vascular biology organotypic vasculature: from …augustin and koh, science 357, eaal2379 (2017) 25...

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VASCULAR BIOLOGY

Organotypic vasculature:From descriptive heterogeneityto functional pathophysiologyHellmut G. Augustin* and Gou Young Koh*

BACKGROUND: Each organ in the humanbody has its own capillary bed to carry out itsdistinctive andversatile functions in response todynamically changing systemic and local needs.Common and specific functions of the micro-vasculature in different organs are executedby organ-specifically differentiated endothelialcells (ECs). Themorphological differentiation ofECs into barrier-forming continuous ECs, fenes-trated ECs, and sinusoidal ECs has long beenrecognized. Nevertheless, the functional prop-erties and underlying molecular mechanismsof organotypic vasculatures have only been un-covered recently.

ADVANCES: This Review covers recent ad-vances in the biology of organotypically differ-

entiated microvascular beds. It describes thekey features of continuous, discontinuous, andsinusoidal ECs, as well as the more specializedECs of Schlemm’s canal and high endothelialvenules. Major transcriptional pathways ofEC specification and differentiation are out-lined, including GATA4 as a key transcrip-tion factor of sinusoidal EC differentiation.Themolecular shear stress–sensingmachinery—which transduces blood flow–mediated bio-physical forces that are essential to maintainthe quiescent, differentiated EC phenotype—isdelineated.In terms of function, this Review also dis-

cusses discoveries in different organs, includ-ing liver, lung, and bone, that have identifiedorganotypically differentiated ECs as a source

of paracrine (“angiocrine”)–acting cytokines,through which they exert active gatekeeperroles on theirmicroenvironment. ECs therebycontrol organ development, homeostasis, andtissue regeneration.On the basis of these general principles of

organotypic vascular differentiation and func-tion, this Review comprehensively covers re-cent landmark discoveries pertaining to theorganotypically specialized (micro)vasculature

in different organs. Fo-cusing on the molecularstructure-functionanalysisof organotypically differen-tiated (micro)vasculatures,it specifically highlightsthe properties of blood

vessels in the brain, eyes, heart, lungs, liver,kidneys, bones, adipose tissue, and endocrineglands. Emphasis is given to the contribu-tion of organotypically differentiated vascu-latures to both physiological organ functionand disease.

OUTLOOK:Research into themechanisms oforganotypic vascular differentiation and func-tion has emerged in recent years as a newbranch of vascular biology, with major impli-cations for our understanding of physiologicaland pathophysiological organ function. Ongoingresearch is aimed at deciphering, in muchhigher resolution (all the way to the single-cell level), the molecular microarchitecture

of organotypic vasculatures, under-standing the multicellular cross-talk through which organotypicvasculatures control their micro-environment, dissecting niche func-tions of organotypic vasculatureswith respect to stem cells andtheir progeny, and unraveling thefate maps of different organotypicvasculatures in health and disease.Future research will not only fo-cus on deciphering the molecularmechanisms and functional con-sequences of organotypic vasculardifferentiation, but will also aimto translate such knowledge for thedevelopment of novel organ- andvessel bed–specific angiotargetedtherapies for multiple diseases thathave hitherto been intractable.▪

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Augustin et al., Science 357, 771 (2017) 25 August 2017 1 of 1

The list of author affiliations is available inthe full article online.*Corresponding author. Email:[email protected] (H.G.A.);[email protected] (G.Y.K.)Cite this article as H. G. Augustin,G. Y. Koh, Science 357, eaal2379 (2017).DOI: 10.1126/science.aal2379

Organotypically differentiated vasculatures take center stage in vascular biology research. Blood vesselsin the body (clockwise from top left, vessels in the brain, retina, heart, adrenal gland, bone, and liver) come indifferent morphologies and have distinct organotypic characteristics that enable them to execute vessel bed–specific functions.They thereby act as gatekeepers of their microenvironments to actively control organ function.

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REVIEW◥

VASCULAR BIOLOGY

Organotypic vasculature:From descriptive heterogeneityto functional pathophysiologyHellmut G. Augustin1,2* and Gou Young Koh3,4*

Blood vessels form one of the body’s largest surfaces, serving as a critical interface betweenthe circulation and the different organ environments.They thereby exert gatekeeperfunctions on tissue homeostasis and adaptation to pathologic challenge. Vascular control ofthe tissuemicroenvironment is indispensable in development, hemostasis, inflammation, andmetabolism, as well as in cancer and metastasis.This multitude of vascular functions ismediated by organ-specifically differentiated endothelial cells (ECs), whose cellular andmolecular heterogeneity has long been recognized. Yet distinct organotypic functionalattributes and the molecular mechanisms controlling EC differentiation and vascular bed–specific functions have only become known in recent years. Considering the involvement ofvascular dysfunction in numerous chronic and life-threatening diseases, a better molecularunderstanding of organotypic vasculatures may pave the way toward novel angiotargetedtreatments to cure hitherto intractable diseases.This Review summarizes recent progress inthe understanding of organotypic vascular differentiation and function.

Reflecting the limited diffusion distance ofoxygen in tissues, every cell of the body is,with few exceptions (e.g., cartilage), within100 to 150 mm of the nearest capillary. Thevasculature thereby forms a systemically

disseminated organ. As a result, essentially allmedical disciplines are affected by research inthe field of vascular biology. However, vascularbiology research is somewhat fragmented andnot as coherently developed aswould be expectedgiven its importance for human health. In fact,it is still not widely appreciated that dysfunctionof the inner lining of blood vessels is the singlemost common cause of human mortality. Thedevastating consequences of hypertensive, ath-erosclerotic, coagulation-related, and pathologicangiogenesis–associateddiseases account formorethan two-thirds of deaths.The anatomical heterogeneity of blood vessels

in different organs has been recognized forcenturies, and research pursued during the past30 years has, in substantial detail, mapped themolecular repertoire of vessel wall–lining endo-thelial cells (ECs) in different vascular beds (1, 2).The functional properties and underlying molec-ular mechanisms of organotypically differenti-

ated ECs have, however, only been uncovered inrecent years. It is increasingly recognized thatECs do not just constitute a barrier-forming cellpopulation acting as a responsive interface; rather,they actively control their microenvironment asgatekeepers of organ development, homeostasis,and tissue regeneration (3). This paradigm shiftwill guide future research aimedat exploiting theorganotypic vasculature as a therapeutic targetfor a broad spectrum of vascular and organ dis-eases. This Review summarizes the latest findingsand advances in themechanistic understandingof organotypic vascular properties and function,highlighting key features of the vasculature indifferent organs in development, maintenance,homeostasis, and disease. It is not aimed atcomprehensively covering the vasculature of allorgans, but it instead focuses on organotypicvasculatures for which there have been majorand groundbreaking discoveries in recent years.We mostly focus on ECs, which should not de-tract from the fact that surrounding pericytesare increasingly recognized asmajor contributorsto organotypic vascular structure and function(4). This Review is restricted to the blood vascu-lar system and does not cover recent advancesin the field of lymphatic biology, including thelandmark discovery of a lymphatic system inthe brain (5, 6), which are reviewed elsewhere(7, 8).

