REVIEWS The quiescent endothelium: signalling pathways regulating organ- specific endothelial normalcy Nicolas Ricard 1, Sabine Bailly2, Christophe Guignabert 3,4 and Michael Simons 1,5 ✉ Abstract | Endothelial cells are at the interface between circulating blood and tissues. This position confers on them a crucial role in controlling oxygen and nutrient exchange and cellular trafficking between blood and the perfused organs. The endothelium adopts a structure that is specific to the needs and function of each tissue and organ and is subject to tissue-specific signalling input. In adults, endothelial cells are quiescent, meaning that they are not proliferating. Quiescence was considered to be a state in which endothelial cells are not stimulated but are instead slumbering and awaiting activating signals. However, new evidence shows that quiescent endothelium is fully awake, that it constantly receives and initiates functionally important signalling inputs and that this state is actively regulated. Signalling pathways involved in the maintenance of functionally quiescent endothelia are starting to be identified and are a combination of endocrine, autocrine, paracrine and mechanical inputs. The paracrine pathways confer a microenvironment on the endothelial cells that is specific to the perfused organs and tissues. In this Review, we present the current knowledge of organ-specific signalling pathways involved in the maintenance of endothelial quiescence and the pathologies associated with their disruption. Linking organ- specific pathways and human vascular pathologies will pave the way towards the development of innovative preventive strategies and the identification of new therapeutic targets. 1Yale Cardiovascular The endothelium forms the innermost layer of blood ves­ of endothelial­ specific genes in different organs are Research Center, Department sels and lymphatic vessels and is best viewed as a multi­ specified during embryonic development and con­ of Internal Medicine, Yale 12 University School of Medicine, functional organ with both systemic and tissue­specific served during mitotic cycles . Transcriptome analysis of New Haven, CT, USA. roles. At the whole­organism level, the endothelium reg­ endothelial cells from different tissues revealed hetero­ 2Université Grenoble Alpes, ulates oxygen and nutrient supply, immune cell traffick­ geneous gene expression signatures even after several 1 2 INSERM, CEA, BIG- Biologie ing and inflammation , haemostasis and coagulation , passages in cell culture, indicating that tissue­ specific du Cancer et de l’Infection, vasomotor tone3, blood vessel permeability4 and epigenetic modifications participate in the regulation of Grenoble, France. angiogenesis5. In addition, the endothelium has a num­ organotypic transcriptomic profiles13,14. However, after 3INSERM UMR_S 999, ber of organ­ specific functions including regulation long­ term cell culture, which removes endothelial cells Pulmonary Hypertension: of organ size and function (myocardial hypertrophy6, from their in vivo microenvironment, approximately Pathophysiology and 7 8 15 Novel Therapies, Hôpital liver size and function , pulmonary alveolar repair and 50% of gene expression patterns are lost , and major Marie Lannelongue, Le kidney function9,10). architectural characteristics, such as fenestrae, also Plessis- Robinson, France. Given this heterogeneity of endothelial cell function, disappear16. 4Université Paris- Saclay, it is not surprising that studies show a remarkable het­ To characterize organotypic endothelial specificity Faculté de Médecine, erogeneity of gene expression profiles in endothelial cells as close to in vivo conditions as possible, many groups Le Kremlin- Bicêtre, France. from different organs11. Interestingly, these expression have utilized microarray or RNA sequencing (RNA­seq) 5 Department of Cell Biology, profiles are functionally matched to local tissue needs. of endothelial cells isolated by flow cytometry without Yale University School of 11,17 Medicine, New Haven, CT, USA. Microenvironment stimuli (shear stress, hypoxia and the cell culture step . Single­cell RNA­seq of endothe­ ✉e- mail: michael.simons@ the presence of specific growth factors, cytokines lial cells isolated from adult male mice identified tran­ yale.edu and hormones) and epigenetics define and continu­ scriptomic signatures of quiescent arterial, venous, https://doi.org/10.1038/ ously optimize local characteristics of endothelial cells. capillary and lymphatic endothelial cells in 11 differ­ s41569-021-00517-4 Epigenetic signatures that regulate the basal expression ent tissues11. Interestingly, lymphatic endothelial cells NATURE REVIEWS | CARDIOLOGY VOLUME 18 | AUGUST 2021 | 565 0123456789();: REVIEWS Key points has been