Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary subcortical vascular dementia. It is caused by mutations in NOTCH3 gene, which encodes a large transmembrane receptor Notch3. The key pathological finding is the accumulation of granular osmiophilic material (GOM), which contains extracellular domains of Notch3, on degenerating vascular smooth muscle cells (VSMCs). GOM has been considered specifically diagnostic for CADASIL, but the reports on the sensitivity of detecting GOM in patients’ skin biopsy have been contradictory. To solve this contradiction, we performed a retrospective investigation of 131 Finnish, Swedish and French CADASIL patients, who had been adequately examined for both NOTCH3 mutation and presence of GOM. The patients were examined according to the diagnostic practice in each country. NOTCH3 mutations were assessed by restriction enzyme analysis of specific mutations or by sequence analysis. Presence of GOM was examined by electron microscopy (EM) in skin biopsies. Biopsies of 26 mutation-negative relatives from CADASIL families served as the controls. GOM was detected in all 131 mutation positive patients. Altogether our patients had 34 different pathogenic mutations which included three novel point mutations (p.Cys67Ser, p.Cys251Tyr and p.Tyr1069Cys) and a novel duplication (p.Glu434_Leu436dup). The detection of GOM by EM in skin biopsies was a highly reliable diagnostic method: in this cohort the congruence between NOTCH3 mutations and presence of GOM was 100%. However, due to the retrospective nature of this study, exact figure for sensitivity cannot be determined, but it would require a prospective study to exclude possible selection bias. The identification of a pathogenic NOTCH3 mutation is an indisputable evidence for CADASIL, but demonstration of GOM provides a cost-effective guide for estimating how far one should proceed with the extensive search for a new or an uncommon mutations among the presently known over 170 different NOTCH3 gene defects. The diagnostic skin biopsy should include the border zone between deep dermis and upper subcutis, where small arterial vessels of correct size are located. Detection of GOM requires technically adequate biopsies and distinction of true GOM from fallacious deposits. If GOM is not found in the first vessel or biopsy, other vessels or additional biopsies should be examined.
Abstract-Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction.In this report, we link Notch signaling to platelet-derived growth factor (PDGF) signaling, a key determinant of VSMC biology, and show that PDGF receptor (PDGFR)- is a novel immediate Notch target gene. PDGFR- expression was upregulated by Notch ligand induction or by activated forms of the Notch receptor. Moreover, upregulation of PDGFR- expression in response to Notch activation critically required the Notch signal integrator CSL. In primary VSMCs, PDGFR- expression was robustly upregulated by Notch signaling, leading to an augmented intracellular response to PDGF stimulation. In newborn Notch3-deficient mice, PDGFR- expression was strongly reduced in the VSMCs that later develop an aberrant morphology. In keeping with this, PDGFR- upregulation in response to Notch activation was reduced also in Notch3-deficient embryonic stem cells. Finally, in VSMCs from a CADASIL patient carrying a NOTCH3 missense mutation, upregulation of PDGFR- mRNA and protein in response to ligand-induced Notch activation was significantly reduced. In sum, these data reveal a hierarchy for 2 important signaling systems, Notch and PDGF, in the vasculature and provide insights into how dysregulated Notch signaling perturbs VSMC differentiation and function. Key Words: PDGF Ⅲ VSMC Ⅲ CADASIL Ⅲ vasculogenesis Ⅲ angiogenesis T he vasculature is formed by an initial aggregation of angioblasts during vasculogenesis, followed by remodeling of the primitive vascular plexus through angiogenesis. 1,2 Recruitment of mural cells, which differentiate to vascular smooth muscle cells (VSMCs) and pericytes, to the endothelial tube is required for stabilization of the vessels. 3 Endothelial and mural cell differentiation is controlled by several key signaling pathways, including PDGF and Notch signaling, 2 and, in this study, we addressed the interrelationship between Notch and PDGF signaling in VSMCs.PDGF signaling is critical for several steps in vascular development and for the homeostasis of blood vessels. There are 4 different genes encoding PDGF ligands (PDGFA through -D), and 2 genes encoding PDGF receptors (PDGFRs) (PDGFR-␣ and -). PDGFRs are receptor tyrosine kinases that, on interaction with ligand, activate several intracellular signaling pathways, including phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling. 4 Loss-of-function analysis has revealed the importance for PDGF-BB/ PDGFR- signaling in vascular development. Mice in which the PDGF-B or PDGFR- gene has been targeted are embryonic lethal, and the phenotypes support a model where endothelial cells, through secretion of PDGF-BB, stimulate proliferation and recruitment of PDGFR- positive mural cells during embryonic development. 5,6 PDGF signaling plays an important role also in restenosis in response to angioplasty. 7 N...
The most common monogenic cause of small-vessel disease leading to ischemic stroke and vascular dementia is the neurodegenerative syndrome cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is associated with mutations in the Notch 3 receptor. CADASIL pathology is characterized by vascular smooth muscle cell degeneration and accumulation of diagnostic granular osmiophilic material (GOM) in vessels. The functional nature of the Notch 3 mutations causing CADASIL and their mechanistic connection to small-vessel disease and GOM accumulation remain enigmatic. To gain insight into how Notch 3 function is linked to CADASIL pathophysiology, we studied two phenotypically distinct mutations, C455R and R1031C, respectively associated with early and late onset of stroke, by using hemodynamic analyses in transgenic mouse models, receptor activity assays in cell culture, and proteomic examination of postmortem human tissue. We demonstrate that the C455R and R1031C mutations define different hypomorphic activity states of Notch 3, a property linked to ischemic stroke susceptibility in mouse models we generated. Importantly, these mice develop osmiophilic deposits and other age-dependent phenotypes that parallel remarkably the human condition. Proteomic analysis of human brain vessels, carrying the same CADASIL mutations, identified clusterin and collagen 18 α1/endostatin as GOM components. Our findings link loss of Notch signaling with ischemic cerebral small-vessel disease, a prevalent human condition. We determine that CADASIL pathophysiology is associated with hypomorphic Notch 3 function in vascular smooth muscle cells and implicate the accumulation of clusterin and collagen 18 α1/endostatin in brain vessel pathology.
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