Characteristics of organotypic capillaries

Capillaries have a diameter of 5 to 10 mmand arelined by a single layer of ECs. There are threemajor types of capillaries: continuous, fenes-trated, and sinusoidal (Fig. 1). Barrier-formingcontinuous capillaries exist ubiquitously in the

human body, except in epithelia and cartilage(Fig. 1A). The distinctive architecture of thesecapillaries permits the diffusion of water, smallsolutes, and lipid-soluble materials into thesurrounding tissues and interstitial fluid withoutany loss of circulating cells and plasma proteins.Larger molecules such as glucose and other nu-trients pass through the EC monolayer by trans-cytosis, a process regulatedby specific transporters.Continuous capillaries exist in a more specializedstate in most of the central nervous system; theECs are firmly bound together by tight junctionsbecause of the necessity of stricter permeabilitythat precludes most large molecules, drugs, andpathogens from passing.Fenestrated capillaries have intracellular pores

(“windows”) with a diaphragm that penetratethe endothelial lining (Fig. 1B). The pores notonly speed up the exchange of water, but alsopermit the passage of solutes as sizable as smallpeptides between plasma and interstitial fluid.This structure is observed with varying perme-ability and numbers of pores in the choroid plex-us of the brain; several endocrine organs such asthe pineal, pituitary, and thyroid glands; thehypothalamus; filtration sites in the kidneys; andabsorptive areas of the intestinal tract.Although closely resembling fenestrated capil-

laries, sinusoidal endothelium has gaps insteadof pores between ECs and is characterized byflattened and irregular shapes and inadequatecoverage by thinner basal lamina (Fig. 1C). Suchcharacteristics lead to free exchange of waterand provide a conduit for large solutes such asplasma proteins between plasma and interstitialfluid. Sinusoids are located in the liver, spleen,bone marrow, and several endocrine organs, in-cluding the pituitary gland and the adrenal me-dulla. Because of the requirement of extensiveexchange in these organs, the blood currentdecelerates in sinusoids to extend the time ofexchange across the sinusoidal barrier. In themeantime, phagocytic cells that are distributedalong the sinusoids of the liver, spleen, and bonemarrow act as wardens that detect and engulfexogenous pathogens, damaged cells, and debris.In comparison, some exchange may occur be-tween blood and interstitial fluid or aqueous fluidby bulk transport—the transcellularmovement ofvesicles that form through endocytosis (trans-cytosis) at the inner endothelial surface.Schlemm’s canal in the peripheral cornea, an

endothelium-lined channel sharing similaritieswith lymphatic vessels, enables sufficient aqueoushumor outflow from the anterior eye chambervia high-rate transcytosis with numerous giantvacuoles (Fig. 1D). High endothelial venules(HEVs), lined with specialized cuboidal ECs,serve as the entry point for lymphocyte migra-tion from the blood into lymph nodes. Somelymphocytes accumulate transiently in “HEVpockets” (Fig. 1E).

Transcriptional regulation of ECspecification and differentiation

Embryonic stem cells at the blastula stageform the primitive streak through a balance of

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Augustin and Koh, Science 357, eaal2379 (2017) 25 August 2017 1 of 12

1Division of Vascular Oncology and Metastasis, GermanCancer Research Center Heidelberg (DKFZ-ZMBH Alliance),69121 Heidelberg, Germany. 2Department of Vascular Biologyand Tumor Angiogenesis, Medical Faculty Mannheim (CBTM),Heidelberg University, D-67167 Mannheim, Germany. 3Centerfor Vascular Research, Institute for Basic Science, Daejeon,Republic of Korea. 4Graduate School of Medical Science andEngineering, Korea Advanced Institute of Science andTechnology, Daejeon, 34141, Republic of Korea.*Corresponding author. Email: [email protected] (H.G.A.);[email protected] (G.Y.K.)

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Wnt/b-catenin, activin/nodal, and BMP (bonemorphogenetic protein) signaling (9, 10), support-ing the differentiation into mesodermal pro-genitor cells. Mesodermal angioblasts give riseto ECs, which is primarily directed by VEGF/VEGFR2 (vascular endothelial growth factor/VEGF receptor 2) signaling. This is associatedwith the up-regulation of EC-specific markerssuch as endoglin, von Willebrand factor, CD31,VE-cadherin, TIE2, EPHB4, and ephrin B2 (11).Differentiated ECs undergo arteriovenous spec-ification by modulating VEGF concentrations (12);high concentrations of VEGF favor arterial speci-fication in a Delta/Notch–dependent manner(NRP1+, DLL4+, and CXCR4+), whereas lowerconcentrations aid venous commitment (NRP2+

and EPHB4+) (13). COUP-TFII (COUP transcriptionfactor 2) controls venous EC specification by sup-pressing Notch signaling (14), which supportsthe concept that venous EC differentiation maybe the developmental default pathway of vascu-lar differentiation. This is also supported by therecent finding that venous-derived angioblastsare the source for organ-specific vessels (15).Likewise, lymphatic specification occurs primar-ily in a PROX1-dependent manner from venousECs (16). TIE2 signaling induces COUP-TFII andvenous identity (17), suggesting that arterio-venous specification may in fact be controlledby the balance of VEGFR and TIE signaling,which is also supported by the observation thatVEGF stimulation down-regulates TIE2 expres-sion and up-regulates expression of the antag-onistic TIE2 ligand angiopoietin 2 (ANGPT2) (18).

Further analysis of EC differentiation path-ways has revealed that inhibition of transforminggrowth factor (TGF)–ß signaling increases VE-cadherin+ ECs and maintains the proliferationand vascular identity of differentiated ECs bysustaining ID1 expression (19). Likewise, Indianhedgehogmediates vasculardifferentiation throughBMP4 signaling (20). ID1 is a helix-loop-helixtranscription factor promoting EC expansion andmaintaining long-term vascular identity. Globalgene expression analysis of developing vascula-ture during mouse embryonic stem cell differen-tiation supports a role of Wnt signaling not justin EC specification but also in EC differentiationand maturation (21). In fact, noncanonical Wntsignaling is required to stabilize the immaturevasculature before it becomes quiescent (22, 23).ETS and forkhead transcription factors are alsoinvolved in driving embryonic EC developmentand differentiation. The ETS transcription factorsETV2, FLI1, and ERG1 act early to specify plu-ripotent cells toward the EC lineage (24). ETV2alone drives vasculogenesis, whereas ETV2 andFLI1 act redundantly during angiogenesis (25).ERG1 promotes vascular differentiation andstability through Wnt/ß-catenin signaling (26).Forkhead transcription factors act later asmodulators of differentiated EC function. Theycontrol EC survival signaling through the PI3K(phosphatidylinositol 3-kinase)/AKT pathwayand couple EC metabolism with their growthstate (27, 28).The transcriptional machinery of early embry-

onic differentiation is overall relatively well un-

derstood, but much needs to be learned aboutthe interplay between developmental vasculartranscription factors. Similarly, little is knownabout the transcriptional programs driving organ-specific EC specification and differentiation.Recently, the transcription factor GATA4 wasidentified as a master regulator of hepatic micro-vascular specification and acquisition of organ-specific vascular competence (29). GATA4mediatesthe down-regulation of continuous EC-associatedtranscripts and up-regulates sinusoidal EC genes.This landmark discovery was only made possiblethrough the generation and use of Stab2-Cremicefor the conditional deletion of GATA4 selectivelyin sinusoidal ECs (29). The functional analysis ofmolecules controlling organotypic EC differenti-ation is hampered by the lack of Cre driver micefor specific subpopulations of ECs.

