 and induces endothelial-to-mesenchymal transition (EndMT) that plays a crucial role in the development of vascular malformations. Nevertheless, the sequence of signaling responses elicited by ablation of CCM genes still remains to be defined. Inhibitors of the TGF-β/BMP-signaling pathways reduce the number and size of the malformations, but not completely (7), suggesting that other signaling pathways may be implicated.

The Wnt/β-catenin pathway, in synergy with TGF-β signaling (8), is responsible for the EndMT switch of endothelial cells giving rise to the heart cushion in the embryo. In addition, the knockdown of CCM1 and CCM3 expression in cultured aortic and artery endothelial cells promotes β-catenin signaling (9, 10), although no direct link with the in vivo model of the disease has been made. Activation of canonical Wnt/β-catenin signaling is critical for brain vascularization and acquisition, by the microvasculature, of blood–brain barrier properties (11–13). Endothelial signaling by β-catenin must be tightly regulated: it is high during blood–brain barrier development, but it is rapidly abrogated postnatally (13). Long-lasting high levels of β-catenin signaling in the vasculature may cause strong alterations in vascular stability and lumen malformations (14).

In the present study, we bring evidence that sustained β-catenin signaling plays an important role in the EndMT and in the development of brain vascular malformations in CCM3-deficient mice. Activation of the β-catenin pathway is followed by TGF-β/BMP signaling that supports further evolution of the vascular lesions. Importantly, pharmacological inhibitors of β-catenin signaling significantly reduce the development of the vascular malformations in CCM3-deficient mice. This work introduces a previously unidentified therapeutic strategy to counteract the formation of brain vascular cavernomas in genetic patients.

Results

Transcription Activity of β-Catenin Precedes TGF-β/BMP Signaling During the Development of CCM Vascular Malformations.

The in vivo mouse system used was generated through a cross of CCM3-flox/flox mice with Cdh5(PAC)-CreERT2 mice to obtain tamoxifen-inducible endothelial-cell–specific expression of Cre recombinase and CCM3 gene recombination (CCM3-ECKO mice). These mice were then crossed with BAT-gal reporter mice, which show β-catenin–activated expression of nuclear β-galactosidase (β-gal). For details, see SI Appendix, Methods, and Fig. S1. CCM3 mutations occur with low frequency (about 10%) in genetic patients (15 and references therein) and determines the most severe CCM (16).

As reported in Fig. 1A, Upper, we could observe a significantly higher β-catenin transcription signal in the nuclei of endothelial cells in CCM3-ECKO mice in comparison with matched controls. This difference was detectable at early stages [3 day postnatal (dpn)] after induction of CCM3 recombination (at 1 dpn). In brain sections of CCM3-ECKO mice, endothelial cells with β-gal–positive nuclei could be found both in pseudonormal vessels and in cavernae of any size (SI Appendix, Fig. S2). In contrast, phospho-Smad1 (p-Smad1) staining in separate sections (Fig. 1A, Lower) and in costaining for β-gal (Fig. 1B) was not enhanced after CCM3 ablation in 3-dpn pups, but it was increased in 9-dpn pups (Fig. 1C). p-Smad1 was significantly high in middle-to-large lesions only (maximal diameter ≥50 μm in 9-dpn pups) (SI Appendix, Fig. S2). Expression of stem-cell/EndMT markers (7) (Klf4, S100a4, and Id1) was high in 3-dpn CCM3-ECKO pups (Fig. 2 A–D, single positive) and was concentrated in endothelial cells with β-gal–positive nuclei (Fig. 2D, colocalization). At 9 dpn, β-gal expression in endothelial cells of CCM3-ECKO pups decreased whereas stem-cell/EndMT markers remained high (Fig. 2D).

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.

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.

In cultured endothelial cells, acute abrogation of CCM3 by siRNA induced early up-regulation of β-catenin target genes and stem-cell/EndMT markers at times when phosphorylation of Smad1 was not yet enhanced (SI Appendix, Fig. S3 A–C). In contrast, lasting CCM3 abrogation induced by CCM3-flox/flox recombination by Adeno-Cre (SI Appendix, Methods) induced higher levels of both p-Smad1 and p-Smad3 (SI Appendix, Fig. S3D). Furthermore, sustained activation of β-catenin signaling by Lef-ΔβCTA induced phosphorylation of both Smad1 and Smad3 (SI Appendix, Fig. S3E), whereas acute stimulation with Wnt3a did not (SI Appendix, Fig. S3G). Taken together, these data suggest a temporal link between β-catenin and TGF-β/BMP signaling.

β-Catenin Signaling in CCM3-Deficient Endothelial Cells Is Activated Through Cell-Autonomous, Wnt-Receptor–Independent Mechanism.

