Figure 4

The pharmacological inhibition of PRC1 protein Cbx7a/CBX7 suppresses CCM-associated cardiac phenotypes in zebrafish ccm2^m201 mutants and lesion burden in a murine pre-clinical CCM3 disease model.

(A) Proteins of the PRC2 are involved in the tri-methylation of histone H3 at Lys27 (H3K27me3). This epigenetic modification recruits CBX7 and other PRC1 proteins to these genomic sites. The RING1A/B ubiquitin ligases of the PRC1 catalyze H2AK119ub modifications, which results in transcriptional downregulation of genes. The two small compound drugs MS351 and MS37452 inhibit the activity of CBX7. (B–D) The preventive pharmacological treatment of zebrafish ccm2^m201 mutants with CBX7 inhibitors suppresses the development of a cardiac ballooning phenotype and the loss of AVC constriction. Shown are maximum projections of confocal image z-stacks of zebrafish hearts at 56 hpf. The endocardium is marked by the expression of Tg(kdrl:EGFP)^s843. The ccm2^m201 mutant heart is ballooned (B) and lacks expression of ALCAM in atrioventricular canal (AVC) endocardial cells. Treatment with the CBX7 inhibitor MS351 (C, C’) or MS37452 (D,D’) strongly improves cardiac morphology (C, D) and normalizes the endocardial expression of ALCAM in AVC endocardial cells (C’,D’; arrows). (E–J) The inhibition of CBX7 with MS37452 in CCM3^iECKO (n = 4) and control mice (n = 10). (E) Treatment with MS37452 does not produce any adverse effects on the brains of the treated mice. (F) CCM3^iECKO animals treated with DMSO show hemorrhages in the region of the cerebellum (arrows). These are markedly reduced in mice treated with MS37452 (H). (G, H) Isolectin B4 (ISOB4) immunostainings reveal fewer lesions in CCM3^iECKO mice that were treated with MS37452, reducing markedly the number of lesions (G, H, arrows). (I) Quantifications of lesion numbers and (J) total lesioned area. Statistical testing is based on unpaired student’s T-test (s.e.m bars indicated). (K) Model figure depicting the control of vasoprotective and pathophysiological gene expression through the interplay of the proteins of the CCM complex, the PRC1 complex protein CBX7, the transcription factor KLF2, and blood flow as a biophysical modulator. Under physiological conditions, CBX7 expression is negatively controlled by blood flow and the CCM complex downregulates KLF2 expression. The loss of CCM becomes detrimental in low-shear stress regions due to the activation of KLF2 expression. In addition, CBX7 expression becomes upregulated due to the lack of blood flow. Together this renders pathophysiological targets, such as WNT9B, TEK, and ANGPT1, available for KLF2-dependent activation. AVC atrioventricular canal, endo endocardium, myo myocardium, Ven ventricle, Atr atrium. Experiments in zebrafish were done in three biological replicates. Scale bars are (B–D) 50 µm; (B’–D’) 20 µm; (G, H) 500 µM. Source data are available online for this figure.