N-Glycans on the extracellular domain of the Notch1 receptor control Jagged-1 induced Notch signalling and myogenic differentiation of S100ß resident vascular stem cells.

Notch signalling, critical for development and postnatal homeostasis of the vascular system, is highly regulated by several mechanisms including glycosylation. While the importance of O-linked glycosylation is widely accepted, the structure and function of N-glycans has yet to be defined. Here, we take advantage of lectin binding assays in combination with pharmacological, molecular, and site-directed mutagenetic approaches to study N-glycosylation of the Notch1 receptor. We find that several key oligosaccharides containing bisecting or core fucosylated structures decorate the receptor, control expression and receptor trafficking, and dictate Jagged-1 activation of Notch target genes and myogenic differentiation of multipotent S100ß vascular stem cells. N-glycans at asparagine (N) 1241 and 1587 protect the receptor from accelerated degradation, while the oligosaccharide at N888 directly affects signal transduction. Conversely, N-linked glycans at N959, N1179, N1489 do not impact canonical signalling but inhibit differentiation. Our work highlights a novel functional role for N-glycans in controlling Notch1 signalling and differentiation of vascular stem cells.

The calcium binding protein S100ß marks hedgehog-responsive resident vascular stem cells within vascular lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening. While medial SMCs contribute, the participation of hedgehog-responsive resident vascular stem cells (vSCs) to lesion formation remains unclear. Using transgenic eGFP mice and genetic lineage tracing of S100ß vSCs in vivo, we identified S100ß/Sca1 cells derived from a S100ß non-SMC parent population within lesions that co-localise with smooth muscle α-actin (SMA) cells following iatrogenic flow restriction, an effect attenuated following hedgehog inhibition with the smoothened inhibitor, cyclopamine. In vitro, S100ß/Sca1 cells isolated from atheroprone regions of the mouse aorta expressed hedgehog signalling components, acquired the di-methylation of histone 3 lysine 4 (H3K4me2) stable SMC epigenetic mark at the Myh11 locus and underwent myogenic differentiation in response to recombinant sonic hedgehog (SHh). Both S100ß and PTCH1 cells were present in human vessels while S100ß cells were enriched in arteriosclerotic lesions. Recombinant SHh promoted myogenic differentiation of human induced pluripotent stem cell-derived S100ß neuroectoderm progenitors in vitro. We conclude that hedgehog-responsive S100ß vSCs contribute to lesion formation and support targeting hedgehog signalling to treat subclinical arteriosclerosis.

Moderate Alcohol Consumption Targets S100ß+ Vascular Stem Cells and Attenuates Injury-Induced Neointimal Hyperplasia.

BACKGROUND: Stem cells present in the vessel wall may be triggered in response to injurious stimuli to undergo differentiation and contribute to vascular disease development. Our aim was to determine the effect of moderate alcohol (EtOH) exposure on the expansion and differentiation of S100 calcium-binding protein B positive (S100ß+ ) resident vascular stem cells and their contribution to pathologic vessel remodeling in a mouse model of arteriosclerosis. METHODS AND RESULTS: Lineage tracing analysis of S100ß+ cells was performed in male and female S100ß-eGFP/Cre/ERT2-dTomato transgenic mice treated daily with or without EtOH by oral gavage (peak BAC: 15 mM or 0.07%) following left common carotid artery ligation for 14 days. Carotid arteries (ligated or sham-operated) were harvested for morphological analysis and confocal assessment of fluorescent-tagged S100 ß + cells in FFPE carotid cross sections. Ligation-induced carotid remodeling was more robust in males than in females. EtOH-gavaged mice had less adventitial thickening and markedly reduced neointimal formation compared to controls, with a more pronounced inhibitory effect in males compared to females. There was significant expansion of S100ß+ -marked cells in vessels postligation, primarily in the neointimal compartment. EtOH treatment reduced the fraction of S100ß+ cells in carotid cross sections, concomitant with attenuated remodeling. In vitro, EtOH attenuated Sonic Hedgehog-stimulated myogenic differentiation (as evidenced by reduced calponin and myosin heavy chain expression) of isolated murine S100ß+ vascular stem cells. CONCLUSIONS: These data highlight resident vascular S100ß+ stem cells as a novel target population for alcohol and suggest that regulation of these progenitors in adult arteries, particularly in males, may be an important mechanism contributing to the antiatherogenic effects of moderate alcohol consumption.

Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Stem Cell Antigen-1 Positive Progenitor Stem Cell Expansion.

BACKGROUND: Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate ethanol (EtOH) on sonic hedgehog (SHh) signaling in regulating possible stem cell antigen-1 positive (Sca1+ ) progenitor stem cell involvement during pathologic arterial remodeling. METHODS: Partial ligation or sham operation of the left carotid artery was performed in transgenic Sca1-enhanced green fluorescent protein (eGFP) mice gavaged with or without "daily moderate" EtOH. RESULTS: The EtOH group had reduced adventitial thickening and less neointimal formation, compared to ligated controls. There was expansion of eGFP-expressing (i.e., Sca1+ ) cells in remodeled vessels postligation (day 14), especially in the neo intima. EtOH treatment reduced the number of Sca1+ cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 and Gli2, and RT-PCR of whole-vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1-eGFP mice with the SHh signaling inhibitor cyclopamine diminished SHh target gene expression, reduced the number of Sca1+ cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1+ adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. CONCLUSIONS: EtOH reduces SHh-responsive Sca1+ progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1+ progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects.