Fasudil Decreases Lesion Burden in a Murine Model of Cerebral Cavernous Malformation Disease
Background and Purpose—Cerebral cavernous malformations (CCMs) are characterized by grossly dilated capillaries, associated with vascular leak and hemorrhage, and occur in sporadic or inherited (autosomal-dominant) forms with mutations in 1 of 3 gene loci (CCM 1, 2 or 3). We previously reported that the CCM1 protein (KRIT1) localizes to endothelial cell–cell junctions and loss of KRIT1 leads to junctional instability associated with activation of RhoA and its effector Rho kinase. Although Rho kinase inhibition has been proposed as potential therapy for CCM, there has been no demonstration of a therapeutic effect on CCM lesion genesis in vivo.
Methods—Our recently generated a model of CCM1 disease (Ccm1+/−Msh2−/−) was treated with the Rho kinase inhibitor fasudil (100 mg/kg/day administered in drinking water from weaning to 5 months of age), or placebo, and blindly assessed CCM lesion burden by systematic survey of animals' brains. For comparison, we also assessed therapeutic effect in previously described Ccm2+/−Trp53−/− mice treated with the same dose and duration of fasudil and placebo.
Results—Fasudil-treated Ccm1+/−Msh2−/− mice had a significantly decreased prevalence of CCM lesions compared with placebo controls. Lesions in treated animals were smaller and less likely associated with hemorrhage, inflammation, and endothelial proliferation and exhibited decreased expression of Rho kinase activation biomarkers. A therapeutic effect was also documented in Ccm2+/−Trp53−/− mice.
Conclusions—This represents the first report of therapeutic benefit of pharmacological therapy in development and progression of CCMs and indicates that Rho kinase activation is a critical step in CCM lesion genesis and maturation.
Cerebral cavernous malformations (CCMs) are common vascular lesions that can cause hemorrhagic stroke, seizures, and focal neurological deficits. Cases occur sporadically or in an inherited, autosomal-dominant form, the most common of which is caused by mutations in CCM1 (KRIT1).1,2
Our group had shown that KRIT1 is localized to and stabilizes endothelial cell (EC) junctions3 and the loss of KRIT1 leads to increased stress fibers, a hallmark of activation of RhoA and its effector Rho kinase (ROCK).3 Inhibition of ROCK in vitro or in vivo reversed the effect of KRIT1 silencing on endothelial junctional stability,4 suggesting it as a potential therapeutic strategy. Although we demonstrated robust activation of ROCK in ECs lining CCM lesions in humans,4 there has been to date no demonstration of a therapeutic effect on the pathogenesis of CCM lesions, the hallmark of the disease.
Based on evidence of a 2-hit mutation mechanism demonstrated in CCM,5–7 we recently showed that Ccm1+/− mice bred in a background of homozygous deletion of Msh2, a mismatch repair complex protein, develop CCM lesions recapitulating the human disease.8 We had previously demonstrated similar CCM lesions in Ccm2 heterozygotes sensitized by loss of tumor suppressor gene Trp53.9 Here, we report that treatment with fasudil, a specific ROCK inhibitor,10,11 decreases overall lesion burden and also inhibits the development of the mature CCM lesions.
The development of Ccm1+/− Msh2−/− mice has been described previously.8 Immediately after weaning (P21), mice were randomly assigned into a treatment group (N=7), receiving fasudil dissolved in their drinking water at a dose of 100 mg/kg/day, or placebo (N=5), receiving fasudil-free drinking water. We verified water consumption by the treated mice, confirming ingestion of fasudil (see Supplemental Methods; http://stroke.ahajournals.org). The dose of fasudil administration in this exploratory study was chosen based on that achieving ROCK inhibition and clinical effects in other murine studies12 and represents the median dose delivered to rodents in >20 published studies. At 5 months of age, the animals were euthanized and their brains harvested and fixed. One animal in the fasudil group and 1 in the placebo group died before completing treatment; their brains were examined postmortem and did not reveal any CCM lesions or tumors. These 2 animals were excluded from analysis. Fasudil-treated animals appeared healthy and did not exhibit overt adverse effects of treatment (see Supplemental Methods).
