RESUMEN
Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in 1 of 3 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-1α 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-1α 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.
Asunto(s)
Astrocitos/fisiología , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Astrocitos/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Noqueados , Modelos Neurológicos , Mutación , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
We describe a method to purify primary brain microvascular endothelial cells (BMEC) from mice bearing floxed alleles of Krit1 (Krit1fl/fl) or Pdcd10 (Pdcd10fl/fl) and an endothelial-specific tamoxifen-regulated Cre recombinase (Pdgfb-iCreERT2), and used these to delete Krit1 or Pdcd10 genes in a time-controlled manner. These BMEC culture models contain a high degree of purity and have been used to identify the major molecular processes involved in loss of Krit1/Pdcd10-induced altered brain endothelial phenotype and function. In addition, these in vitro models of cerebral cavernous malformations (CCMs) enable molecular, biochemical, and pharmacological studies that have contributed significantly to understand the pathogenesis of CCMs. The findings using this in vitro CCMs model have been validated in mouse CCM models and observed in human CCMs. In this chapter, we summarize procedures for isolation and purification of BMEC from transgenic mice, as well as our experience to genetically inactivate CCM genes in the brain endothelium.