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Correspondingly, we demonstrate that it is not a marker for tumour EC in multiple malignancy types. Imaging of EC from different vascular beds reveals nestin subcellular distribution is shear-modulated.
Our study challenges the dogma that nestin is a marker of proliferation, and provides insight into its regulation and function in EC. Furthermore, our systems-based approach can be applied to investigate body-wide expression profiles of any candidate protein. Nestin is a type-IV intermediate filament IF , first identified in the neuroepithelial stem cells of the embryonic rat neural tube 1. Since then it has been widely described as a marker for stem or progenitor cells, mainly in the developing central nervous system 2 , 3 , but also heart 4 , 5 , bone marrow 6 , and others.
Following cell differentiation, nestin is reportedly replaced by other cell-type specific IF 7 — 9 , and its expression is predominantly restricted to areas of regeneration in healthy adult tissues 3 , such as skeletal muscle 10 , hair follicles 11 , dopaminergic neurons 12 and neural stem cells 13 , Nestin is also expressed in kidney podocytes 15 , 16 and the neuromuscular junction In the adult cardiovascular system, nestin is expressed in actively proliferating endothelial cells EC , but is considered absent from mature vasculature 6 , 18 , Thus, it has been suggested as a potential therapeutic target for tumour-associated angiogenesis 18 , 20 — 23 and a prognostic marker 24 — There are few studies of nestin expression in humans, with current knowledge almost entirely extrapolated from observations in animal models.
Here, we perform a transcriptome and antibody-based profiling analysis of 37 human organs, which reveals body-wide EC-enriched nestin expression, independent of proliferative status. In addition, we analyse different tumour types to reveal that nestin is not a specific marker of tumour-associated EC, and its expression level is not an independent prognostic factor. We use primary human EC from four vascular beds to examine nestin spatial profiles under static and flow culture, and demonstrate an unexpected role for nestin in the inhibition of EC proliferation.
Correspondingly, we show reduced nestin expression in atherosclerotic plaque neovessels. Transcripts per kilobase million TPM values were calculated for 22, mapped protein-coding genes. Correlations were observed between NES and test transcripts corr. The most significant over-represented groups were related to vascular or EC function Fig. S1A—F , in this replication dataset. This analysis indicated that NES transcript expression is enriched in EC across organs, and we used protein profiling to confirm that nestin protein was specifically expressed in the EC compartment of the human adult cerebral cortex, adrenal gland, thyroid, skeletal muscle, lung, tonsil, spleen, pancreas, salivary gland, esophagus, stomach, duodenum, colon, urinary bladder, adipose, placenta, ovary, testis and prostate Fig.
Nestin was also expressed in EC of the heart, kidney, and breast, although in these tissues, consistent with previous reports 32 — 34 , non-EC expression was observed in cardiomyocytes, renal glomeruli, and myoepithelial cells, respectively Fig. Nestin is a pan-EC enriched protein in the human adult.
RNA-seq data from individual samples from 37 different human tissues were used to generate Spearman pair wise correlation values between NES transcript expression and transcripts for all other mapped protein coding genes. Correlation values and corresponding p-values are shown in the top left and bottom right of each scatter plot, respectively. Human tissue sections from D cerebral cortex, adrenal gland, thyroid, skeletal muscle, lung, tonsil, spleen, gallbladder, pancreas, salivary gland, esophagus, stomach, duodenum, colon, urinary bladder, adipose, placenta, ovary, testis, prostate and E heart, kidney and breast were stained for protein encoded by NES.
See also Supplemental Fig. The vast majority of the adult human vasculature is quiescent 35 , but as nestin expression is currently considered largely restricted to progenitor or proliferating cells, we investigated the proliferative status of nestin-positive EC. As nestin was expressed in the EC of all these tissues, its expression is therefore not dependent on proliferative status.
Nestin expression is not restricted to proliferating EC. Nestin has been described as a specific marker for vessels in various tumour types 22 , 39 , 40 , with prognostic potential 25 , 41 — Here, we used transcriptomic and protein profiling of five tumour types to determine if EC nestin expression is modified in malignant tissue, and to determine its value as a prognostic marker for survival.