Recombination of the CCM3 gene in vitro in freshly isolated brain endothelial cells or in a lung endothelial cell line from CCM3-flox/flox mice (SI Appendix) showed active β-catenin [dephosphorylated on Ser37 and Thr41 (17)] in the nucleus (by immunofluorescence microscopy in SI Appendix, Fig. S4 A and B, KO, arrowheads; confirmed by cell fractionation in SI Appendix, Fig. S4 C and D). This effect paralleled a strong alteration of adherens junctions with delocalization of both β-catenin and VE-cadherin (SI Appendix, Fig. S4 A and B).

Enhanced β-catenin transcription activity in CCM3-deficient endothelial cells was further confirmed by (i) increased expression of β-catenin target genes abrogated by a dominant-negative mutant of TCF4 (dnTCF4) (18) (Fig. 3A) and (ii) activation of Tcf/Lef-dependent transcription of the exogenous luciferase gene as measured in Top/Fop Flash reporter assays (see controls in SI Appendix, Fig. S7B).

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).

Furthermore, transcription of genes related to acquisition and maintenance of the stem-cell/EndMT phenotype (19) [Klf4, Ly6a, S100a4, Id1, Cdh2, and Acta2 (20)] was significantly enhanced under basal conditions in the CCM3-knockout endothelial cells (Fig. 3B) and inhibited by dnTCF4 (18) (Fig. 3B and SI Appendix, Fig. S8B).

Activation of β-catenin–mediated transcription in CCM3-knockout endothelial cells was cell-autonomous because (i) it was observed in absence of exogenous Wnt; (ii) the porcupine inhibitors IWP2 and IWP12 (21, 22), as well as Dkk1, a competitor of Wnt coreceptor Lrp5/6 (21, 22), did not inhibit transcription of typical β-catenin targets (SI Appendix, Fig. S5 A–D); (iii) Lrp6 phosphorylation was not increased (SI Appendix, Fig. S5 E and F); and (iv) stimulation by exogenous Wnt3a did not induce expression of stem-cell/EndMT markers, whereas the constitutively active form of β-catenin, Lef-ΔβCTA (23), did (SI Appendix, Fig. S5 G and H).

Taken together, these data indicate that in CCM3-knockout endothelial cells enhanced nuclear localization and transcription activity of β-catenin do not depend on a classical ligand–receptor interaction.

We reported previously that silencing or dismantling of VE-cadherin from endothelial junctions can up-regulate β-catenin signaling (18). Consistently, we observed here that silencing VE-cadherin by siRNA activated the expression of EndMT markers (S100a4 and Id1) in addition to typical β-catenin targets (Axin2, Ccnd1, and Nkd1, SI Appendix, Fig. S5I) and promoted nuclear localization of active β-catenin (SI Appendix, Fig. S6A). Moreover, VE-cadherin knockdown did not enhance the phosphorylation of Smad1 (SI Appendix, Fig. S6B).

These data suggest that in CCM the first trigger of β-catenin signaling is the dismantling of VE-cadherin junctions that, in turn, causes the release of β-catenin in the cytoplasm and nuclear translocation. This process precedes and possibly contributes to the activation of TGF-β/BMP signaling for lesion progression (7).

Sulindac Sulfide Reduces β-Catenin Transcription Activity and Expression of Stem-Cell/EndMT Markers in Endothelial Cells of CCM3-ECKO Mice.

In the attempt to translate the results described above into therapeutic opportunities, we investigated the effects of inhibitors of β-catenin signaling on expression of target genes in CCM3-knockout endothelial cells. We tested a range of pharmacological agents already used in humans and able to affect β-catenin signaling: sulindac sulfide, sulindac sulfone (24, 25), silibinin, curcumin, and resveratrol (26) (SI Appendix, Fig. S7A).

Sulindac sulfide and sulfone were the most effective inhibitors of the β-catenin target genes (SI Appendix, Fig. S7A). We concentrated our efforts on sulindac sulfide. This drug inhibited β-catenin signaling by Top/Fop Flash reporter assay (SI Appendix, Fig. S7B) and active β-catenin nuclear localization in CCM3-knockout endothelial cells (SI Appendix, Figs. S7C and S8C). Furthermore, protein expression and staining of β-catenin–dependent stem-cell/EndMT markers were also reduced in cultured CCM3-knockout endothelial cells by treatment with sulindac sulfide (SI Appendix, Figs. S8C and S9).

The inhibitory effects of sulindac sulfide on β-catenin signaling paralleled the effect of the drug in restoring a correct organization of β-catenin and VE-cadherin at adherens junctions by immunofluorescence costaining (SI Appendix, Fig. S7C magnification in i and ii; quantification in SI Appendix, Fig. S7D) and by coimmunoprecipitation (SI Appendix, Fig. S7 E and F).

