von Willebrand Factor Permeates Small Vessels in CADASIL and Inhibits Smooth Muscle Gene Expression

Abstract

Background and Purpose CADASIL (cerebral autosomal dominant arteriopathy subcortical infarcts and leukoencephalopathy) is a genetic disorder hallmarked by ischemic stroke and vascular dementia. Characteristic pathological changes in the vasculature include thickening of small arteries and accumulation of heterogeneous material within the vessel wall. We tested whether endothelial von Willebrand factor (vWF) accumulates in CADASIL vessels and whether exposure of smooth muscle cells to vWF alters the expression of smooth muscle gene expression. Methods Brain sections obtained at autopsy from six North American CADASIL patients were examined using immunohistochemistry for vWF and IgG. Rat aortic smooth muscle cells (A7R5 cells) were tested for binding to infrared-tag labeled vWF. Finally, A7R5 cells were exposed to vWF, and expression of mature smooth muscle marker genes was analyzed by quantitative reverse transcriptase PCR. Results vWF is expressed in the penetrating arterial walls in all CADASIL samples. IgG, a marker of serum extravasation, was present only in a minority of arterial walls. vWF binds to smooth muscle cells in vitro, and low concentrations of vWF rapidly activate c-fos, EGR, TSP1, and c-myc while specifically inhibiting RNA encoding smooth muscle actin, calponin, and SM22. Conclusions These data demonstrate that vWF, likely produced by the endothelium, permeates the vessel wall of CADASIL brains. Exposure of smooth muscle cells to vWF results in reduction of specific RNAs required for normal vascular homeostasis. This is the first report of accumulation of a protein within CADASIL vessels that inhibits vascular gene expression and implicates a role for vWF beyond hemostasis.

Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state

“…Another possibility is that other factors (e.g. Notch-binding matrix proteins 10, 15–18 ), not active in our expression system, bind specifically to NOTCH3 with a reduced cysteine. Subsequently, this factor could facilitate the unmasking of N-terminal epitopes in mutant NOTCH3 protein.…”

“…Brain tissue and genetic mutations in NOTCH3 from six CADASIL patients have been described before 10 . The study was granted an exemption from IRB review.…”

The Small Leucine-Rich Proteoglycan BGN Accumulates in CADASIL and Binds to NOTCH3

“…Formalin fixed frontal lobes sections were acquired from the Brain Bank of the National Institute for Developmental and Childhood Disorders at the University of Maryland and the Alzheimers Disease Research Core at the University of Michigan and have been previously described [6, 29]. In addition, two CADASIL brains from patients with ischemic stroke and dementia with the NOTCH3 mutations R141C and R153C were studied [28]; both of these patients were Caucasian males in their 60s who died of complications of the disease.…”

Vascular accumulation of the small leucine-rich proteoglycan decorin in CADASIL

“…Six rains from CADASIL patients with cysteine-altering NOTCH3 mutations have been previously described[5,6]. Two additional CADASIL brains with mutations R141C and R153C in NOTCH3 were also studied.…”

“…Brain arteries affected in CADASIL are markedly thickened and exhibit significant smooth muscle cell loss and fibrosis in the vascular media [2,3]. The role of protein pathology in CADASIL is underscored by the striking molecular genetics of CADASIL mutations (which nearly invariably involve cysteine residues) and marked hyalinization of brain arteries that includes complex macromolecules such as NOTCH3 [4], multiple forms of collagen[5], von Willebrand factor [6], TIMP3 [7], and vitronectin [7]. Staining of CADASIL brains demonstrates intense periodic acid Schiff (PAS) reactive arteries [3], suggesting the accumulation of glycosylated molecules within thickened vessel walls.…”