The circadian protein BMAL1 supports endothelial cell cycle during angiogenesis.

AIMS: The circadian clock is an internal biological timer that co-ordinates physiology and gene expression with the 24-h solar day. Circadian clock perturbations have been associated to vascular dysfunctions in mammals, and a function of the circadian clock in angiogenesis has been suggested. However, the functional role of the circadian clock in endothelial cells (ECs) and in the regulation of angiogenesis is widely unexplored. METHODS AND RESULTS: Here, we used both in vivo and in vitro approaches to demonstrate that ECs possess an endogenous molecular clock and show robust circadian oscillations of core clock genes. By impairing the EC-specific function of the circadian clock transcriptional activator basic helix-loop-helix ARNT like 1 (BMAL1) in vivo, we detect angiogenesis defects in mouse neonatal vascular tissues, as well as in adult tumour angiogenic settings. We then investigate the function of circadian clock machinery in cultured EC and show evidence that BMAL and circadian locomotor output cycles protein kaput knock-down impair EC cell cycle progression. By using an RNA- and chromatin immunoprecipitation sequencing genome-wide approaches, we identified that BMAL1 binds the promoters of CCNA1 and CDK1 genes and controls their expression in ECs. CONCLUSION(S): Our findings show that EC display a robust circadian clock and that BMAL1 regulates EC physiology in both developmental and pathological contexts. Genetic alteration of BMAL1 can affect angiogenesis in vivo and in vitro settings.

How to Generate a Vascular-Labelled Transgenic Zebrafish Model to Study Tumor Angiogenesis and Extravasation.

The use of transgenic animals carrying exogenous DNA integrated in their genome is a routine in modern-day laboratories. Nowadays, the zebrafish system represents the most useful tool for transgenesis studies mainly due to easy accessibility and manipulation of the eggs, which are produced in high numbers and over a relatively short generation time. The zebrafish transgenic technology is very straightforward when coupled with angiogenesis studies allowing easy in vivo observation of the vertebrate embryonic vasculature. Here, we describe the most common technique to generate vascular-labelled transgenic zebrafish embryos and their applications to study tumor angiogenesis and visualize tumor extravasation.

Cancer-Induced Metabolic Rewiring of Tumor Endothelial Cells.

Cancer is a leading cause of death worldwide. If left untreated, tumors tend to grow and spread uncontrolled until the patient dies. To support this growth, cancer cells need large amounts of nutrients and growth factors that are supplied and distributed to the tumor tissue by the vascular system. The aberrant tumor vasculature shows deep morphological, molecular, and metabolic differences compared to the blood vessels belonging to the non-malignant tissues (also referred as normal). A better understanding of the metabolic mechanisms driving the differences between normal and tumor vasculature will allow the designing of new drugs with a higher specificity of action and fewer side effects to target tumors and improve a patient's life expectancy. In this review, we aim to summarize the main features of tumor endothelial cells (TECs) and shed light on the critical metabolic pathways that characterize these cells. A better understanding of such mechanisms will help to design innovative therapeutic strategies in healthy and diseased angiogenesis.

Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis.

Angiogenesis, the active formation of new blood vessels from pre-existing ones, is a complex and demanding biological process that plays an important role in physiological as well as pathological settings. Recent evidence supports cell metabolism as a critical regulator of angiogenesis. However, whether and how cell metabolism regulates endothelial growth factor receptor levels and nucleotide synthesis remains elusive. We here shown in both human cell lines and mouse models that during developmental and pathological angiogenesis, endothelial cells (ECs) use glutaminolysis-derived glutamate to produce aspartate (Asp) via aspartate aminotransferase (AST/GOT). Asp leads to mTORC1 activation which, in turn, regulates endothelial translation machinery for VEGFR2 and FGFR1 synthesis. Asp-dependent mTORC1 pathway activation also regulates de novo pyrimidine synthesis in angiogenic ECs. These findings identify glutaminolysis-derived Asp as a regulator of mTORC1-dependent endothelial translation and pyrimidine synthesis. Our studies may help overcome anti-VEGF therapy resistance by targeting endothelial growth factor receptor translation.

Oxidative pentose phosphate pathway controls vascular mural cell coverage by regulating extracellular matrix composition.

Vascular mural cells (vMCs) play an essential role in the development and maturation of the vasculature by promoting vessel stabilization through their interactions with endothelial cells. Whether endothelial metabolism influences mural cell recruitment and differentiation is unknown. Here, we show that the oxidative pentose phosphate pathway (oxPPP) in endothelial cells is required for establishing vMC coverage of the dorsal aorta during early vertebrate development in zebrafish and mice. We demonstrate that laminar shear stress and blood flow maintain oxPPP activity, which in turn, promotes elastin expression in blood vessels through production of ribose-5-phosphate. Elastin is both necessary and sufficient to drive vMC recruitment and maintenance when the oxPPP is active. In summary, our work demonstrates that endothelial cell metabolism regulates blood vessel maturation by controlling vascular matrix composition and vMC recruitment.

LPHN2 inhibits vascular permeability by differential control of endothelial cell adhesion.

Dynamic modulation of endothelial cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein-coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

Role of amino acid metabolism in angiogenesis.

The role of endothelial metabolism represents a crucial element governing the formation and the differentiation of blood vessels, termed angiogenesis. Besides glycolysis and fatty acid oxidation, endothelial cells rely on specific amino acids to proliferate, migrate, and survive. In this review we focus on the metabolism of those amino acids and the intermediates that hold an established function within angiogenesis and endothelial pathophysiology. We also discuss recent work which provides a rationale for specific amino acid-restricted diets and its beneficial effects on vascular tissues, including extending the life span and preventing the development of a variety of diseases.