0differentiation of arterial ,venous and lymphatic endothelial cells from vascular progenitors

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Yamashita,53Shogoin Kawahara-cho, Sakyo-ku,Kyoto606-8507,Japan.Tel.:(+81) 75-751-3853;fax:(+81)75-751-4824;e-mail:juny@frontier.kyoto-u.ac.jp.D2007,Elsevier Inc.All rights reserved. 1050-1738/07/$-see front matter ECs have been reported.These discov-eries are now opening the gate for research to elucidate the molecular and cellular mechanisms for vascular devel-opment and diversification.In this review,molecular mechanisms for arte-rial,venous,and lymphatic EC differ-entiation will be reviewed and discussed with our recent studies that use embry-onic stem(ES)cell system.Arterial EC DifferentiationA transmembrane ligand ephrinB2and its receptor tyrosine kinase EphB4were the first reported markers for arterial and venous ECs,respectively(Wang et al. 1998).Segregated expression pattern of ephrinB2and EphB4on arterial and venous ECs,respectively,in yolk sac before the onset of blood flow clearly demonstrated that there was a genetic basis of arterial-venous specification mechanisms.Disruption of ephrinB2or EphB4in mouse embryos showed sim-ilar vascular morphologic defects(Wang et al.1998,Gerety et al.1999),indicating that the ephrinB2-EphB4system is essential to establish mature blood vessel system with arterial-venous identity. Currently,various molecular markers for arterial,venous,and lymphatic ECs have been reported(Table1).Notch signaling plays a central role in arterial EC differentiation.Loss of Notch or gridlock,a downstream target gene of Notch,led to a loss of ephrinB2expres-sion in arteries in zebrafish(Zhong et al. 2001,Lawson and Weinstein2002). Double knockout mice of Hey1and2, mammalian homologues of gridlock, showed vascular abnormality withreduced arterial EC markers(Fischer et al.2004).Disruption of the recombi-nation recognition sequence binding pro-tein at the J j site(RBP-J),an essential downstream molecule for Notch activa-tion,or a Notch ligand,Delta-like4,in mice showed impaired arterial develop-ment(Krebs et al.2004,Duarte et al.2004).Forced activation of Notch in zebrafish led to ectopic expression of ephrinB2in venous ECs and sup-pressed EphB4expression(Lawson and Weinstein2002).These results indicate that Notch signaling promotes arterial EC differentiation and suppresses venous EC fate.Arterial-venous specification mechanisms in zebrafish were further demonstrated to be a regulatory signaling cascade of sonic hedgehog-vascular endothelial growth factor(VEGF)-Notch-ephrinB2(Lawson and Weinstein 2002).Arterial EC differentiation was reported to be regulated by other factors, such as transforming growth factor b families(Seki et al.2003),VEGF-A (Mukouyama et al.2002),mechano-chemical stimuli including shear stress (Coultas et al.2005),and oxygen concen-tration(Claxton and Fruttiger2005).Venous EC DifferentiationVenous EC differentiation has been considered as a default pathway of EC differentiation.That is,insufficient Notch activation during angioblast dif-ferentiation to ECs leads ECs to venous fate(Thurston and Yancopoulos2001). Recently,COUP-TFII,an orphan nuclear receptor transcription factor,was reported to repress Notch signalingthrough suppressing neuropilin1expression to maintain vein identity(You et al.2005).Double mutant ofangiopoietin1and Tie1tyrosine kinasereceptor showed specific ablation ofthe venous system(Loughna and Sato2001).These results suggest the exis-tence of a specific regulation systemfor venous development.Lymphatic EC DifferentiationLymphatic ECs are differentiated fromvenous ECs.A subset of venous ECsexpressing LYVE1,which is competentto lymphatic differentiation,are com-mitted into lymphatic ECs with theexpression of Prox-1homeobox tran-scription factor(Oliver and Harvey2002).Prox-1is considered as the mostTable1.Arterial,venous,and lymphatic EC markers in the mouseArtery Vein LymphaticAlk1(Seki et al.2003)COUP-TFII(You et al.2005)CCL21(Jain and Padera2003)Bmx(Rajantie et al.2001)EphB4(Wang et al.1998)LYVE1(Jain and Padera2003)CD44(Fischer et al.2004)Neuropilin2(Yuan