CCM lesion burden was assessed in the remaining brains of fasudil-treated Ccm1+/−Msh2−/− mice (n=6) and placebo (n=4) as described previously9 and in the Supplemental Methods. Serial 1-mm-thick coronal slices of each brain were microsectioned at 5 μm and stained with hematoxylin and eosin, and CCM lesions were counted and catalogued blinded to treatment status. A Stage 1 CCM lesion was defined as an isolated dilated capillary with a maximal diameter accommodating at least 25 red blood cells (cavern), whereas a Stage 2 CCM lesion was defined as a cluster of ≥2 contiguous caverns as described in previous reports.8,9 Immunohistochemical stains for iron deposition, B-cell infiltration (the primary inflammatory cell marker in CCM lesions8), proliferative index, and phosphorylated myosin light chain as a biomarker of ROCK activation have been described in detail in this model.8 A second biomarker of ROCK activation, phosphorylated myosin binding subunit (myosin-binding subunit of myosin phosphatase/myosin phosphatase target), was used for verification13 (see Supplemental Methods).
Another comparison group of Ccm2+/−Trp53−/− mice underwent a similar treatment regimen and were assessed for confirmatory effect on lesion burden in the second CCM genotype. This model has been described previously,9 and details about the CCM2 treatment group and the analysis of lesion burden in this group and in the combined cohort (both genotypes) are presented in the Supplemental Methods and Data.
For the statistical assessment of treatment effect on the primary outcome (lesion counts per brain), we tested the assumption of Poisson distribution in each cohort and we used parametric testing (Poisson mean likelihood test) only where Poisson distribution was demonstrated. Results of lesion counts and cavern counts were also analyzed by the more stringent nonparametric Wilcoxon rank-sum test. Results with both parametric and nonparametric comparisons are reported for instances where Poisson distribution was confirmed and only the nonparametric comparison when Poisson distribution was not confirmed. Two-tailed Student t test was used for comparison of lesion size data, where we verified normal distribution. For categorical outcomes, nonparametric comparison tests (Fisher exact) was used. For each comparison, statistical significance was assumed at the 0.05 level. Statistical methods, including justification of Poisson distribution and data normality, where appropriate, are further detailed in the Supplemental Methods.
Fasudil-treated Ccm1+/−Msh2−/− mice exhibited a significantly lower CCM lesion burden (Figure 1). Twelve CCM lesions were identified in the 4 mice in the placebo group and only 5 CCM lesions in the 6 fasudil-treated mice (P=0.01 by parametric comparison with verified Poisson distribution of lesion counts and 0.06 with the more stringent nonparametric Wilcoxon rank-sum test). The prevalence of single cavern (Stage 1) and multicavernous (Stage 2) CCM lesions was reduced in the treated group as compared with placebo, and the number of total caverns per brain was significantly reduced (P=0.030 by Wilcoxon rank-sum test). The maximal diameter of Stage 2 CCM lesions was significantly smaller in the fasudil group than in the placebo group (142 μm [1 lesion] versus 425±146 μm; P=0.03) but not the size of individual caverns that comprise the lesions (Figures 1 and 2). There were no brain tumors noted in either cohort on systematic histological screening.
Phenotypic features of lesions in the fasudil and placebo cohorts are presented in the Table and illustrated in Figure 2. Extravascular iron deposits, indicative of chronic hemorrhage, were present in 4 of the 12 lesions (in 3 of the 4 placebo mice) and in none of the lesions in the fasudil group (P=0.03). Infiltration of B cells was present in the same lesions exhibiting iron deposits, in the placebo group, and in none of the lesions in the fasudil group (P=0.03). The EC proliferative index (ratio of Ki67-immunopositive EC/total number of ECs lining caverns in CCM lesions) was 7.2% in the placebo group and remarkably 0% in the fasudil group (8 of 111 versus 0 of 59 ECs; Fisher exact test, P=0.05).