Flow-dependent maintenance of theorganotypic vasculature

Blood flow–mediated biophysical forces, erro-neously called shear “stress,” are essential tomaintain the quiescent, differentiated EC phe-notype (30, 31). Shear stress–inducednitric oxide(NO) production and release was identifiedmorethan 20 years ago as a regulator of vascular toneand thereby a contributor to vessel maintenance(32, 33). More recently, hemodynamic forceshave been shown to control hematopoiesis in aNO-dependent manner (34, 35).Substantial progress has been made in re-

cent years in the molecular characterization ofthe shear stress–sensing machinery. ECs convert


Fig. 1. Three major and two specialized types of capillaries. Endothelialcells (ECs) are shown in light pink, encircled in black. (A) Barrier-formingcontinuous capillaries are found inmost organs, including the brain and retina.Solutes are transported through the ECmonolayer by regulated transcytosis.(B) Fenestrated capillaries have intracellular pores that are covered witha diaphragm; this structure is found in the endocrine glands, intestine,and kidneys, rendering them permeable by fluid and small molecules.(C) Sinusoidal capillaries have larger intercellular gaps and a discontinuous

basement membrane, allowing free exchange of materials; this structure isgenerally found in the liver, bone marrow, and spleen. (D) Schlemm’s canalenables sufficient aqueous humor outflow from the anterior eye chamber bymeans of high-rate transcytosis with numerous giant vacuoles. (E) Highendothelial venules (HEVs), linedwith cuboidal ECs, serve as an entry point forlymphocyte migration from the blood into lymph nodes. Some lymphocytesaccumulate transiently in “HEV pockets.” The orange dashed arrowsindicate the main directions of transcytosis, flow, or lymphocyte migration.

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mechanical stimuli into biochemical signalsthrough mechanotransducers, including recep-tor tyrosine kinases, ion channels, integrins, andjunctional proteins (VEGFR2, PECAM-1, and VE-cadherin). VEGFR3 plays a role in the mechano-sensory complex to determine a preferred levelof fluid shear stress, or “set point,” for differenttypes of vessels (arteries, veins, and lymphatics)(36). PIEZO1 and syndecan 4 are flow sensors ofECs (37). PI3K/AKT and MAPK/ERK (mitogen-activated protein kinase) signaling pathways ac-tivate flow-dependent transcription factors KLF2(Krüppel-like factor 2) and NRF2 (nuclear factorerythroid 2–like 2) to maintain endothelial phe-notypes (38, 39) and metabolic state (40). Re-cently, Hippo signaling–mediated transcriptionalactivities of YAP (Yes-associated protein) and TAZ(transcriptional coactivator with PDZ-bindingmotif) have emerged as EC regulators of lam-inar versus turbulent flow (41). Accordingly, theatheroprotective effects of unidirectional lam-inar flow are mediated by integrin-dependentinhibition of YAP/TAZ-JNK (c-Jun N-terminalkinase) signaling (41).

Perfusion-independent functions oforganotypic endothelium

It is increasingly recognized that capillary ECsare not merely passive conduits for the deliveryof oxygen or nutrients, but also support organdevelopment andadult organ regeneration throughelaboration of tissue-specific paracrine growthfactors, called angiocrine factors or angiokines(3). For example, liver sinusoidal ECs up-regulateHGF (hepatocyte growth factor) and down-regulate TGFß after partial hepatectomy, therebycontrolling liver regeneration in a paracrineman-ner (42, 43). Sinusoidal ECs of bone marrowsecrete Notch ligands and IGFBPs (insulin-likegrowth factor–binding proteins) for reconstitut-ing hematopoiesis after chemotherapy and ir-radiation (44). Lung capillary ECs produceMMP14(matrix metalloproteinase 14) after pneumonec-tomy, which unmasks cryptic EGF (epidermalgrowth factor)–like ligands and promotes regen-erative alveolarization by stimulating the prolif-eration of epithelial progenitor cells (45). Duringtumor progression, ECs create distinct vascularniches that have instructive roles in promotingtumor growth at the primary and metastaticsites (46). Such tumor EC–derived activity maycontext-dependently act in a pro- and antitumor-igenicway. For example, tumorEC–derived IGFBP7blocks IGF1 and thereby inhibits the expansionof IGF1 receptor–expressing tumor stemlike cells,whereas chemotherapy suppresses EC produc-tion of IGFBP7, thereby inducing a feedforwardloop that converts naive tumor cells to chemo-resistant tumor stem cells (47). A similar EC-controlled feedforward mechanism has beenshown to drive glioblastomamultiforme progres-sion through the hypoxia-independent inductionof HIF-1a (hypoxia-inducible factor 1a) by phos-phorylation of profilin-1 (48).Molecular analysis of EC gatekeeper functions

in the microenvironment is still in the earlystages, andmuchneeds tobe learned. For example,

what is the role of aberrant production ofangiokines by locally diseased ECs in the controlof organ function? How do differences in youngand agedECs contribute to changes in angiocrinesignaling during aging?Howare epigeneticmech-anisms such as epigenetic memory involved inthe regulation of EC angiocrine functions?Recentadvances in omics techniques allowing the pro-filing of single-cell transcriptomes (49) makeit possible to address these questions, whichwill generate insights into the mechanisms andfunctional consequences of organotypic EC dif-ferentiation at a hitherto unparalleled level ofresolution.

Blood-brain barrier–forming vasculature

Supported by pericytes and astrocytes, the brainendothelium forms a particularly tight layercalled the blood-brain barrier (BBB) (50, 51).ECs of the BBB have specialized tight inter-cellular junctions, no fenestrae, and an extreme-ly low rate of transcytosis. These special featuresprotect the brain from harmful substances butare also an obstacle for the delivery of thera-peutic drugs into the brain. BBB ECs expressspecialized molecular transporters and receptorssuch as GLUT-1 (glucose transporter type 1) andmembers of the ABC (adenosine triphosphate–binding cassette) transporter family. For exam-ple, docosahexaenoic acid (DHA) is an omega-3fatty acid essential for normal brain growth andcognitive function. It cannot be de novo syn-thesized in the brain and must be transportedacross the BBB. Although the mechanisms forDHA uptake into the brain had been enigmaticuntil recently, MFSD2A has been identified asthe major transporter (52), as well as a negativeregulator of transcytosis (53).The formation and maintenance of the BBB