et al.2002)Neuropilin2(Jain and Padera2003) Connexin37(Duarte et al.2004)Podoplanin(Breiteneder-Geleff et al.1999) Connexin40(Mukouyama et al.2002)Prox1(Wigle et al.2002)CXCR4(Yurugi-Kobayashi et al.2006)VEGFR3(Makinen et al.2001)Delta-like4(Duarte et al.2004)Depp(Shin and Anderson2005)ephrinB2(Wang et al.1998)IGFBP-5P(Shin and Anderson2005)Jagged1(Villa et al.2001)Neuropilin1(Mukouyama et al.2002)Notch1,4(Villa et al.2001)Unc5b(Lu et al.2004)Figure 1.Endothelial diversification from ES-cell-derived vascular progenitors.Vascularendothelial growth factor is essential to induce CD31+/VE-cadherin+ECs from VEGFR2+vascular progenitor cells.Vascular endothelial growth factor alone with serum inducesephrinB2À(EphB4+)venous ECs.When AM/cAMP and Notch signaling is present togetherwith VEGF,ephrinB2+arterial ECs are induced.For lymphatic EC differentiation,in additionto VEGF-C and angiopoietins,some factors secreted from OP9stroma cells are required.Lymphatic ECs are possibly differentiated directly from VEGFR2+progenitors or throughvenous ECs.specific and functional lymphatic EC marker.Targeted disruption of Prox-1 in mice led to a failure in the budding of lymphatic ECs and lymphatic vessel development(Wigle et al.2002).VEGF-C and VEGF-D signaling mediated by VEGF receptor3(VEGFR3)is a major regulator of lymphatic EC proliferation, migration,and survival that induce lym-phangiogenesis(Makinen et al.2001). Insufficient VEGFR3signal activation resulted in hypoplastic lymphatic vessels (Karkkainen et al.2004).Angiopoietin signaling is also important in lymphatic vessel development.Angiopoietin1is reported to induce lymphatic vessel for-mation(Morisada et al.2005,Tammela et al.2005).Mice lacking angiopoietin2 exhibit major lymphatic vessel defects (Gale et al.2002).Other factors such as platelet-derived growth factor BB(Cao et al.2004),hepatocyte growth factor (Kajiya et al.2005),interleukin3(Groger et al.2004),and so on,are reported to be involved in lymphangiogenesis and/or lymphatic EC differentiation.Arterial,Venous,and Lymphatic ECs from ES CellsPreviously,Yamashita et al(2000) established a novel ES cell differentia-tion system for vascular cells.Vascular endothelial growth factor receptor 2(VEGFR2),also designated as Flk1, is the earliest functional differentiation marker for blood and ECs,and an indicator of the lateral plate mesoderm. VEGFR2+cells are induced from undif-ferentiated ES cells and purified by fluorescent-activated cell sorting.When VEGFR2+cells are recultured on type-IV collagen-coated dishes with serum and VEGF,CD31+/vascular endothelial (VE)cadherin+ECs,and a-smooth muscle actin+MCs are selectively induced.Induced vascular cells can form mature vascular-like structure with endothelial tube and surrounding MCs in vitro and can contribute to newly formed vessels as ECs and MCs in vivo(Yamashita et al.2000,Yurugi-Kobayashi et al.2003).Recently,the author’s group suc-ceeded in inducing arterial,venous,and lymphatic ECs from ES cells with the use of this ES cell differentiation system and demonstrated molecular and cellular mechanisms of EC diversifica-tion from vascular progenitor cells(Yurugi-Kobayashi et al.2006,Konoet al.2006)(Figure1).When VEGFR2+cells were cultured with VEGF andserum on type-IV collagen,N90%ofinduced ECs became ephrinB2ÀvenousECs.When cyclic adenosine monophos-phate(cAMP)pathway was stimulated bya cAMP homolog,8bromo-cAMP,or acAMP-elevating humoral factor,adreno-medullin(AM),together with VEGF andserum,Notch signaling was activated inECs,and ephrinB2+arterial ECs wereefficiently induced in up to~70%ofinduced ECs.Loss of function of Notchsignaling with disruption of RBP-J in EScells,or with addition of c-secretaseinhibitor,which inhibits cleavage ofintracellular domain of Notch(NICD)toform active RBP-J-NICD complex,com-pletely abolished arterial EC inductionby cAMP activation.These results indi-cate that Notch signaling is also essentialin arterial EC differentiation in this EScell system.On the other hand,gain-of-function of Notch with forced Notchactivation by the constitutively activeNotch1did not induce arterial ECs