Blood biomarkers in a mouse model of CADASIL

Abstract

Mutations in NOTCH 3 are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a neurological disorder characterized by stroke, and vascular cognitive impairment and dementia. Loss of vascular smooth muscle cells (VSMC) and accumulation of granular osmiophilic material (GOM) deposits are hallmarks of CADASIL. There are no therapies for CADASIL and experimental endpoints to examine the preclinical efficacy of potential drugs are lacking. This study aims to use a mouse carrying the C455R mutation in Notch 3 to identify biomarkers associated with CADASIL. Mass spectrometry and antibody arrays were used to explore the aorta and blood proteomes of CADASIL mice, ELISA assays were utilized for biomarker validation, a ligand-dependent assay was applied to examine the relationship between Notch signaling and biomarker expression, and retinal histology was performed for quantification of VSMC loss in arteries. Two-hundred day-old mice with the C455R CADASIL mutation in Notch 3 mice display robust VSMC loss in retinal arteries and had increased plasma levels of collagen18α1/endostatin (col18α1) and high-temperature requirement A serine peptidase 1 (HTRA1) and reduced levels of Notch 3 extracellular domain (N3ECD), compared to control wild type mice. Measurements of plasma endostatin, HTRA1 and N3ECD, along with VSMC quantification in retinal arteries, may serve as surrogate endpoints for assessing efficacy in preclinical therapeutic studies of CADASIL using mice.

Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL

“…6-mo-old C455R mice had reduced mural cell coverage in retinal arteries and arterioles ( Fig. 1, B and C ), reduced plasma levels of N3ECD, and increased plasma levels of HTRA1, IGFBP-1, and endostatin/collagen 18α1, compared with control mice ( Primo et al, 2016 ). Rescue of mural cell coverage in small or large vessels was included as required, prespecified criteria for efficacy because mural cell loss is a hallmark of SVD and because we showed this variable depended on Notch3 signaling in our models ( Fig.…”

“…N3ECD is present in mouse and human plasma and its levels are reduced in mice carrying the C455R Notch3 mutation ( Primo et al, 2016 ). We tested whether N3ECD was detectable in cell culture supernatants and whether its levels changed upon activation.…”

“…We then tested the A13 Notch3 agonist antibody in vivo using morphological and molecular signatures previously reported in C455R mice ( Primo et al, 2016 ) as primary end points to assess the efficacy of treatment. 6-mo-old C455R mice had reduced mural cell coverage in retinal arteries and arterioles ( Fig.…”

“…3 ). Endostatin/collagen 18α1 expression is regulated by Notch3 transcriptional activity ( Primo et al, 2016 ). Age of the animal also affects the levels of this biomarker in plasma ( Primo et al, 2016 ).…”

“…For that purpose, we used mouse models with Notch3 mutations ( Arboleda-Velasquez et al, 2008 , 2011 ) and a Notch3 agonist antibody ( Li et al, 2008 ). To examine the efficacy of the treatment, we leveraged a roster of morphological and blood biomarkers previously characterized in a CADASIL mouse model, including mural cell coverage in arteries and changes in plasma levels of Notch3 extracellular domain (N3ECD), high-temperature requirement A serine peptidase 1 (HTRA1), collagen 18α1/endostatin, and insulin-like growth factor binding protein 1 (IGFBP-1; Primo et al, 2016 ).…”