Mice treated with fasudil showed decreased phosphorylated myosin light chain staining in ECs lining CCM lesions (Table; Figure 2), indicating that fasudil inhibited ROCK activity within the lesion proper. The prevalence of phosphorylated myosin light chain staining in ECs in CCM lesions was greater in the placebo group (72.5%/16.5%/11% of cavern-lining ECs with negative/faint/intense staining, respectively), as compared with the fasudil group (93%/7%/0% of cavern-lining ECs with negative/faint/intense staining, respectively). Fasudil-treated CCM lesions also exhibited less prevalent staining for another biomarker of ROCK activation, phosphorylated myosin binding subunit (phosphatase/myosin phosphatase target 1). The prevalence of phosphorylated myosin binding subunit staining in ECs in CCM lesions was greater in the placebo group (70.6%/17.0%/12.4% of cavern-lining ECs with negative/faint/intense staining, respectively) as compared with the fasudil group (94.5%/5.5%/0% of cavern-lining ECs with negative/faint/intense staining, respectively).
A beneficial effect was also apparent in the blinded analysis of lesion burden in Ccm2+/−Trp53−/− mice treated with the same dose, treatment onset, and duration of fasudil or placebo. Sixty CCM lesions were identified in the brains of 4 placebo mice (15 lesions per brain) versus 15 lesions in 2 fasudil-treated mice (7.5 lesions per brain). These results are summarized in the Supplemental Data. When combining the 2 cohorts, the difference in the number of CCM lesions and caverns per mouse with both genotypes were significant (P=0.049 and P=0.022, respectively) even by the most stringent nonparametric comparisons.
Fasudil treatment appears to reduce lesion genesis in the CCM1 murine model, as judged by significantly fewer overall CCM lesions in treated animals and fewer individual caverns. Fasudil also appears to prevent lesion maturation into complex clinically significant lesions. The CCM lesions in fasudil-treated mice were smaller and lacked features of hemorrhage, B-cell infiltration, and endothelial cell proliferation present in more mature CCM lesions.8 The inhibition of phosphorylated myosin light chain and phosphorylated myosin binding subunit staining indicated effective blocking of ROCK activity by fasudil in the disease-target ECs lining CCM lesions. The fasudil treatment was well tolerated with both groups exhibiting comparable weights and only 1 death in each group (1 of 5 receiving placebo and 1 of 7 receiving fasudil), an attrition rate not different from that previously reported in larger cohorts of untreated animals in this model8 and a result consistent with the known safety of fasudil in humans.12 Other systemic effects of fasudil, and potential mechanistic impact beyond ROCK inhibition in CCM lesions, were not examined in this preliminary study and will require more careful documentation and specific hypothesis testing in future studies.
Importantly, increased EC RhoA or ROCK activity has also been reported as a consequence of reduced expression of CCM2 or CCM3,4,14,15 the genes mutated in other forms of inherited CCM. Thus, this therapeutic approach may be broadly applicable to the other familial forms of this disease and possibly to sporadic CCM lesions.4 Indeed, a benefit on lesion burden was also apparent in our CCM2 murine model. Future studies should explore potential differences in the therapeutic effect in various genotypes and with fasudil treatment onset at different ages and for different duration. One or more of the features demonstrated here (lesion burden, size, phenotypic modification) may be more relevant in specific clinical settings. This proof of principle will also need to be confirmed in longitudinal studies that probe the potential therapeutic effect on established CCM lesions. Future research should also explore whether a similar CCM therapeutic effect can be accomplished with other agents such as statins, whose effects independent of lipid-lowering (ie, pleiotropic effects) include ROCK inhibition and which are broadly used chronically in human populations with cardiovascular disease.12
These data represent the first report of therapeutic benefit of pharmacological intervention in CCM genesis and maturation in vivo and indicate that ROCK inhibition is a potential therapy for CCM disease.
Sources of Funding
This work was supported by grants from the National Institutes of Health R01-NS06748 (I.A.A., D.A. Marchuk), K01-HL092599 (R.A.S.), and R01-HL106489 (M.H.G.).
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.625467/-/DC1.
- Received May 6, 2011.
- Revision received August 8, 2011.
- Accepted September 8, 2011.
- © 2012 American Heart Association, Inc.
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