require several specific molecules, includingGPR124 (G protein–coupled receptor 124) (54, 55).GPR124 functions as a coactivator of Wnt7a- andWnt7b-stimulated canonical Wnt signaling via aFrizzled receptor and LRP co-receptor acting inconcert with Norrin/Frizzled4 signaling to con-trol vascular development of the central nervoussystem and BBB maintenance (56). Ablation ofNorrin/Frizzled4 signaling results in BBB defects,which are ameliorated by stabilizing b-catenin (57).BBB breakdown is a hallmark of several neuro-

logical disorders including ischemic stroke,multiplesclerosis, and Alzheimer’s disease (58–60). Recentadvances in the pathogenesis of cerebral cavernousmalformations (CCMs) have provided importantinsights into BBB maintenance and breakdownmechanisms. CCMs have a prevalence of up to0.5% in the general population. Loss of functionin any of the three genesKRIT1 (CCM1),CCM2, andPDCD10 (CCM3) causes familial CCMs. Mechanis-tically, mutations in the three nonhomologousgenes converge in the gain of MEKK3 (MAPK/ERK kinase kinase 3)–KLF2/4 signaling in earlyCCM lesions, perturbing the quiescent EC pheno-type (61). EC quiescence during CCM progressionis also controlled by the vascular destabilizingfunctionsofANGPT2,whoseUNC13B- andVAMP3(vesicle-associatedmembraneprotein 3)–dependent

exocytosis is actively suppressed by PDCD10 (62).Vascular destabilization induced by EC-specificdeletion of KRIT1 (CCM1) promotes endothelial-to-mesenchymal transition through up-regulationofBMP6,TGFb, andBMPsignaling (63). Endothelial-to-mesenchymal transition is a result of KLF4transcriptional activity, which promotes TGFb/BMP signaling through the production of BMP6(64). Recently, ECTLR4 (Toll-like receptor 4) andthe gut microbiome were identified as criticalupstream stimulants of CCM formation (65).

Blood vessels in the eye

Dysfunction and concomitant angiogenesis ofthe retinal vasculature is a major cause of blind-ing diseases, including diabetic retinopathy andage-related macular degeneration (AMD). Inmice, retinal vessels grow postnatally in a highlyorganized pattern, allowing the study of physi-ological developmental angiogenesis in a quasi–two-dimensional setting. This model has beenwidely used to decipher the molecular and cel-lular mechanisms of sprouting angiogenesis, tipand stalk cell formation, artery versus vein spec-ification, and vascular remodeling and pruning.Retinal vessels exist as superficial, intermediate,and deep vascular plexuses (Fig. 2A). Upon for-mation of the superficial plexus, vertical sproutinginto the deep layers initiates, sequentially form-ing intermediate and deep vascular plexuses. Thehypoxia-driven VEGF gradient acts as an angio-genic cue that stimulates tip cells to extend,along with preformed astrocytes, during post-natal development. Premature babieswho receiveoxygen therapy often develop retinopathy of pre-maturity as a result of disorganized vessel growthsuch as vasoregression and abnormal angiogen-esis (Fig. 2A). EC-derived Wnt ligands and FGD5are closely involved in vascular pruning and re-gression (22, 66) (Fig. 2A). In contrast, integrinavb8 expressed in astrocytes and SLIT2 secretedfrom surrounding neuronal cells stimulate andstabilize the three retinal vascular plexuses (67–69).Surrounded by pericytes, glial cells, and neurons,the retinal vessels form the blood-retinal barrier(BRB), which prevents the diffusion of toxic com-pounds and microorganisms and proinflam-matory leukocytes from surrounding tissues.Patients with diabetic retinopathy exhibit mul-tiple hallmarks of vasculopathies such as pericytedropout, microaneurysm, nonperfused vessels,and abnormal angiogenesis in the retina (Fig. 2A).Inadequate pericyte coverage impairs retinalvascular growth and BRB function, leading toANGPT2 up-regulation and sensitization toVEGF, which triggers the progression of BRBbreakdown (70).The choroid has the largest blood flow by

weight of all organs andan extremely anastomosedcapillary network called the choriocapillaris. ECsof the choriocapillaris have numerous fenestra-tions that permit the passage of large moleculesinto the extravascular space. The distribution ofchoroidal pericytes is extremely diverse, rangingfromcomplete coverage inprimaryarteries to sparsedispersion in the choriocapillaris (71). Althoughthis distributionmay contribute to the structural



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plasticity of the choriocapillaris in response tophysiological changes, sparse pericyte ensheath-ment could be related to neovascular (NV-) AMD.NV-AMD is characterized by choroidal neo-vascularization, an outgrowth of leaky vesselsfrom the choriocapillaris, penetrating throughBruch’smembrane,which gradually disintegratesduring AMD progression; this leads to serous orhemorrhagic leakage, retinal edema, and subse-quent visual dysfunction (Fig. 2B). In NV-AMD,loss of the choriocapillaris causes ischemia inretinal pigment epithelium (RPE), followed bythe production of hypoxia-inducible angiogenicfactors, including VEGF, in RPE and adjacentcells (72). Consequently, VEGF blockade is thetreatment of choice at present for NV-AMD.VEGF is, however, also required to maintain thechoroidal vasculature, and repeated treatmentwith VEGF blockers could aggravate hypoxiaand oxidative stress by inducing damage of thechoriocapillaris, leading to vision loss caused bydysfunction of photoreceptors (73). The numberof patients with NV-AMD is rapidly increasing.Thus, safer and more effective treatments areneeded.Corneal avascularity is essential for the cor-

nea’s transparency and unperturbed vision.Avascularity is presumably due to an environmentrich in antiangiogenic and antilymphangiogenicmolecules, including truncated soluble VEGFR1,VEGFR2, and VEGFR3 secreted from surround-ing cells (74, 75). Corneal neovascularization, theexcessive ingrowth of blood and lymphatic vesselsfrom the limbus, occurs in several pathologic con-ditions, such as herpes simplex stromal keratitis,contact lense–induced keratitis, and graft rejec-tion after keratoplasty (76) (Fig. 2C). Althoughanti-VEGF therapy has shown promising effectsin patients with corneal neovascularization, fur-ther investigation is needed to limit cornealscarring and loss of transparency.Schlemm’s canal is an endothelium-lined

channel that encircles the cornea and providesa specialized vascular bed for aqueous humorflow, which constantly refreshes the anterior eyechamber between the cornea and the lens (Figs.1D and 2D). Aqueous humor is produced by theciliary body and is drained through a trabecularmeshwork into Schlemm’s canal and aqueousand episcleral veins. Schlemm’s canal shares mor-phological and functional attributes of lymphaticvessels (77–79). Molecularly, Schlemm’s canal isan intermediate vessel type between lymphaticand blood vessels, expressing PROX1, VEGFR3,and integrin a9, but not LYVE-1 or podoplanin(77–79). The primitive Schlemm’s canal originatesfrom the choroidal vein, and Schlemm’s canalendothelium is postnatally respecified to acquirelymphatic traits by up-regulating PROX1 (79).Schlemm’s canal dysfunction, including reducedgiant vacuole formation, results in impaired aque-ous humor drainage and increased intraocularpressure, ultimately leading to glaucoma (Fig. 2D).Intriguingly, primary congenital glaucoma is alsodetected in mice with genetic deletion of Angpt1and Angpt2 or deletion of the angiopoietin recep-tor gene Tie2 (80). Tie2 mutations have been