Furthermore, the small GTPase Rap1, which controls the organization of endothelial adherens junctions and is upstream of CCM1 activity (27), was inhibited in CCM3-knockout endothelial cells, but was restored by sulindac sulfide treatment (SI Appendix, Fig. S7 G and H).

Treatment of CCM3-ECKO mice with sulindac sulfide reduced the expression of the β-catenin reporter gene in brain endothelial cells at different stages after CCM3 ablation (Fig. 4 A and B) and, in parallel, inhibited junction dismantling (Fig. 4C). Sulindac sulfide also decreased the expression of the stem-cell/EndMT markers (SI Appendix, Figs. S10–S13) and limited increased endothelial cell proliferation in the brain lesions (SI Appendix, Fig. S14).

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.)

Sulindac Sulfide Reduces Development of Vascular Lesions in the Brain and Retina of CCM3-ECKO Mice.

Sulindac sulfide significantly reduced the number and size of the superficial CCM vascular malformations in the cerebellum and in the deeper layers of the brain (Fig. 5 A–C). The vessels of the retina of the CCM3-ECKO mice showed vascular malformations with multiple lumens concentrated at the periphery of the vascular network. Such lesions develop from veins, which present an enlarged lumen (compare Vehicle in WT and CCM3-ECKO in Fig. 5D). Sulindac sulfide partially normalized this aberrant vascular network of CCM3-ECKO mice (Fig. 5 D and E) and reduced the enlargement of the most central tract of the veins (diameter 89.5 ± 7.1 μm in Vehicle-CCM3-ECKO versus 35 ± 7.8 μm in sulindac sulfide CCM3-ECKO, mean ± SD) of 30 measurements in seven retinas each) (Fig. 5F). In addition, sulindac sulfide significantly prolonged the survival of CCM3-ECKO pups in both acute and subacute gene-recombination schedules (details in the figure legend) (P = 0.0046 and P = 0.0032, log-rank test, respectively, Fig. 5 G and H).

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).

Sulindac sulfide has been reported to inhibit cyclooxygenase, potentially leading to inhibition of platelet aggregation (24) and, possibly, increasing the hemorrhagic tendency of these patients. However, sulindac sulfone, which does not inhibit cyclooxygenase (24), showed a comparable activity to sulindac sulfide in inhibiting β-catenin signaling and expression of stem-cell/EndMT markers in cultured CCM3-knockout endothelial cells (SI Appendix, Figs. S7A and S16). As with sulindac sulfide, the reduction of active β-catenin in the nucleus corresponded to the increased localization of this mediator at cell junctions together with VE-cadherin (SI Appendix, Fig. S16). Sulindac sulfone also limited the expression of stem-cell/EndMT markers and strongly reduced the number of lesions in the brain of the CCM3-ECKO mice (SI Appendix, Figs. S17 and S18).

In conclusion, both sulindac metabolites inhibit β-catenin signaling and, in parallel, exert therapeutic effects in hindering the development of brain vascular cavernomas in a preclinical model.

Discussion

Here, we report that endothelial-cell–selective deletion of the CCM3 gene activates β-catenin transcription signaling in vivo. This response develops early after CCM3 deletion, before activation of TGF-β/BMP signaling (7), and contributes to the pathogenesis of CCM3 cavernomas in this model. Furthermore, pharmacological inhibition of β-catenin transcription activity by sulindac sulfide and sulindac sulfone reduced the number and size of the cerebral vascular malformations in this murine model.

CCM malformations develop largely, although not exclusively, in the central nervous systems in patients and in mouse models (5, 6). The Wnt pathway is a well-established determinant for the specification of the blood–brain barrier, and it is under tight control during physiological angiogenesis of the central nervous system (11–13).

Our data support the hypothesis of a cell-autonomous deregulated activation of the β-catenin pathway in response to CCM3 deletion and delineate an unconventional mechanism of a β-catenin–driven transcription program instructing endothelial dedifferentiation.

We report that in CCM3-deficient endothelial cells activation of β-catenin–mediated transcription is independent of both endogenous Wnt ligand and Wnt-receptor stimulation.

We show that, in the absence of CCM3, endothelial adherens junctions are dismantled, as observed after ablation of both CCM1 (27, 28) and CCM2 (5). In sparse endothelial cells and in VE-cadherin–null endothelial cells, when junctions are disorganized, β-catenin dissociates from cell–cell junctions and accumulates to the nucleus (18). We observe here that in CCM3-deficient cells the amount of β-catenin associated with VE-cadherin is indeed reduced by 35–50% concomitantly with the increase of this active mediator in the nucleus.