Protein profiling and survival analysis was performed as part of our HPA pathology atlas NES expression was associated with poor outcome in renal cancer Fig. Nestin expression is not specific to tumour EC.
Protein profiling confirmed increased nestin staining in GBM, compared to normal brain, but this expression was not restricted to EC. Taken together, these data show that nestin is a marker of EC in both normal and tumour tissues with the exception of GBM , and thus is not specifically expressed by tumour-associated EC. Nestin transcription and sub-cellular protein distribution was characterised in human EC from four different vascular beds.
Under static conditions, EC grew in the characteristic cobblestone pattern and nestin was localised in a cytoplasmic perinuclear pattern around the nuclear membrane in all EC types, with some wider cytoplasmic expression Fig. Exposure to laminar shear stress caused the EC to elongate in shape and induced the formation of a highly filamentous network in all EC Fig. A time-course assay revealed shear stress-induced nestin spatial reorganisation was induced by 4 hours of flow exposure Fig.
To determine the relative contribution of de novo NES transcription vs. EC nestin is regulated by laminar shear stress in vitro. Nestin has a short N-terminal head domain that inhibits self-assembly, but it can form heterodimers with other IF proteins 46 — Vimentin VIM is the principle IF in EC 49 , 50 and can co-assemble with nestin in other cell types 8 , 46 , 47 , 51 therefore, we investigated the relationship between nestin and vimentin in EC.
Like nestin, vimentin protein was localised around the nuclear membrane under static conditions and underwent shear stress induced sub-cellular redistribution. Vimentin and nestin showed a high degree of subcellular co-localisation under static and flow exposed conditions Fig.
No correlation existed between vimentin and the cytoskeletal protein actin Corr. These data indicate that common regulatory processes underlie the spatial distribution of EC nestin and VIM under static and laminar shear stress. We demonstrated that EC nestin expression in vivo is not restricted to cells that express markers of replication Fig.
We used serum starvation to inhibit EC division in vitro 52 to further investigate the relationship between nestin and proliferative status. This data further supports that proliferation is not a pre-requisite for EC nestin expression. D Representative phase contrast images were captured at 0 h, 24 h and 48 h post wound creation 48 h, 72 h and 96 h post-transfection, respectively. Individual data points from 96 h post-transfection are shown for velocity and migration F and H , respectively.
Green boxes indicate measurements taken at the same time point post-transfection 96 h. Nestin inhibition did not affect the expression or subcellular organisation of vimentin under either static or flow conditions Fig. This accelerated gap closing could be due to: 1 increased EC motility or 2 increased EC proliferation.
To determine the relative contribution of each, the position and number of individual EC were recorded over a period of 96 hours following transfection N1, N2 and C siRNA. Taken together, these results indicate that inhibition of nestin increases cellular proliferation and thus, nestin expression inhibits the capacity for EC division. The cellular redistribution of nestin under flow and the effects of nestin knockdown on wound healing and proliferation led us to investigate nestin expression in the context of atherosclerosis.
IHC staining showed that nestin in normal arteries was expressed both in adventitial micro-vessels and luminal lining, and occasional smooth muscle cells Fig. The neovessels of atherosclerotic plaques had weak nestin staining, or were negative altogether Fig.
Nestin expression is lower in atherosclerotic plaque vs. Arrows highlight blood vessel EC. Two single nucleotide polymorphisms SNPs in the NES gene rs and rs were previously reported as associated with early onset coronary heart disease CHD 54 , however no data linking these SNPs to nestin expression cell type, or degree of has been reported.
Four haplotypes were inferred from the 3 selected SNPs. Here, we used a systems-based approach, combining RNAseq analysis and protein profiling, to identify nestin as a highly EC-enriched intermediate filament in the human adult, and to characterise its expression in malignancy and atherosclerosis.