eventogether with VEGF and cAMP activa-tion.These results indicate that,as theauthors previously demonstrated,VEGFis essential for EC differentiation fromVEGFR2+progenitor cells.For venousEC differentiation,VEGF alone withserum is sufficient.For arterial ECinduction,VEGF and Notch signaling isessential but not sufficient in this model.The AM/cAMP pathway can activateNotch signaling and is another indispen-sable signaling to induce arterial ECs.Coordinated signaling of VEGF,Notch,and cAMP are sufficient to constructivelyinduce arterial ECs from vascular pro-genitors in vitro.Induction of lymphatic ECs from EScells were reported from several groupsin2006(Liersch et al.2006,Kreuger et al.2006,Kono et al.2006).Liersch et al.(2006)and Kreuger et al.(2006)inducedprox-1+lymphatic ECs in embryoidbodies and aggregates of ES cells,withVEGF-A and VEGF-C treatment.Konoet al.(2006)induced prox-1+lymphaticECs from VEGFR2+progenitors by cul-turing the progenitors on OP9mousestroma cells,which have been reportedto induce ECs from VEGFR2+cellsmainly through VEGF production(Hirashima et al.1999).Prox-1+lym-phatic ECs appeared from1.5-to3-dayculture on OP9cells.Appearance ofLYVE1+ECs preceded prox-1expression,suggesting that this differentiation modelmay mimic the sequential differentiationmodel of lymphatic ECs from LYVE1+lymphatic-competent venous ECs in vivo(Oliver and Harvey2002,Jain and Padera2003).Nevertheless,whether lymphaticECs can be differentiated only fromvenous ECs or they can bypass venousEC stage and be differentiated directlyfrom vascular progenitors is still left to bedistinctively resolved with other exper-imental strategies.Functional relevan-cies of ES-cell-derived lymphatic ECsshould be further evaluated,for example,by transplantation experiments ofinduced lymphatic ECs.Inhibition ofVEGF-C and angiopoietin signaling byaddition of VEGFR3-and Tie2-immuno-globlin fusion proteins almost com-pletely a bolished lymphatic E Cinduction from VEGFR2+cells on OP9cells,indicating that VEGF-C and angio-poietin signaling are essential to lym-phatic EC differentiation.On the otherhand,treatment of VEGFR2+cells withVEGF-C or angiopoietin-1on type-IVcollagen-coated dishes failed to inducelymphatic ECs,whereas conditionedmedia of OP9cells showed an inductionactivity of lymphatic ECs even on colla-gen-coated dishes.Addition of otherreported lymphatic-inducing factors,interleukin3and platelet-derived growthfactor BB,also failed to induce lymphaticECs in the absence of OP9cells.Theseresults indicate that in addition to VEGF-C and angiopoietin,some secreted fac-tors from OP9cells are required toconstructively induce lymphatic ECsfrom vascular progenitors.Now,three major cell types of ECs—arterial,venous,and lymphatic ECs—have been successfully induced from EScells.Further studies to dissect outmolecular and cellular mechanisms ofEC diversification are required.Significance of ES CellDifferentiation ModelThis novel vascular cell differentiationsystem can systematically and construc-tively induce arterial,venous,and lym-phatic ECs from common progenitorVEGFR2+cells.This strategy could pro-vide novel insights into vascular develop-ment by adding a novel view point asconstructive developmental biology.Genetic animal models have been accepted as one of the most potent and significant experimental strategies in developmental biology.One needs to be carefully aware that the results we can usually observe in the animal models are the final consequence of gene modifica-tion after complicated molecular and cellular interaction in the context of body plan.A transgenic mouse model express-ing NICD in adult ECs showed ectopic expression of ephrinB2expression in a limited region of venous ECs(Carlson et al.2005).This result looks inconsistent with in vitro experiments that use ES cells that forced Notch activation in EC pro-genitors did not induce ephrinB2expres-sion(Yurugi-Kobayashi et al.2006).One possible speculation is that,as the timing of Notch activation was different between the former in vivo model and the latter in vitro model,Notch signaling may possess differentiation stage-specific