Fig. 2. Features of eye-specific vasculatures and related diseases. (A) Retinal vessels constitutea highly organized network with superficial (s), intermediate (i), and deep (d) vascular plexi.Patients with retinopathy of prematurity (ROP) exhibit both vascular regression and abnormalangiogenesis, whereas those with diabetic retinopathy (DR) exhibit multiple hallmarks of vasculopathiesin the retina. On the right, green lines along red blood vessels indicate pericytes. (B) An outgrowthof leaky vessels from choriocapillaris is a typical feature of neovascular aged-related maculardegeneration (NV-AMD), leading to retinal edema and vision loss. RPE, retinal pigment epithelium.(C) Abnormal outgrowth of blood (red) and lymphatic (green) vessels into the avascular, transparentcornea can be seen in patients with keratitis or corneal graft rejection. (D) Dysfunction ofSchlemm’s canal. Reduced giant vacuole (GV) formation causes impaired aqueous humor drainageand increased intraocular pressure, leading to open-angle glaucoma. Blue lines in boxes indicatethe direction and relative amount of aqueous humor flow.


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identified in patients with primary congenitalglaucoma (81), potentially paving the way fornovel therapeutic strategies for glaucoma.

Heart vasculature

The origin of cardiac capillary ECs has been thesubject of intense study in recent years. Theproepicardiumwas proposed as the source of car-diac capillaries, but this has been challenged byrecent lineage-tracing analyses in mice. Only asubset of the proepicardium contributes to asmall portion of cardiac capillaries (82–84), whereasthe sinus venosus and the endocardium serve asprimary sources. The sinus venosus provides ECprogenitors for cardiac capillaries, which furthercontribute to a substantial portion of the cardiaccapillary bed in the lateral free walls of the ven-tricles (85–87) (Fig. 3, A and C). Endocardialprogenitors give rise to cardiac capillaries withinthe ventricular septum and ventral wall of theembryonic heart (87, 88) (Fig. 3B). Interestingly,endocardial cells are converted to ECs of newlyformed cardiac capillaries in the inner ventricu-lar wall during trabecular compaction in the neo-natal heart (89).The molecular mechanisms of cardiac vascu-

lar morphogenesis remain poorly understood.

Myocardium-derived VEGF regulates the endo-cardial contribution to the origin of cardiaccapillaries (88). Epicardial VEGF-C promotessprouting angiogenesis from the sinus venosus(87). Moreover, myocardium-derived ANGPT1controls coronary vein formation (90). Muralcells [pericytes and smoothmuscle cells (SMCs)]provide structural support to adjacent endothe-lium and maintain vessel lumen. Unlike cardiaccapillary ECs, the majority of cardiac mural cellsare derived from epicardial cells (82, 83). Lineagetracing has also revealed that cardiac pericytesdevelop into SMCsof coronary arteries in responseto Notch signaling during arterial remodeling;this study also showed the presence of undiffer-entiated pericytes in the adult heart, which canbe used to produce SMCs during the regenerationof coronary arteries (91).Coronary atherosclerosis causes myocardial

infarction and subsequent heart failure, one ofthe leading causes of death worldwide. Athero-sclerotic plaques with neointima formation arethe most prominent pathologic substrate. Yetgrowing evidence suggests that ECs play a piv-otal role in the pathogenesis of atherosclerosis(92, 93). Laminar shear stress maintains endo-thelial quiescence and suppresses atherosclerosis

through activation of KLF2, which induces endo-thelial NO synthase (eNOS) and thrombomodulin(94). KLF2 coordinates atheroprotective commu-nication between ECs and SMCs by regulatingmicroRNA-143 (miR-143) and mi-145 expression,thereby controlling SMCs (95). Endothelial miR-126 limits atherosclerosis by targeting the non-canonical Notch ligand DLL1 (96). Unidirectionallaminar shear stress also induces phosphorylationand suppression of YAP/TAZ through integrinsignaling (41). Disturbed flow accelerates theprogression of atherosclerosis by transcriptionalactivation of YAP/TAZ signaling and proinflam-matory gene expressions, which can be reversedby YAP/TAZ inhibition (41).Alterations of EC metabolism are critical for

atherosclerosis progression (97). Forkhead box O(FOXO) transcription factors are AKT substratesregulating cell growth, differentiation, andmetab-olism. FOXO1 couples the metabolic activity ofECs to their growth state (27). ECmetabolism, inturn, is strongly influenced by systemic metabo-lism; for example, insulin resistance activatesendothelial FOXO1 and FOXO3, thereby augment-ingproinflammatory cytokineexpression,decreasedNO production, and disturbed atheroprotectiveinsulin signaling (98). Conversely, enhancing the


Fig. 3. Development of the cardiac capillary bed. (A) Migrating ECs fromthe sinus venosus form a capillary bed over the dorsal surface of the growingheart and infiltrate into the myocardium of the ventricular wall (C).(B) Endocardial cells contribute to the establishment of most coronary

vessels in the ventricular septum. (C) During capillary invasion into themyocardium, a subset of the capillary bed develops into coronary arteries,which are covered by pericytes that differentiate into smooth musclecells (SMCs). The majority of pericytes are derived from epicardial cells.


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insulin sensitivity of ECs leads to a paradoxicaldecline in EC function, resulting in a proathero-sclerotic imbalance of NO and superoxide causedby increased tyrosine phosphorylation of eNOSand excess NOX2-derived superoxide (99).

Blood-gas barrier in the lungs

The circulatory system of the lungs is composedof a thin layer of capillary ECs underlying anexpansive surface of alveolar epithelial cells.The development of the lungs requires precisetemporal and spatial organization of capillaryECs and alveolar epithelial cells. Proper capillarydensity and positioning is specified early in de-velopment and is required for viability at birth(100). Angiocrine factors secreted fromprimitivelung capillaries contribute to specifying endo-derm and mesoderm progenitor differentiationinto primitive lung epithelial and vascular pre-cursor cells (101, 102). Likewise, BMP4-BMPR1Asignaling triggers calcineurin/NFATc1–dependentexpression of TSP1 (thrombospondin 1) in lungECs to drive alveolar lineage–specific differenti-ation of bronchoalveolar stem cells (103). Duringpostnatal development of the lung, a high level ofvascular refinement, remodeling, and matura-tion is imperative to determine the tissue-specificarchitecture of branched organs, which is inde-pendent of perfusion, flow, or blood-borne sub-stances (104).Defective lung vascular development can result

in alveolar capillary dysplasia (ACD) and bron-chopulmonary dysplasia (BPD). ACD is a lethaldisorder in humans characterized by a failure ofalveolar capillary formation, often accompaniedby misalignment of pulmonary veins. BPD is achronic lung disease affecting premature infants(<1000 g) that is caused by impaired alveolarizationand dysmorphic vascular development associ-atedwith prematurity (105). The development oftherapeutic strategies to repair the respiratorycapacity in patients with lung disorders is hand-icapped by the limited understanding of lungregeneration mechanisms. Reconstitution of thealveolar-capillary interface is pivotal for adultlung regeneration (45). Transplantation of prop-erly activated lung capillary ECs or injection oflung-specific angiocrine mediators could there-fore emerge as a therapeutic modality to driveepithelial cell repopulation and improve respira-tory function (3, 45).