We demonstrate that the subcellular distribution of nestin is regulated by shear forces and that it has a role in the inhibition of EC proliferation. We identified nestin as EC-enriched throughout all adult vascular beds. However, current dogma dictates that nestin is a neovascularisation marker, absent from non-proliferating adult EC 3 , 18 , 19 , 57 , 58 ; a concept underpinned by studies showing angiogenic EC in the human embryo and corpus luteum were nestin positive 40 , whilst nestin was largely absent from the vasculature of the adult rat 57 and mouse However, until now there has been no systematic study of nestin expression in the human adult, although there are isolated reports of nestin-positive EC being detected in adult human brain 59 and pancreas There is divergence between the sequence and promoter regions of the NES gene between species 61 , 62 , which could account for the discrepancies between our observations and those in animal models 19 , 57 ; indeed, it is well documented that key EC genes can vary between rodents and humans 63 — We found no evidence that nestin expression profiles in kidney, bladder, lung and stomach tumours were distinct from established EC markers.
Previous reports suggest nestin is a specific marker for angiogenic EC in various tumour types, and could be useful as a therapeutic target 18 , 21 , 22 , 40 , 58 , We did find that higher nestin expression was associated with unfavourable kidney cancer prognosis, consistent with previous studies However, we found a similar association with PECAM1 and CD34, suggesting that these EC genes represent a proxy for the degree of vascularisation, a factor previously linked with outcome Nestin expression was markedly different in glioblastoma GBM compared to the other tumour types, with high levels of expression outside the vasculature; as previously reported 20 , 68 , Genes with highly EC restricted expression across tissue beds tend to be important for vascular stability or EC-specific functions 72 — We demonstrated that nestin subcellular spatial arrangement is subject to shear stress regulation in primary EC from multiple vascular beds.
Such redistribution, from a perinuclear aggregate to a filamentous network, is characteristic of IF polymerisation. IF polymerisation status is associated with exposure to stimulus such as hypoxia 75 , mechanical, chemical or toxin induced stress 76 — 79 and changes in phosphorylation 76 — 78 , Nestin has many potential phosphorylation sites 81 , the most well studied associated signalling pathway being the regulation of the kinase CDK5 and its co-activator p35 16 , 17 , 80 , Although this signalling pathway has not been studied in EC, CDK5 has previously been linked to the regulation of EC migration and angiogenesis We observed subcellular co-localisation between EC nestin and vimentin under static conditions and following shear stress induced redistribution, however the subcellular distribution of vimentin did not appear to be disrupted by nestin inhibition.
Vimentin is the most well studied EC IF to date but, unlike nestin, it is not highly EC-specific in the adult 49 , 50 , Vimentin is important for EC-specialised functions, such as lymphocyte recruitment 85 , basement membrane interactions 86 and the formation of Von Willebrand factor strings on the EC surface The functional relevance of nestin-vimentin co-localisation in such processes is currently unknown.
We found that silencing of the NES gene in in vitro increased EC proliferation, but did not affect migration. Previous studies also reported that NES silencing had no effect on EC migration 21 , but reported inhibition of EC proliferation 21 , Experimental differences could contribute to this apparent discrepancy, including the use of immortalised murine and human EC lines 21 , in contrast to human primary EC, and the use of VEGF as a proliferation stimulus, following serum starvation 88 , compared to the measurement of spontaneous replication.
NES silencing can also increase proliferation capacity in terminally differentiated human podocytes 32 , indicating that nestin may also influence cell cycle progression in other cell types where it is constitutively expressed.
We found a reduced expression of NES mRNA in human carotid atherosclerotic plaques, compared to normal arteries, corroborated by reduced or absent neovessel EC nestin protein expression. The role of nestin in human atherosclerosis is not well studied, although the disease-associated modified shear stress is well known to affect several processes in which we have here demonstrated a functional role of nestin, such as cytoskeletal rearrangement, wound repair and proliferation 89 , This haplotype includes two genetic variants previously linked to early onset CAD 54 , but the connection with cardiovascular risk remains an open question, especially as no genetic variation at the NES locus had emerged in large genome-wide genetic association studies for such diseases.
Our study challenges the current dogma that nestin is restricted to areas of tissue regeneration in the adult, showing that it is an integral body-wide EC-enriched protein.
Examining the transcriptional networks controlling nestin expression in EC vs.
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