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Appropriate therapeutic modulation of endothelial proliferation and sprouting is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The current view is that an increase in growth factor concentration, and the resulting mitogenic activity, increases both endothelial proliferation and sprouting. Here, we modulate mitogenic stimuli in different vascular contexts by interfering with the function of the VEGF and Notch signalling pathways at high spatiotemporal resolution in vivo. Contrary to the prevailing view, our results indicate that high mitogenic stimulation induced by VEGF, or Notch inhibition, arrests the proliferation of angiogenic vessels. This is due to the existence of a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21, determining whether endothelial cells sprout, proliferate, or become quiescent. The identified mechanism should be considered to achieve optimal therapeutic modulation of angiogenesis. High mitogenic stimuli have been suggested to promote endothelial cell proliferation and sprouting during angiogenesis. Here Pontes-Quero et al., by interfering with levels of VEGF and Notch signalling in single endothelial cells in vivo, find that high mitogenic stimuli instead arrest angiogenesis due to a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21.

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis. The Cre-Lox system allows high spatiotemporal control of genetic modifications. Here the authors present iSuRe-Cre that significantly increases Cre activity in reporter expressing cells, which ultimately increases the efficiency and reliability of Cre-dependent reporter and gene function analysis.

The original version of this Article contained errors in Fig. 8. In panel a, the labels ‘VEGF’, ‘Notch’, ‘p21’, and ‘P-ERK’ were inadvertently omitted. This has been corrected in the PDF and HTML versions of the Article.

RNA editing by the adenosine deaminase ADAR1 controls cathepsin S expression in endothelial cells, a mechanism that is implicated in determining cathepsin S levels in patients with atherosclerotic vascular diseases. Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA ( CTSS ), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3′ untranslated region (3′ UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx + regions, which form a long stem–loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1 ) to the 3′ UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases.

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and, therefore, do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here we present the iFlpMosaics toolkit comprising a large set of new genetic tools and mouse lines that enable recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wild-type and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by fluorescence-activated flow cytometry and single-cell RNA sequencing. iFlpMosaics facilitate the induction and analysis of genetic mosaics in any quiescent or progenitor cell, and for any given single or combination of floxed genes, thus enabling a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis and disease. iFlpMosaics are a compendium of mouse lines and genetic tools for tissue-agnostic generation and analysis of genetic mosaics.

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and therefore do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here, we present a large set of new genetic tools and mouse lines that enable Flp recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wildtype and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by FACS and scRNA-seq. This technology facilitates the induction and analysis of genetic mosaics in any cell type and for any given single or combination of floxed genes. iFlpMosaics enables a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis, and disease.