Liver vasculature

Endowed with two separate, albeit connected,vascular systems—the arterial and the portalvasculature—the liver has a distinctive vascularsupply. The arterial system primarily serves nu-tritive purposes, whereas the portal system feedslipid droplet–rich, poorly oxygenated blood fromthe intestine to the liver. Both systems drain bloodthrough the sinusoidal vasculature (Figs. 1C and 4).The sinusoidal vasculature has a characteristicmorphology,with fenestrations grouped into sieveplates, specialized junctional complexes, and anincompletebasementmembrane. Sinusoidal fenes-trae allow free flow of blood (except cells) into thespace of Disse, with direct access to the surface

of hepatocytes. They also enable T lymphocytesto send foot processes into the space of Disse,allowing immunosurveillanceof hepatocyteswith-out extravasation (106).Liver sinusoidal ECs (LSECs) make up a het-

erogeneous cell population (107) that servesimportant scavenger functions, clearing macro-molecular wastemolecules from the circulation.LSECs also act as antigen-presenting cells andmediate clearance of immune complexes, viruses,lipopolysaccharides, and other molecules (108).These functions are executed by different scav-enger receptors [e.g., STAB1 and STAB2 (stabilin1 and 2)] and other molecules mediating highlyefficient endocytosis (109). Perturbation of scav-enger receptor function (e.g., by genetic deletionof STAB1 and STAB2) has systemic consequencesand causes glomerulofibrotic nephropathy (110).The liver vasculature has been extensively stud-

ied for its ability to control organ function in anangiocrine manner. During embryonic develop-ment, paracrine signals from ECs control earlysteps of organogenesis (111). The transcriptionfactor GATA4 drives LSEC specification, whichis an indispensable requirement not just for nor-mal liver development, but also for liver coloniza-tion by hematopoietic progenitor cells.Mice withtargeted deletion of GATA4 in LSECs die from ane-mia in late embryonic development (29). Likewise,LSEC-expressed PLVAP (plasmalemma vesicle–associated protein) is essential for the seeding offetal monocyte-derivedmacrophages to tissues inmice. PLVAP formsdiaphragms in the fenestrae ofliver sinusoidal endothelium during embryogen-esis and selectively regulates the egress of fetallivermonocytes to the systemic vasculature (112).Lineage-tracing experiments in adult mice

have identified AXIN2-positive pericentral hep-atocytes as a self-renewing reservoir of cells thatreplace hepatocytes during homeostatic renewal.Proliferation of these cells is controlled by Wntligandsproducedby central veinECs (113). Centralvein ECs are also the primary source of theWntsignaling enhancer RSPO3, thereby controllingliver zonation (114). It remains to be seen howmorphogeneticWnt signaling gradients originatefrom the central vein against the primary direc-tion of blood flow, but the retrograde flow in thespace of Disse is likely involved in this process(Fig. 4). As a result, hepatocytes receive theircharacteristic zonal alignment along the sinus-oids, which has beenmapped at single-cell reso-lution to reveal that around 50% of liver genesare nonuniformly expressed, in line with this zo-nation (115). Future complementary single-LSECmapping will enable the construction of a bio-informatic signaling cross-talk map to shed newlight on the molecular mechanisms of stromal-parenchymal interaction. For example, BMP2secreted by LSECs acts on hepatocytes to controllocal and systemic iron metabolism (116). Thisexample likely reflects only the tip of the iceberg,and it can be expected that numerous other EC-derived instructive gatekeeper functions will beidentified in the future.Beyond homeostatic maintenance, LSECs reg-

ulate the response to liver damage (117). Partial

hepatectomy leads to rapid global proliferationof the remaining hepatocytes. Angiocrine signalsincluding HGF and Wnt2, secreted from LSECsbefore the induction of angiogenesis, stimulatehepatocyte proliferation (118, 119). Similarly, down-regulation of ANGPT2 in LSECs after partialhepatectomy leads to down-regulation of LSECTGFß production (43). TGFß is a potent neg-ative regulator of hepatocyte proliferation. Assuch, LSEC down-regulation of TGFß enablesliver regeneration by removing an angiocrinebreak. Intriguingly, recovery of LSEC ANGPT2production during the later steps of liver regen-eration controls LSEC VEGFR2 expression in anautocrine manner, thereby facilitating the angi-ogenic response to hepatocyte-derived VEGF (43).As such, the dynamic temporal regulation of asingle LSEC-derived cytokine controls hepatocyteand LSEC proliferation as a dynamic rheostat atdifferent stages of liver regeneration.Long-term liver damage results in fibrosis,

which is a major risk factor for the developmentof hepatocellular carcinoma (HCC). Fibrosis andHCC lead to LSEC transdifferentiation with lossof LSEC markers and sinusoidal fenestrae, aprocess known as capillarization, during whichLSECs lose their protective properties andpromoteangiogenesis and vasoconstriction (120, 121). Asa result, LSEC dedifferentiation is associatedwith a switch from vessel co-option and intus-susceptive angiogenesis to sprouting angiogen-esis (122, 123). LSEC-derived angiocrine signalscontribute to regulating the balance betweenliver regeneration and fibrosis (42). The regu-latory loops between the vascular and parenchy-mal compartments are, however, not restrictedto LSEC-hepatocyte interactions, but also involvehepatic stellate cells (HepSCs), which are thespecialized pericytes of the liver. For example,HepSC-expressed endosialin promotes fibro-genesis and reduces hepatocyte proliferationduring liver damage (124). Conversely, activatedHepSCs limit HCC progression in an endosialin-dependent manner (125).

Kidney microvasculature

The renal vasculature supports kidney functionin distinctly different compartments, includingglomeruli, cortical peritubular capillaries, andvasa recta bundles. Correspondingly, differenttypes of capillaries are found in different partsof the kidney. Glomerular ECs are the prototypeof fenestrated endothelium. For efficient filtra-tion of serum to Bowman’s space, glomerularcapillaries form compact loops to maximize thecontact surface of blood flow to the glomerularfiltration unit. The basement membrane ofglomerular ECs is fused to that of podocytes tofacilitate filtration, whereas the outer surface ofglomerular ECs is wrapped by foot processesof podocytes to form an additional barrier offiltration. VEGF/VEGFR2 signaling is essentialfor embryonic development and maintenanceglomeruli. VEGF blockademay cause proteinuriaand thrombotic microangiopathy (126). Increasedlevels of VEGF have been detected in diabeticnephropathy, although whether VEGF actually



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contributes to disease progression remains in-conclusive (127). Moreover, VEGF secreted fromproximal tubules contributes to themaintenanceof peritubular capillaries and blood pressure(128). Soluble extracellular domain of VEGFR1(sFlt1) is produced by glomerular podocytes anddynamically regulates local VEGF by acting asa decoy receptor of VEGF (129). Inhibition ofpodocyte-secreted sFlt1 causes disintegration ofthe glomerular barrier, leading to proteinuria andrenal failure such as nephrotic syndrome as aresult of cytoskeleton reorganization in podocytes(129). In addition to VEGF/VEGFR2 signaling,ANGPT1/TIE2 signaling is critical for glomerulardevelopmentandmaintenance.Genetic inactivationof ANGPT1 results in dilated glomerular capillaryloops (130). ANGPT1 secreted by podocytes andmesangial cells acts protectively against diabetickidney injury (131).