roles in arterial EC induction.However,it also may be possible to speculate that some cAMP-stimulating signals already existed in the region where ectopic ephrinB2 induction was observed,and forced Notch stimulation then could induce ephrinB2expression by cooperating with regionally pre-existing cAMP signals.By carefully comparing and referring with constructive in vitro differentiation mod-els and in vivo genetic animal models, these experimental approaches would complementarily contribute to further profound understanding of the under-lying mechanisms.This system possesses another ad-vantage in that it is amenable to puri-fication and collection of cells at various intermediate differentiation stages from ES cells to specified ECs. Global analysis of gene expression pro-files in arterial,venous,and lymphatic ECs,together with VEGFR2+progeni-tors and undifferentiated ES cells, would provide comprehensive under-standing of genetic as well as epigenetic mechanisms for sequential process of cell differentiation and diversification from pluripotent cells to terminally differentiated ECs.Differentiation stud-ies that use ES cells would play various important roles in developmental biol-ogy as well as regenerative medicine and would provide not only cell sources for cell therapy but also seeds for developing other therapeutic strategies, including gene therapy and novel drugs. PerspectivesUnderstanding the cellular and molecu-lar mechanisms of vascular developmentand diversification would contribute tothe exploration of various options ofclinical application.Arterial EC differ-entiation machineries may provide arte-rial-specific vascular regeneration forischemic heart or brain diseases.Lym-phatic or tumor-vessel-specific antian-giogenic therapy may be a potentanticancer therapeutic strategy.It isknown that aberrant angiogenesis isinvolved in the development of patho-logic situations in various diseases suchas diabetic retinopathy,inflammatory orautoimmune diseases,cardiac valve dis-eases(Yoshioka et al.2006),and so on.Elucidation of tissue-specific mecha-nisms of vascular diversification in moredetail would be required to develop novelspecific vascular-targeting strategies forthese diseases.To figure out the wholescheme and machinery of EC diversifica-tion with the use of various scientificapproaches should be one of the mostimportant research targets for 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This review examines the evidence linking K ATP channels to the regulation of coronary vascular tone and the potential clinical implica-tions of pharmacologic therapies that act on K ATP channels.(Trends Cardiovasc Med2007;17:63–68)D2007,Elsevier Inc.The coronary circulation provides bloodsupply to the heart,a dynamic and highlymetabolically active organ,allowing it togenerate the pressure required to perfusethe systemic circulation.The myocar-dium is dependent on aerobic metabo-lism and cannot function effectivelywithout a consistent supply of oxygenand other metabolic substrates.This isevident when these conditions are notsatisfied during the pathological states ofmyocardial ischemia or infarction,whichmay result in deleterious effects on pumpfunction and circulatory perfusion.Under conditions of increased myocar-dial metabolism and oxygen demand,there is only limited capacity for a furtherincrease in oxygen extraction becausethis is already at near maximal levels.Inthis setting,myocardial oxygen supply isdependent on increased delivery by aug-mentation of coronary blood flow(CBF).H.M.Omar Farouque is at the Department ofCardiology,Austin Health,Heidelberg,Victo-ria,3084,Australia,and University of Mel-bourne,Melbourne,3052,Australia.Ian T.Meredith is at the Cardiovascular ResearchCentre,Monash Medical Centre,Melbourne,3168,Australia,and Monash University,Mel-bourne,3800,Australia.*Address correspondence to:Dr.H.M.Omar Farouque,Department of Cardiology,Austin Health,145Studley Road,PO Box5555,Heidelberg,Victoria,3084,Australia.Tel.:(+61)3-94963034;fax:(+61)3-9459-0971;e-mail:omar.farouque@.au.D2007,Elsevier Inc.All rights reserved.1050-1738/07/$-see front matter。