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1.
Arterioscler Thromb Vasc Biol ; 44(6): 1246-1264, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38660801

RESUMEN

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.


Asunto(s)
Receptor 1 de Quimiocinas CX3C , Quimiocina CX3CL1 , Modelos Animales de Enfermedad , Hemangioma Cavernoso del Sistema Nervioso Central , Transducción de Señal , Animales , Femenino , Humanos , Masculino , Ratones , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/genética , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hipoxia/metabolismo , Hipoxia/complicaciones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/genética
2.
J Clin Invest ; 131(13)2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-34043589

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/biosíntesis
3.
FASEB Bioadv ; 3(5): 334-355, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33977234

RESUMEN

The transmembrane protein heart of glass1 (HEG1) directly binds to and recruits Krev interaction trapped protein 1 (KRIT1) to endothelial junctions to form the HEG1-KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel-like factors 4 and 2 (KLF4 and KLF2), which are implicated in endothelial vascular homeostasis; however, the effect of acute inhibition of the HEG1-KRIT1 interaction remains incompletely understood. Here, we report a high-throughput screening assay and molecular design of a small-molecule HEG1-KRIT1 inhibitor to uncover acute changes in signaling pathways downstream of the HEG1-KRIT1 protein complex disruption. The small-molecule HEG1-KRIT1 inhibitor 2 (HKi2) was demonstrated to be a bona fide inhibitor of the interaction between HEG1 and KRIT1 proteins, by competing orthosterically with HEG1 through covalent reversible interactions with the FERM (4.1, ezrin, radixin, and moesin) domain of KRIT1. The crystal structure of HKi2 bound to KRIT1 FERM revealed that it occupies the same binding pocket on KRIT1 as the HEG1 cytoplasmic tail. In human endothelial cells (ECs), acute inhibition of the HEG1-KRIT1 interaction by HKi2 increased KLF4 and KLF2 mRNA and protein levels, whereas a structurally similar inactive compound failed to do so. In zebrafish, HKi2 induced expression of klf2a in arterial and venous endothelium. Furthermore, genome-wide RNA transcriptome analysis of HKi2-treated ECs under static conditions revealed that, in addition to elevating KLF4 and KLF2 expression, inhibition of the HEG1-KRIT1 interaction mimics many of the transcriptional effects of laminar blood flow. Furthermore, HKi2-treated ECs also triggered Akt signaling in a phosphoinositide 3-kinase (PI3K)-dependent manner, as blocking PI3K activity blunted the Akt phosphorylation induced by HKi2. Finally, using an in vitro colocalization assay, we show that HKi6, an improved derivative of HKi2 with higher affinity for KRIT1, significantly impedes recruitment of KRIT1 to mitochondria-localized HEG1 in CHO cells, indicating a direct inhibition of the HEG1-KRIT1 interaction. Thus, our results demonstrate that early events of the acute inhibition of HEG1-KRIT1 interaction with HKi small-molecule inhibitors lead to: (i) elevated KLF4 and KLF2 gene expression; and (ii) increased Akt phosphorylation. Thus, HKi's provide new pharmacologic tools to study acute inhibition of the HEG1-KRIT1 protein complex and may provide insights to dissect early signaling events that regulate vascular homeostasis.

4.
Methods Mol Biol ; 2152: 139-150, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32524550

RESUMEN

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.


Asunto(s)
Separación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Separación Celular/métodos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Integrasas , Ratones , Ratones Transgénicos , Recombinación Genética
5.
Blood ; 133(3): 193-204, 2019 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-30442679

RESUMEN

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1 ECKO ) or Pdcd10 (Pdcd10 ECKO ), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10 ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10 ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.


Asunto(s)
Anticoagulantes/metabolismo , Proteínas Reguladoras de la Apoptosis/fisiología , Hemorragia Cerebral/diagnóstico , Endotelio Vascular/patología , Hemangioma Cavernoso del Sistema Nervioso Central/complicaciones , Proteína KRIT1/fisiología , Proteínas de la Membrana/fisiología , Proteína C/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Trombomodulina/sangre , Adulto , Animales , Coagulación Sanguínea , Estudios de Casos y Controles , Hemorragia Cerebral/sangre , Hemorragia Cerebral/etiología , Receptor de Proteína C Endotelial/metabolismo , Endotelio Vascular/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Humanos , Factor 4 Similar a Kruppel , Ratones , Ratones Noqueados , Transducción de Señal , Adulto Joven
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