Bone vasculature

Long bones are highly vascularized, except inthe growth plate and articular cartilage. Thevasculature has a hierarchical organization, witharterial feeding into a capillary network anddrainage into a large central vein in the diaphy-sis. Nutrient arteries penetrate the medullarycavity through the bone cortex and run towardthe metaphysis as interconnected columnar cap-illaries. Columnar capillaries make an arch nearthe growth plate, forming a sinusoidal networkthat drains into the central vein. Endochondral os-

sification is an example of angiogenic-osteogeniccoupling at the primary ossification center (embry-onic day 14 inmice), growth plate, and secondaryossification center (postnatal day 6 in mice)(Fig. 5A).Endochondral ossification begins with co-

invasion of preexisting cartilage template byosteoclasts, blood vessels, and osteoprogenitors(132). The hypoxic environment of avascularcartilage induces HIF signaling in hypertrophicchondrocytes (Fig. 5A). This initiates the expres-sion of target genes regulating angiogenesis,cellular survival, proliferation, and extracellularmatrix (ECM)production (133).MMPs, particularlyMMP9 andMMP13, are secreted from osteoclastsand ECs to degrade ECM and enhance VEGFsignaling. Because ECs and osteoblastic-lineagecells express VEGFR2, VEGF-mediated signalingpromotes migration, proliferation, and survivalof these cell types (134, 135). Conversely, angiocrinesignals from ECs (e.g., Noggin and Wnt ligands)regulate chondrocytematurationandosteogenesis(136, 137) (Fig. 5B). Interestingly, DLL4-Notchsignaling in the bone enhances angiogenesis andosteogenesis (44), whereasNotch activity inhibitsangiogenesis in other organs. Specification ofbone ECs entails distinct cell-matrix interactionsinvolving a specialized EC subtype, termed typeE, which supports osteoblast lineage cells andlater gives rise to other EC subpopulations (138).Adult bone capillaries can be divided into

two types, H and L, on the basis of their marker

expression and function (136) (Fig. 5B). ECs intype H vessels express high levels of CD31 andendomucin. Type H vessels are primarily in themetaphysis and endosteum,where they are closelyassociated with PDGFRß (platelet-derived growthfactor receptor ß)–expressing perivascular cellsand osterix-expressing osteoprogenitor cells. Incontrast, ECs of type L vessels express lowerlevels of CD31 and endomucin. Type L vesselsare highly branched and form the sinusoidalvascular network in the diaphysis, where theyare surrounded by hematopoietic cells (139).Because arteries exclusively supply type H vessels,type L vessels receive blood from type H vessels;thus, each type of vessel has a distinct metabolicenvironment. Type H vessels in the metaphysisand endosteum are relatively well oxygenated,whereas type L vessels in the diaphysis are in alow-oxygen environment because of the lack of adirect arterial supply (44, 136). The blood velocityis also higher in typeHvessels, suddenly droppingafter the branching point in type L sinusoidalvessels (140). Variations in oxygen tension andpermeability create distinctly different micro-environments for hematopoietic stemcells (HSCs)in the bone marrow. Less permeable arterialblood vessels maintain HSCs in a state withlow reactive oxygen species, whereas the morepermeable sinusoids promote HSC activationand serve as sites for immature and matureleukocyte trafficking to and from the bonemarrow (141).


Fig. 4. Positional relationship between blood vessels and hepato-cytes in the liver. Lipid droplet–rich blood from the portal vein (PV) mixeswith blood from the hepatic artery (HA) and flows through the sinusoidalvasculature into the central vein (CV). Fenestrated liver sinusoidal ECs(LSECs; inset) are separated by the space of Disse from the hepatocytes,

which are zonated along the sinusoidal vasculature. Liver zonation iscontrolled by EC-derived signals involving Wnt ligands and the Wntsignaling enhancer RSPO3. Liver regeneration is controlled by angiocrinesignals secreted from ECs. BD, bile duct; Hep, hepatocyte; KC, Kupffercell; HepSC, hepatic stellate cell.


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Bone undergoes constant remodeling that isregulated by the balanced activity of osteoblasts(bone formation) and osteoclasts (bone resorp-tion). Disruption of this balance may cause age-related and disease-associated bone losses that

increase the risk of fracture (142). Bone resorp-tion increases with aging, primarily as a resultof hormone changes, whereas bone formationgradually decreases. In addition, type H vesselsand perivascular osteoprogenitors are reduced

in aged animals without a substantial reductionin the total number of ECs (136). Decreasedblood flow in aged mice may be associated withdecreased bone formation and loss of type Hvessels (Fig. 5B). Furthermore, type H vessels


Fig. 5. Bone vasculatures. (A) Angiogenic-osteogenic coupling duringendochondral ossification. At embryonic day 14 (E14) in mice, hypertrophicchondrocyte (HC) in the primary ossification center (POC) initiatesexpressions of HIF-signaling target genes including Vegf and producesextracellular matrix (ECM) proteins. Osteoclasts, ECs, and osteoprogenitors(Osp) co-invade into the cartilage matrix. Matrix metalloproteinases (MMPs)are secreted from osteoclasts and ECs to degrade ECM and enhance VEGFsignaling. After birth, continuous degradation of cartilage matrix followed byco-invasion of ECs and osteoprogenitors leads to longitudinal growth. Atpostnatal day 6 (P6), the center of the epiphysis becomes a secondary

ossification center (SOC), and vascularization begins. Noggin from ECspromotes chondrocyte maturation and osteogenesis, whereas Notchactivation of ECs enhances angiogenesis and osteogenesis. PVC, perivascularcell. (B) In adolescent murine bone, type H vessels are located in themetaphysis, whereas type L vessels constitute a sinusoidal network. Bloodflow velocity suddenly drops after the branching points in type L vessels.Type H vessels and type L vessels are surrounded by distinct subpopulationsof perivascular cells. In aged murine bone, blood flow and type H vesselswith perivascular osteoprogenitors decrease, which is associated withreduced bone formation at old age.


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are reduced in a premenopausal mouse modelof osteoporosis (143). Aging results in the strongreduction of HSC niche–forming vessels. EC Notchsignaling supports the expansion of HSC niches,and activation of ECNotch signaling restoresHSC-supporting vascular niches (44, 144). Thus, phar-macological rejuvenation of blood vessels maybecome an attractive therapeutic goal to preventage-related or disease-related loss of bone mass.

