Incongruence between transcriptional and vascular pathophysiological cell states.

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

Role of Notch in endothelial biology.

The Notch signalling pathway is one of the main regulators of endothelial biology. In the last 20 years the critical function of Notch has been uncovered in the context of angiogenesis, participating in tip-stalk specification, arterial-venous differentiation, vessel stabilization, and maturation processes. Importantly, pharmacological compounds targeting distinct members of the Notch signalling pathway have been used in the clinics for cancer therapy. However, the underlying mechanisms that support the variety of outcomes triggered by Notch in apparently opposite contexts such as angiogenesis and vascular homeostasis remain unknown. In recent years, advances in -omics technologies together with mosaic analysis and high molecular, cellular and temporal resolution studies have allowed a better understanding of the mechanisms driven by the Notch signalling pathway in different endothelial contexts. In this review we will focus on the main findings that revisit the role of Notch signalling in vascular biology. We will also discuss potential future directions and technologies that will shed light on the puzzling role of Notch during endothelial growth and homeostasis. Addressing these open questions may allow the improvement and development of therapeutic strategies based on modulation of the Notch signalling pathway.

Arterialization requires the timely suppression of cell growth.

The formation of arteries is thought to occur by the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow1,2. The initial steps of arterial specification require both the VEGF and Notch signalling pathways3-5. Here, we combine inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous differentiation and mobilization. We show that endothelial cells with high levels of VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; however, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that increased levels of VEGF and Notch signalling in pre-arterial capillaries suppresses MYC-dependent metabolic and cell-cycle activities, and promotes the incorporation of endothelial cells into arteries. Mosaic lineage-tracing studies showed that endothelial cells that lack the Notch-RBPJ transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when the function of MYC was suppressed. Thus, the development of arteries does not require the direct induction of a Notch-dependent arterial differentiation programme, but instead depends on the timely suppression of endothelial cell-cycle progression and metabolism, a process that precedes arterial mobilization and complete differentiation.

Endothelial sprouting, proliferation, or senescence: tipping the balance from physiology to pathology.

Therapeutic modulation of vascular cell proliferation and migration is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The general view is that an increase in vascular growth factor levels or mitogenic stimulation is beneficial for angiogenesis, since it leads to an increase in both endothelial proliferation and sprouting. However, several recent studies showed that an increase in mitogenic stimuli can also lead to the arrest of angiogenesis. This is due to the existence of intrinsic signaling feedback loops and cell cycle checkpoints that work in synchrony to maintain a balance between endothelial proliferation and sprouting. This balance is tightly and effectively regulated during tissue growth and is often deregulated or impaired in disease. Most therapeutic strategies used so far to promote vascular growth simply increase mitogenic stimuli, without taking into account its deleterious effects on this balance and on vascular cells. Here, we review the main findings on the mechanisms controlling physiological vascular sprouting, proliferation, and senescence and how those mechanisms are often deregulated in acquired or congenital cardiovascular disease leading to a diverse range of pathologies. We also discuss alternative approaches to increase the effectiveness of pro-angiogenic therapies in cardiovascular regenerative medicine.

Neutrophil infiltration regulates clock-gene expression to organize daily hepatic metabolism.

Liver metabolism follows diurnal fluctuations through the modulation of molecular clock genes. Disruption of this molecular clock can result in metabolic disease but its potential regulation by immune cells remains unexplored. Here, we demonstrated that in steady state, neutrophils infiltrated the mouse liver following a circadian pattern and regulated hepatocyte clock-genes by neutrophil elastase (NE) secretion. NE signals through c-Jun NH2-terminal kinase (JNK) inhibiting fibroblast growth factor 21 (FGF21) and activating Bmal1 expression in the hepatocyte. Interestingly, mice with neutropenia, defective neutrophil infiltration or lacking elastase were protected against steatosis correlating with lower JNK activation, reduced Bmal1 and increased FGF21 expression, together with decreased lipogenesis in the liver. Lastly, using a cohort of human samples we found a direct correlation between JNK activation, NE levels and Bmal1 expression in the liver. This study demonstrates that neutrophils contribute to the maintenance of daily hepatic homeostasis through the regulation of the NE/JNK/Bmal1 axis.

Every day, the body's biological processes work to an internal clock known as the circadian rhythm. This rhythm is controlled by 'clock genes' that are switched on or off by daily physical and environmental cues, such as changes in light levels. These daily rhythms are very finely tuned, and disturbances can lead to serious health problems, such as diabetes or high blood pressure. The ability of the body to cycle through the circadian rhythm each day is heavily influenced by the clock of one key organ: the liver. This organ plays a critical role in converting food and drink into energy. There is evidence that neutrophils ­ white blood cells that protect the body by being the first response to inflammation ­ can influence how the liver performs its role in obese people, by for example, releasing a protein called elastase. Additionally, the levels of neutrophils circulating in the blood change following a daily pattern. Crespo, González-Terán et al. wondered whether neutrophils enter the liver at specific times of the day to control liver's daily rhythm. Crespo, González-Terán et al. revealed that neutrophils visit the liver in a pattern that peaks when it gets light and dips when it gets dark by counting the number of neutrophils in the livers of mice at different times of the day. During these visits, neutrophils secreted elastase, which activated a protein called JNK in the cells of the mice's liver. This subsequently blocked the activity of another protein, FGF21, which led to the activation of the genes that allow cells to make fat molecules for storage. JNK activation also switched on the clock gene, Bmal1, ultimately causing fat to build up in the mice's liver. Crespo, González-Terán et al. also found that, in samples from human livers, the levels of elastase, the activity of JNK, and whether the Bmal1 gene was switched on were tightly linked. This suggests that neutrophils may be controlling the liver's rhythm in humans the same way they do in mice. Overall, this research shows that neutrophils can control and reset the liver's daily rhythm using a precisely co-ordinated series of molecular changes. These insights into the liver's molecular clock suggest that elastase, JNK and BmaI1 may represent new therapeutic targets for drugs or smart medicines to treat metabolic diseases such as diabetes or high blood pressure.

Genetic Tools to Study Cardiovascular Biology.

Progress in biomedical science is tightly associated with the improvement of methods and genetic tools to manipulate and analyze gene function in mice, the most widely used model organism in biomedical research. The joint effort of numerous individual laboratories and consortiums has contributed to the creation of a large genetic resource that enables scientists to image cells, probe signaling pathways activities, or modify a gene function in any desired cell type or time point, à la carte. However, as these tools significantly increase in number and become more sophisticated, it is more difficult to keep track of each tool's possibilities and understand their advantages and disadvantages. Knowing the best currently available genetic technology to answer a particular biological question is key to reach a higher standard in biomedical research. In this review, we list and discuss the main advantages and disadvantages of available mammalian genetic technology to analyze cardiovascular cell biology at higher cellular and molecular resolution. We start with the most simple and classical genetic approaches and end with the most advanced technology available to fluorescently label cells, conditionally target their genes, image their clonal expansion, and decode their lineages.

Publisher Correction: High mitogenic stimulation arrests angiogenesis.

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.

High mitogenic stimulation arrests angiogenesis.

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.

iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications.

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.

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.