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.

Disease-Relevant Single Cell Photonic Signatures Identify S100ß Stem Cells and their Myogenic Progeny in Vascular Lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100ß+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- ß1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100ß+ vSCs and identified the presence of S100ß+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.

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.

Resident multipotent vascular stem cells exhibit amplitude dependent strain avoidance similar to that of vascular smooth muscle cells.

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4-6%, 2-8% and 0-10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications.

Label-free discrimination analysis of de-differentiated vascular smooth muscle cells, mesenchymal stem cells and their vascular and osteogenic progeny using vibrational spectroscopy.

The accumulation of vascular smooth muscle (SMC)-like cells and stem cell-derived myogenic and osteogenic progeny contributes significantly to arteriosclerotic disease. This study established whether label-free vibrational spectroscopy can discriminate de-differentiated 'synthetic' SMCs from undifferentiated stem cells and their myogenic and osteogenic progeny in vitro, compared with conventional immunocytochemical and genetic analyses. TGF-ß1- and Jagged1-induced myogenic differentiation of CD44+ mesenchymal stem cells was confirmed in vitro by immunocytochemical analysis of specific SMC differentiation marker expression (α-actin, calponin and myosin heavy chain 11), an epigenetic histone mark (H3K4me2) at the myosin heavy chain 11 locus, promoter transactivation and mRNA transcript levels. Osteogenic differentiation was confirmed by alizarin red staining of calcium deposition. Fourier Transform Infrared (FTIR) maps facilitated initial screening and discrimination while Raman spectroscopy of individual cell nuclei revealed specific spectral signatures of each cell type in vitro, using Principal Components Analysis (PCA). PCA fed Linear Discriminant Analysis (LDA) enabled quantification of this discrimination and the sensitivity and specificity value was determined for all cell populations based on a leave-one-out cross validation method and revealed that de-differentiated SMCs and stem-cell derived myogenic progeny in culture shared the greatest similarity. FTIR and Raman spectroscopy discriminated undifferentiated stem cells from both their myogenic and osteogenic progeny. The ability to detect stem cell-derived myogenic progeny using label-free platforms in situ may facilitate interrogation of these important phenotypes during vascular disease progression.

An Emerging Role for SNARE Proteins in Dendritic Cell Function.

Dendritic cells (DCs) provide an essential link between innate and adaptive immunity. At the site of infection, antigens recognized by DCs via pattern-recognition receptors, such as Toll-like receptors (TLRs), initiate a specific immune response. Depending on the nature of the antigen, DCs secrete distinct cytokines with which they orchestrate homeostasis and pathogen clearance. Dysregulation of this process can lead to unnecessary inflammation, which can result in a plethora of inflammatory diseases. Therefore, the secretion of cytokines from DCs is tightly regulated and this regulation is facilitated by highly conserved trafficking protein families. These proteins control the transport of vesicles from the Golgi complex to the cell surface and between organelles. In this review, we will discuss the role of soluble n-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs) in DCs, both as facilitators of secretion and as useful tools to determine the pathways of secretion through their definite locations within the cells and inherent specificity in opposing binding partners on vesicles and target membranes. The role of SNAREs in DC function may present an opportunity to explore these proteins as novel targets in inflammatory disease.