Blood vessels in adipose tissue

Adipose tissue is one of the most plastic organs,constantly expanding and shrinking dependingon energy status. Adipogenesis is highly depen-dent on angiogenesis (145, 146) (Fig. 6A). Thevasculature of adipose tissue provides externalcues for the development and differentiation ofadipose progenitors as a vascular niche (147, 148).In turn, adipocytes produce angiogenic factorsduring adipose tissue expansion and remodeling(149). Adipocyte-derived VEGF and high VEGFR2–expressing adipose vasculatures play crucial rolesin adipogenesis, lipogenesis, acclimation to cold

exposure, andmetabolic functions (150). DecreasedVEGF-B signaling in rodent models of type 2 dia-betes has been reported to restore insulin sensitiv-ity and improve glucose tolerance (151).More recentwork has challenged these findings, suggestingthat VEGF-B gene transduction inhibits obesity-associated inflammation and improves metabolichealthwithout changes in bodyweight or ectopiclipid deposition (152).The vasculature in adipose tissue serves as a

rich source for paracrine-acting hormones, growthfactors, and inflammation-related cytokines. Inobesity, expanded white adipose tissue (WAT)displays capillary rarefaction and hypoxia, whichcorrelate with inflammatory cytokine expressionand macrophage infiltration (150) (Fig. 6A). Incontrast, hypervascularization of adipose tissuecan be associated with browning (149) (Fig. 6A).Brown adipose tissue (BAT) is a highly vascular-ized organ with abundant mitochondria thatproduce heat through UCP1 (highly enricheduncoupling protein 1)–mediated uncoupled res-piration. An increase in lipid accumulation caused

by a high-fat diet or the absence of heat stress(thermoneutrality) reduces the rate of thermo-genesis, leads to deposition of excess calories aslipids, and causes whitening, which is the tran-sition from BAT to WAT characteristics (153)(Fig. 6A).The adipose endothelium is a gatekeeper be-

tween blood and adipose tissue. EC-regulatedfatty acid (FA) transport and uptake limits ex-cessive lipid accumulation, lipid overflow in thebloodstream, and, consequently, insulin resist-ance (154). FA transporters (FATPs) mediatetransendothelial FA transport, and FATP3 isthe only FATP that is expressed specifically in bloodvessels (155) (Fig. 6B). Moreover, EC-expressedGPIHBP1 (glycosylphosphatidylinositol-anchoredhigh-density lipoprotein-binding protein 1) is cru-cial for the lipolytic processing of triglyceride-rich lipoproteins (156) (Fig. 6B). Thus, endothelialmetabolic functions and the mechanisms involvedin the cross-talk between ECs and adipocytes couldhave important implications for the treatmentof obesity, diabetes, and metabolic syndrome.

Endocrine glands

Endocrine glands are endowed with a densenetwork of capillaries, which provide substratesfor hormone synthesis and transport releasedhormones (157). Their fenestrated capillariesfacilitate proper transport of low-molecular-weight hydrophilic molecules, including synthe-sized hormones. ECs and pericytes in endocrineglands secrete a battery of growth factors, cyto-kines, and ECM molecules, which control prolif-eration, differentiation, maintenance, and evenregeneration of endocrine cells. Maintenance ofendocrinevascular integrity is tightly regulatedbyVEGF/VEGFR2 signaling (158). Stimulating hor-mones such as TSH, ACTH, and FSH increasetranscellular transport by increasing EC contentand the number of intact fenestrae on each ECthrough up-regulation of VEGF in glandularepithelial cells of the thyroid, adrenal glands,and ovaries (luteinizing granulosa cells). Con-versely, low levels of stimulating hormones de-crease transcellular transport by reducing ECcontent and thenumber of fenestrae on eachECas a result of reduced expression of VEGF in glan-dularepithelialcells.However,themolecularmech-anisms of fenestrae formation and maintenancehave not been unraveled in great detail. Long-term inhibition of VEGF or VEGFR2 eventuallyleads to the reduction of EC fenestrae, resultingin hypofunction of endocrine glands (158–160).The safe margin of VEGF or VEGFR2 blockade—forexample, in the treatmentofcancerpatients—forproper endocrine functionandmaintenanceof EC fenestrae needs to be better defined.

Conclusion and perspective

Blood pressure regulation, coagulation, inflam-mation, atherosclerosis, and angiogenesis arethe five major branches of vascular biologyresearch. The recent advances in the understand-ing of themolecular mechanisms driving organo-typic vascular differentiation and function reflectthe emergence of a sixth branch. Organotypic


Fig. 6. Specialized features of adipose capillary bed. (A and B) Angiogenesis governs adipogenesisto differentiate preadipocytes into mature adipocytes. These mature adipocytes can develop intoeither hypertrophic adipocytes as a result of a high-fat diet–induced increase in lipid accumulation orinto beige adipocytes upon cold exposure or b3-adrenergic receptor agonism. These processes areaccompanied by vascular changes; a high-fat diet decreases vascular density, whereas browning bycold exposure increases vascular density in adipose tissues. This plastic transition of adipocytes frombrown or beige adipocytes to white adipocytes and vice versa is termed whitening and browning,respectively. (C) Expression of vascular fatty acid transporters (FATPs) induces subsequent transportof FAs across the EC layer into cells of metabolic organs. GPIHBP1 shuttles lipoprotein lipase (LPL)from interstitial spaces to the bloodstream, which subsequently hydrolyzes triglycerides (TGs) intoFAs, possibly mediated by FATP3 and FATP4 during transendothelial FA transport.


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vascular research will be aimed at (i) unraveling,inmuch greatermechanistic detail, themolecularrepertoire of organotypically differentiated cellsof the vessel wall (not restricted just to ECs) allthe way to the single-cell level; (ii) elucidating themultidirectional molecular cross-talk of vessel wallcells with the cells of their microenvironment,as well as systemic effects controlled by organo-typic vasculatures; (iii) dissecting niche functionsof organotypic vasculatures on stem cells andtheir progeny; and (iv) resolving the fate maps ofdifferent organotypic vasculatures in healthand disease. It will thereby lay the foundationfor novel diagnostic and capillary bed–specificangiotargeted therapies.

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ACKNOWLEDGMENTS

We regret that, because of space limitations, we were not able to citeall of the original research articles and related references on thistopic. Work in the authors’ laboratories is supported by funds fromthe Deutsche Forschungsgemeinschaft, the Helmholtz Association,the European Union, the Fondation Leducq, and the Institute for BasicScience funded by the Ministry of Science, ICT and Future Planning,Korea (grant IBS-R025-D1).

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Organotypic vasculature: From descriptive heterogeneity to functional pathophysiologyHellmut G. Augustin and Gou Young Koh

originally published online August 3, 2017DOI: 10.1126/science.aal2379 (6353), eaal2379.357Science

, this issue p. eaal2379Scienceand homeostasis, but also for disease states ranging from inflammation to cancer.surfaces within the body. Vascular control of the tissue microenvironment is vital, not only for normal tissue developmentin different organs. Differentiated endothelial cells develop as a sort of cobblestone monolayer to form one of the largest

review molecular mechanisms of vascular development and functionet al.between the circulation and tissues. Augustin to exogenous cytokines. However, recent work has shown that blood vessels serve as a highly dynamic interface

Blood vessels have long been considered as passive conduits for blood and circulating cells that, at best, respondDynamic vascular surfaces

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REFERENCES

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