Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation

Abstract

Background: Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM.

Methods and results: We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%.

Conclusions: By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Keywords: cerebrovascular disorders; endothelium; genetics; hemorrhage; stroke.

Figure 1

Project work-flow. An overview of the four different screening assays used to identify two promising compounds from among 2100 initial candidates.

Figure 2

Primary screen - rescue of structural phenotypes associated with loss of CCM2. (A) Western blot analysis of siCCM2 knockdown. (B) Immunofluorescence images of endothelial cells treated with siCTRL or siCCM2 stained for DNA (blue), actin (green), and VE-cadherin (red). (C) DNA (top) and VE-cadherin (bottom) raw images segmented into nuclei and cell objects, respectively. (D) Result of scoring positive and negative control images using rules generated by machine-learning algorithms in CellProfiler Analyst software. Scale bars = 50 µm.

Figure 3

Secondary screen - rescue of functional phenotypes associated with loss of CCM2. (A) Baseline transendothelial resistance of unperturbed siCTRL and siCCM2 monolayers. N=6 (siCTRL) and N=75 (siCCM2). Graph depicts each value, the mean, and SEM. *** indicates P<0.001 as evaluated by t-test. (B) When added to siCCM2-treated cell monolayers, seven compounds identified using automated analysis in the primary screen partially or fully rescued resistance measures after 24-hours of treatment. Each compound was tested individually with two replicates. A representative replicate is plotted.

Figure 4

Tertiary screen - rescue of dermal leak phenotype associated with a murine model of CCM. (A) Timeline of dermal permeability experiment. Drugs were administered into intradermal wheals in the shaved backs of either Ccm2 control mice (Ccm2^f/+) or endothelial cell knockout mice (Ccm2^f/−; +/Tg(Pdgfb-iCreER^T2). 90 minutes later, Evans blue dye (EBD) was administered systemically by tail vein injection. After an additional 30 minutes, skin from the site of each drug injection was removed and the amount of EBD assayed. (B) Quantification of dermal permeability assay described in (A). N=4–12 mice per group were tested. Graph depicts each value and the mean. * indicates P<0.05 as evaluated by mixed effects linear regression with Benjamini-Hochberg adjusted significance test.

Figure 5

Quaternary screen – rescue of lesion burden in a murine model of CCM. (A) Timeline of treatment and analysis of cholecalciferol or tempol in Ccm2 ecKO mice. (B) The number of CCM lesions as measured by MRI in ecKO mice. Experiment represents N=8–12 mice per group. Graph depicts each value, the mean, and SEM. * indicates P<0.05 as evaluated by one-way ANOVA and Dunnett’s multiple comparison test, comparing each active diet against the control diet. An effect of Tempol achieved marginal significance compared to control, denoted by # (P=0.0518). (C) For each mouse, the count of lesions within the designated size range (horizontal axis) was computed and compared between treatment groups using exact Wilcoxon Mann-Whitney tests, with p-values adjusted for 2 comparisons within each bin. The median count of lesions of each size among the mice for each treatment is depicted. * denotes P<0.05, and # denotes P<0.1 after multiplicity adjustment. (D) Three-dimensional reconstruction of the brain (grey/cream) and lesions (red) are shown for a representative brain from each treatment arm (the mouse with the median number of lesions for each treatment group is shown). The top, middle, and bottom brains are from mice treated with standard, VD3-enhanced, or tempol enhanced diets, respectively.

Figure 6

Treatments illuminate pathophysiology of disease. 60 minute treatment with 100 nM cholecalciferol, but not its precursor 7-DHC, rescues CCM2 knockdown-induced (A) RHOA, (B) pMLC, and (C) ARF6 activation. N=3 for each group in A–C. Graphs depict each value, the mean, and SEM. * indicates P<0.05, ** indicates P<0.01, and *** denotes P<0.001 as evaluated by one-way ANOVA and Dunnett’s Multiple Comparison Test. (D) Cholecalciferol rescues CCM2-induced activation of ARF6 within 5 minutes. (E) Quantification of (D). N=5–8. Graph depicts each value, mean and SEM.= Multiple t-tests were used to evaluate significance with the Sidak-Bonferroni multiple comparison. (F) Knockdown of CCM2 in EA-hy926 endothelial cells induces increased reactive oxygen species. (G) Knockdown of CCM2 in EA-hy926 endothelial cells induces a reduction of FOX01 expression.

Figure 7

CCM mice display endothelial-dependent vascular dysfunction. (A) Middle cerebral arteries isolated from endothelial-specific CCM2 knockout mice (KO) subjected to acetylcholine-induced vasodilation. (B) Sodium Nitroprusside treatment (an NO-donor) of middle cerebral arteries of both mice genotypes demonstrates the normal function of the smooth muscle in both wild-type and knockout mice. (C) L-name, a specific inhibitor of eNOS, eliminates the ability of endothelial cells to produce NO. (D) Tempol completely rescues acetylcholine-induced vasodilation in endothelial-specific CCM2 knockout mice. For A–D, N=9–19. Graphs depict mean and SEM. Two-way ANOVA with one factor being genotype and the second factor being acetylcholine dose as a repeated measures factor with Sidak’s multiple comparison test were used to evaluate significance. (E) Systolic blood pressure (P=0.093)(F) diastolic blood pressure (P=0.051), (G) and mean blood pressure (P=0.052) trended toward an increase in ecKO mice, consistent with decreased vasodilatory function. (H) Heart rate trended toward a reduction (P=0.051) in the endothelial-specific CCM2 knockout mice. For E–H, N=5–10. Graphs depict each value, median and interquartile range. Wilcoxon Mann-Whitney exact analysis was used to evaluate significance.