SUMMARY
Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no medical therapies exist. CCMs arise from loss of an adaptor complex that negatively regulates MEKK3-KLF2/4 signaling in brain endothelial cells, but upstream activators of this disease pathway remain unknown. Here, we identify endothelial TLR4 and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by gram negative bacteria or lipopolysaccharide accelerates CCM formation, while genetic or pharmacologic blockade of TLR4 signaling prevents CCM formation in mice. Polymorphisms that increase expression of TLR4 or its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signaling in the pathogenesis of a cerebrovascular disease, as well as novel strategies for its treatment.
Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by lossof-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2 and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1a in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1a target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.
Background and Purpose
We sought to compare the effect of chronic treatment with commonly tolerated doses of Fasudil, a specific RhoA kinase (ROCK) inhibitor, and simvastatin (with pleiotropic effects including ROCK inhibition) on cerebral cavernous malformation (CCM) genesis and maturation in two models that recapitulate the human disease.
Methods
Two heterozygous murine models, Ccm1+/-Msh2-/- and Ccm2+/-Trp53-/-, were treated from weaning to 4-5 months of age with Fasudil (100 mg/kg/day), simvastatin (40 mg/kg/day) or with placebo. Mouse brains were blindly assessed for CCM lesion burden, non-heme iron deposition (as a quantitative measure of chronic lesional hemorrhage) and ROCK activity.
Results
Fasudil, but not simvastatin, significantly decreased mature CCM lesion burden in Ccm1+/-Msh2-/- mice, and in meta-analysis of both models combined, when compared to mice receiving placebo. Fasudil and simvastatin both significantly decreased the integrated iron density per mature lesion area in Ccm1+/-Msh2-/- mice, and in both models combined, compared to mice given placebo. ROCK activity in mature lesions of Ccm1+/-Msh2-/- mice was similar with both treatments. Fasudil, but not simvastatin, improved survival in Ccm1+/-Msh2-/- mice. Fasudil and simvastatin treatment did not affect survival or lesion development significantly in Ccm2+/-Trp53-/- mice alone, and Fasudil benefit appeared limited to males.
Conclusion
ROCK inhibitor Fasudil was more efficacious than simvastatin in improving survival and blunting the development of mature CCM lesions. Both drugs significantly decreased chronic hemorrhage in CCM lesions. These findings justify the development of ROCK inhibitors, and the clinical testing of commonly used statin agents in CCM.
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