given to the signalling events that main­ tain endothelial normalcy and quiescence. The latest • Quiescent endothelial cells require active maintenance to preserve normalcy in advances in this area are the subject of this Review. a tissue- specific manner. • Dysregulation of signalling pathways involved in endothelial normalcy maintenance FGF signalling leads to endothelial dysfunction and vascular pathologies. The fibroblast growth factor (FGF) signalling cascade • Endothelial quiescence and normalcy are important for disease resilience. includes a family of 18 ligands, four receptor tyrosine • Identification of organ- specific signalling pathways that maintain endothelial kinases (RTK) (FGFR1–FGFR4) and several accessory normalcy and quiescence will lead to new therapeutic targets supporting disease molecules such as Klotho proteins and syndecans22. resilience and treatment of associated vascular pathologies. After FGFs bind to their high­ affinity RTKs, several intracellular pathways are activated, including the from all the tissues cluster together, suggesting that the phosphoinositide 3­ kinase (PI3K)–AKT pathway and molecular signature of lymphatic endothelial cells is not mitogen­ activated protein kinase (MAPK) pathways tissue­specific. By contrast, arterial and venous endothe­ mediated by extracellular signal­ regulated kinase 1 lial cells from a specific tissue clustered together, show­ (ERK1) and ERK2 (REF.22). ing that vascular endothelial cell heterogeneity comes The extensive structural overlap and cross­ reactivity mainly from tissue specificity rather than arterial, capil­ among FGF ligands and receptors represent a real chal­ lary or venous identity. Moreover, capillary endothelial lenge to identifying the function of FGF signalling in the cells that are involved in gas, ion, metabolite and hor­ endothelium. Results from studies in mice with knock­ mone exchange between the blood and tissues have the out of individual Fgf genes or individual Fgfr genes are highest heterogeneity among tissues11. hard to interpret because of functional redundancy, Structural differences in the capillary endothelium whereas attempts to use FGFR chemical inhibitors are were first described in the 1960s with the use of elec­ hampered by the low specificity and cross­ reactivity tron microscopy18. Three major types of capillaries of these compounds. Successful strategies to circum­ exist (continuous, fenestrated and discontinuous). The vent the redundancy in the FGF family and investigate capillary type of an organ is related to its functions19. FGF signalling include mice with knockout of multiple Most organs have barrier­ forming, continuous capillar­ Fgfr genes (Fgfr1−/−Fgfr2−/− (REF.22) and Fgfr1−/−Fgfr3−/− ies (lungs, brain, skin and heart) with tightly connected (REF.23)), the use of soluble FGFR traps that target vari­ endothelial cells surrounded by a continuous basement ous FGF family members24, and endothelial cell­ specific membrane. This architecture permits diffusion of water, expression of a dominant­negative FGFR1 construct that small solutes and lipid­ soluble materials, while preclud­ can inactivate all four FGF receptors25. Mice with con­ ing the passage of cells or pathogens. By contrast, fenes­ ditional endothelial cell­ specific deletion of Fgfr1 and trated capillaries have intracellular pores (windows) with Fgfr2 are viable, with no vascular developmental defects a diaphragm and are found in renal glomeruli, exocrine and no alterations in vascular homeostasis26. However, glands, endocrine glands and intestinal mucosa. These postnatal endothelial cell­ specific knockout of Fgfr1 in fenestrae increase permeability to fluids and solutes, but mice with global knockout of Fgfr3 results in impaired not macromolecules20. Sinusoids are fenestrated capillar­ development of blood and lymphatic vessels23. A soluble ies with gaps instead of pores between endothelial cells receptor trap strategy was tested with the use of a solu­ and a thinner basement membrane than in continuous ble FGFR1 trap (sFGFR1) that binds to a large number or fenestrated endothelia and are present in the liver, of FGFs24. In this study, transient FGF inhibition was spleen and bone marrow. The gaps found in sinusoids achieved in vivo in mice via adenovirus­ mediated sys­ facilitate selective exchange of materials. temic expression of sFGFR1. This FGF inhibition led to Structural differences notwithstanding, normal an increase in vascular permeability and, eventually, pul­ endothelial cells everywhere are quiescent. This quies­ monary and myocardial haemorrhages, demonstrating cent state is defined by minimal or absent endothelial the necessity for FGF signalling in the maintenance of proliferation and migration, minimal or no vascular