Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT‐PCR analysis, we observed increased Transforming growth factor‐β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ‐neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ‐neutralizing antibody in ECs co‐cultured with VSMCs. ECs co‐cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co‐cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co‐cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial progressive degenerative disorder and is caused by mutations in NOTCH3 gene. Previous study reported that mutant NOTCH3 is more prone to form aggregates than wild-type NOTCH3 and the mutant aggregates are resistant to degradation. We hypothesized that aggregation or accumulation of NOTCH3 could be due to impaired lysosomal-autophagy machinery in VSMC. Here, we investigated the possible cause of accumulation/aggregation of NOTCH3 in CADASIL using cerebral VSMCs derived from control and CADASIL patients carrying NOTCH3 R133C mutation. Thioflavin-S-staining confirmed the increased accumulation of aggregated NOTCH3 in VSMC R133C compared to VSMC WT. Increased levels of the lysosomal marker, Lamp2, were detected in VSMC R133C , which also showed co-localization with NOTCH3 using double-immunohistochemistry. Increased level of LC3-II/LC3-I ratio was observed in VSMC R133C suggesting an accumulation of autophagosomes. This was coupled with the decreased co-localization of NOTCH3 with LC3, and Lamp2 and, further, increase of p62/SQSTM1 levels in VSMC R133C compared to the VSMC WT. In addition, Western blot analysis indicated phosphorylation of pERK , p-S6RP, and p-P70 S6K. Altogether, these results suggested a dysfunction in the autophagy-lysosomal pathway in VSMC R133C. The present study provides an interesting avenue of the research investigating the molecular mechanism of CADASIL.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by NOTCH3 mutations. Typical CADASIL is characterised by subcortical ischemic strokes due to severe arteriopathy and fibrotic thickening of small arteries. Arteriolar vascular smooth muscle cells (VSMCs) are the key target in CADASIL, but the potential mechanisms involved in their degeneration are still unclear. Focusing on cerebral microvessels in the frontal and anterior temporal lobes and the basal ganglia, we used advanced proteomic and immunohistochemical methods to explore the extent of inflammatory and immune responses in CADASIL subjects compared to similar age normal and other disease controls. There was variable loss of VSMC in medial layers of arteries in white matter as well as the cortex, that could not be distinguished whether NOTCH3 mutations were in the epidermal growth factor (EGFr) domains 1–6 or EGFr7-34. Proteomics of isolated cerebral microvessels showed alterations in several proteins, many associated with endoplasmic reticulum (ER) stress including heat shock proteins. Cerebral vessels with sparsely populated VSMCs also attracted robust accrual of perivascular microglia/macrophages in order CD45+ > CD163+ > CD68+cells, with > 60% of vessel walls exhibiting intercellular adhesion molecule-1 (ICAM-1) immunoreactivity. Functional VSMC cultures bearing the NOTCH3 Arg133Cys mutation showed increased gene expression of the pro-inflammatory cytokine interleukin 6 and ICAM-1 by 16- and 50-fold, respectively. We further found evidence for activation of the alternative pathway of complement. Immunolocalisation of complement Factor B, C3d and C5-9 terminal complex but not C1q was apparent in ~ 70% of cerebral vessels. Increased complement expression was corroborated in > 70% of cultured VSMCs bearing the Arg133Cys mutation independent of N3ECD immunoreactivity. Our observations suggest that ER stress and other cellular features associated with arteriolar VSMC damage instigate robust localized inflammatory and immune responses in CADASIL. Our study has important implications for immunomodulation approaches to counter the characteristic arteriopathy of CADASIL.

Typical cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the human NOTCH3 gene. Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy is characterized by subcortical ischemic strokes due to severe arteriopathy and fibrotic thickening of small vessels. Blood regulating vascular smooth muscle cells (VSMCs) appear as the key target in CADASIL but the pathogenic mechanisms remain unclear. With the hypothesis that brain glucose metabolism is disrupted in VSMCs in CADASIL, we investigated post-mortem tissues and VSMCs derived from CADASIL patients to explore gene expression and protein immunoreactivity of glucose transporters (GLUTs), particularly GLUT4 and GLUT2 using quantitative RT-PCR and immunohistochemical techniques. In vitro cell model analysis indicated that both GLUT4 and -2 gene expression levels were down-regulated in VSMCs derived from CADASIL patients, compared to controls. In vitro studies further indicated that the down regulation of GLUT4 coincided with impaired glucose uptake in VSMCs, which could be partially rescued by insulin treatment. Our observations on reduction in GLUTs in VSMCs are consistent with previous findings of decreased cerebral blood flow and glucose uptake in CADASIL patients. That impaired ability of glucose uptake is rescued by insulin is also consistent with previously reported lower proliferation rates of VSMCs derived from CADASIL subjects. Overall, these observations are consistent with the development of severe cerebral arteriopathy in CADASIL, in which VSMCs are replaced by widespread fibrosis.