Diverse cell labeling by vascular endothelial cadherin-driven lineage tracing in adult mice
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ABSTRACT:
SUBMITTER: Brad Morrison
PROVIDER: S-BSST278 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease. To address whether endothelial cells transdifferentiate into non-vascular cell types, we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells (VEcad-CreERT2; B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze). Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4-5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include: cerebellar granule neurons, ependymal cells, skeletal myocytes, pancreatic beta cells, pancreatic acinar cells, tubular cells in the renal cortex, duodenal crypt cells, ileal crypt cells, and hair follicle stem cells. As Nestin expression has been reported in a subset of endothelial cells, Nes-CreERT2 mice were also utilized in these conditions. The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells. Interestingly, Nestin tracing also labeled skeletal myocytes, ileal crypt cells, and sparsely marked cerebellar granule neurons. Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools. This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets. The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee (approval No. 006-AC15-018) on October 31, 2018.
| S-EPMC7513977 | biostudies-literature
Project description:Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.
| S-EPMC2798002 | biostudies-literature
Project description:Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a 'Brainbow' of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish.
| S-EPMC3678346 | biostudies-literature
Project description:Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces. Trimerization of the bacterially produced protein appears to be an artifact that arises from a lack of glycosylation. We also present the 2.1-Å-resolution crystal structure of the VE-cadherin EC1-2 adhesive region, which reveals homodimerization via the strand-swap mechanism common to classical cadherins. In common with type II cadherins, strand-swap binding involves two tryptophan anchor residues, but the adhesive interface resembles type I cadherins in that VE-cadherin does not form a large nonswapped hydrophobic surface. Thus, VE-cadherin is an outlier among classical cadherins, with characteristics of both type I and type II subfamilies.
| S-EPMC3084036 | biostudies-literature
Project description:Lymphocytes emigrate from the circulation to target tissues through the microvascular endothelial cell (EC) barrier. During paracellular transmigration cell-cell junctions have been proposed to disengage and provide homophilic and heterophilic interaction surfaces in a zip-like process. However, it is not known whether ECs modulate junction proteins during this process. Here we show that tyrosine phosphorylation of adherens junction vascular endothelial cadherin (VEC) is required for successful transendothelial lymphocyte migration. We found that adhesion of lymphocytes or activation of the endothelial intercellular adhesion molecule 1 (ICAM1) led to tyrosine phosphorylation of VEC. Substitution of tyrosine for phenylalanine in VEC at positions 645, 731 or 733 produced ECs that were significantly less permissive to lymphocyte migration. We also found that these same tyrosine residues are involved in ICAM1-dependent changes of VEC phosphorylation. ICAM1 activation enhanced transendothelial permeability, suggesting the occurrence of junction disassembly. In agreement, the expression of VEC mutated at Y645F, Y731F or Y733F predominantly affected lymphocyte transmigration in paracellular areas. Taken together, these results demonstrate that phosphorylation of adherens junctions constitutes a molecular endpoint of lymphocyte-induced vascular EC signaling and may be exploited as a new target of anti-inflammatory therapies.
| S-EPMC3810954 | biostudies-literature
Project description:Vascular endothelial (VE)-cadherin is the predominant component of endothelial adherens junctions essential for cell-cell adhesion and formation of the vascular barrier. Endocytic recycling is an important mechanism for maintaining the expression of cell surface membrane proteins. However, little is known about the molecular mechanism of VE-cadherin recycling and its role in maintenance of vascular integrity.Using calcium-switch assay, confocal imaging, cell surface biotinylation, and flow cytometry, we showed that VE-cadherin recycling required Ras-related proteins in brain (Rab)11a and Rab11 family-interacting protein 2. Yeast 2-hybrid assay and coimmunoprecipitation demonstrated that direct interaction of VE-cadherin with family-interacting protein 2 (at aa 453-484) formed a ternary complex with Rab11a in human endothelial cells. Silencing of Rab11a or Rab11 family-interacting protein 2 in endothelial cells prevented VE-cadherin recycling and VE-cadherin expression at endothelial plasma membrane. Furthermore, inactivation of Rab11a signaling blocked junctional reannealing after vascular inflammation. Selective knockdown of Rab11a in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide or polymicrobial sepsis.Rab11a/Rab11 family-interacting protein 2-mediated VE-cadherin recycling is required for formation of adherens junctions and restoration of VE barrier integrity and hence a potential target for clinical intervention in inflammatory disease.
| S-EPMC4732894 | biostudies-literature
Project description:INTRODUCTION: Deregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)-? signaling. Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer. METHODS: The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis. RESULTS: VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of ?-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression was forced. CONCLUSIONS: Our results highlight the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents the re-expression of E-cadherin and localization of ?-catenin at the plasma membrane of mesenchymal mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.
| S-EPMC4053141 | biostudies-other
Project description:AimsCardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells.Methods and resultsWe observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass.ConclusionOur genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.
| S-EPMC7797216 | biostudies-literature
Project description:Many inflammatory diseases are associated with elevated blood concentration of fibrinogen (Fg) leading to vascular dysfunction. We showed that pathologically high (4 mg/ml) content of Fg disrupts integrity of endothelial cell (EC) layer and causes macromolecular leakage affecting tight junction proteins. However, role of adherence junction proteins, particularly vascular endothelial cadherin (VE-cadherin) and matrix metalloproteinase-9 (MMP-9) in this process is not clear. We tested the hypothesis that at high levels Fg affects integrity of mouse brain endothelial cell (MBEC) monolayer through activation of MMP-9 and downregulation of VE-cadherin expression and in part its translocation to the cytosol. The effect of Fg on cultured MBEC layer integrity was assessed by measuring transendothelial electrical resistance. Cellular expression and translocation of VE-cadherin were assessed by Western blot and immunohistochemical analyses, (respectively). Our results suggest that high content of Fg decreased VE-cadherin expression at protein and mRNA levels. Fg induced translocation of VE-cadherin to cytosol, which led to disruption of cell-to-cell interaction and cell to subendothelial matrix attachment. Fg-induced alterations in cell layer integrity and their attachment were diminished during inhibition of MMP-9 activity. Thus Fg compromises EC layer integrity causing downregulation and translocation of VE-cadherin and through MMP-9 activation. These results suggest that increased level of Fg could play a significant role in vascular dysfunction and remodeling.
| S-EPMC3190052 | biostudies-literature
Project description:Using genetic fate-mapping with Cux2-Cre and Cux2-CreERT2 mice we demonstrated that the neocortical ventricular zone (VZ) contains radial glial cells (RGCs) with restricted fate potentials (Franco et al., 2012). Using the same mouse lines, Guo et al. (2013) concluded that the neocortical VZ does not contain lineage-restricted RGCs. We now show that the recombination pattern in Cux2-Cre/CreERT2 mice depends on genetic background and breeding strategies. We provide evidence that Guo et al. likely reached different conclusions because they worked with transgenic sublines with drifted transgene expression patterns. In Cux2-Cre and Cux2-CreERT2 mice that recapitulate the endogenous Cux2 expression pattern, the vast majority of fate-mapped neurons express Satb2 but not Ctip2, confirming that a restricted subset of all neocortical projection neurons belongs to the Cux2 lineage. This Matters Arising paper is in response to Guo et al. (2013), published in Neuron. See also the Matters Arising Response paper by Eckler et al. (2015), published concurrently with this Matters Arising in Neuron.
| S-EPMC4455040 | biostudies-literature