Furthermore, by treating CCM3-knockout endothelial cells in vitro and in vivo with sulindac metabolites, we were able to restore junction organization and β-catenin association to VE-cadherin while reducing β-catenin nuclear signaling. Overall, these data strongly suggest that a significant aspect of the function of the CCM complex is that of stabilizing endothelial cell junctions maintaining β-catenin at the membrane, preventing, in this way, an uncontrolled β-catenin transcriptional signaling.

We have recently demonstrated that after endothelial-cell–selective ablation of CCM1 in mice the TGF-β/BMP pathway is activated and sustains the progression of the pathology (7). We confirm here these observations in CCM3-ECKO mice. In addition, we observed that activation of β-catenin–driven transcription and nuclear localization precedes the initiation of TGF-β/BMP signaling in CCM3-ablated endothelial cells both in vivo and in vitro and that β-catenin contributes to such stimulation. These findings delineate a sequence of signaling steps in response to ablation of CCM3 in which, as an early event and concurrently with disorganization of adherens junctions, β-catenin concentrates in the nucleus to drive the expression of a dedifferentiation program, which comprises expression of stem-cell/EndMT markers and activation of TGF-β/BMP signaling for the progression of the vascular lesions. TGF-β/BMP signaling can then follow β-catenin signaling and, possibly, contribute to the decrease in β-catenin signaling that we observed in vivo in endothelial cells of late stage vascular malformations (29). Similar sequential activation of different signaling pathways after early β-catenin signaling has been reported in epithelial transformation in colon cancer (30).

Considering future pharmacological interventions, we have identified two sulindac metabolites, sulindac sulfide and sulindac sulfone, that significantly inhibit β-catenin–stimulated transcription of stem-cell/EndMT markers and the development of vascular lesions. These agents are nonsteroidal anti-inflammatory drugs that have shown significant inhibition of cancer progression in different preclinical models and have been used in the treatment of colon cancer in human patients (24). The mechanism of action of these drugs is complex (31) and includes, among various targets, inhibition of proteasome-dependent degradation of β-catenin, down-regulation of β-catenin transcription, and inhibition of β-catenin nuclear localization through inhibition of cGMP-PDE5 and activation of PKG (25, 31). We report here a comparable activity of the two compounds in constraining CCM cavernomas in mice, but the sulfone metabolite is more promising for further pharmacological development because it is devoid of cyclooxygenase and platelet inhibitory activity (32, 24).

In conclusion, the targeting of β-catenin signaling with specific pharmacological tools represents a promising strategy for the reduction or prevention of cavernoma development in genetic CCM patients, and it deserves consideration for clinical trials.

Materials and Methods

Endothelial-Cell–Specific Recombination in CCM3-flox/flox Mice.

The CCM3-flox/flox mice were generated at TaconicArtemis. Two P-lox sequences were inserted that flank exons 4 and 5 of the murine CCM3 gene to produce a loss-of-function mutation after excision by Cre recombinase. Detailed description of the breedings of these mice to generate endothelial-specific recombination of CCM3 and β-catenin–driven expression of β-galactosidase as well as Cre-recombinase-reporter expression of enhanced yellow green fluorescent protein is presented in SI Appendix.

Treatment with Sulindac Sulfide and Sulindac Sulfone.

Both sulindac sulfide (Sigma) and sulindac sulfone (Sigma) were dissolved in DMSO and further diluted 1:50 in corn oil. They were administered intragastrically daily (30 mg/kg body weight), starting 1 d after the induction of recombination unless otherwise described. The control mice were treated in parallel with Vehicle only [corn oil plus 2% (vol/vol) DMSO]. Matched littermate pups were treated in parallel with either the drug or the Vehicle. Animal experimentation has been approved by the FIRC Institute of Molecular Oncology Institutional Animal Care and Use Committee and was performed according to the guidelines of the Italian Ministry of Health regulating animal experimentation.

In Vitro Isolation, Culture, and Recombination of Endothelial Cells from the CCM3-flox/flox Mice.

Endothelial cells from the CCM3-flox/flox mice (8–10 wk old) were isolated from the brain as previously described (13). CCM3 ablation was with AdenoCre viral vector as described in SI Appendix.

Antibodies.

The full list of the antibodies used is reported in SI Appendix.

Western Blotting and Immunoprecipitation.

Standard procedures were used to extract and analyze the protein content by Western blotting and immunoprecipitation (28). Nuclear fractionation was as described in SI Appendix.

Assessment of Lesion Burden.

For the classification and counting of lesions, entire brains were sectioned, stained, and analyzed as described in the SI Appendix according to McDonald et al. (33).