Modeling human endothelial cell transformation in...

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IntroductionVascular neoplasias are a spectrum of disorders that include rare,aggressive malignancies such as angiosarcoma, as well as commonlyoccurring infantile hemangiomas that are usually indolent but cangive rise to life-threatening complications when vital organs areinvolved (Drolet et al., 1999; Szajerka and Jablecki, 2007; Koch et al.,2008; Young et al., 2010). The unifying pathological feature of theseconditions is perturbation of endothelial cell (EC) proliferation anddisorganization of endothelial tissue architecture. In a non-pathological state, mature ECs have limited proliferative ability andform an organized monolayer along the inner surface of blood andlymphatic vessels. These cells are derived from a hierarchy of highlyproliferative progenitors that are thought to reside within the bonemarrow and are ontologically related to hematopoietic stem cells(Asahara et al., 1997; Asahara et al., 1999). An array of cytokines andchemokines direct the recruitment and homing of endothelialprogenitors from the bone marrow, via the peripheral circulation,to the inner walls of blood and lymph vessels where they differentiateand contribute to many normal physiological processes, such aswound healing, inflammation, coagulation, cell migration andhematopoiesis (Shi et al., 1998; Asahara et al., 1999; Krishnaswamyet al., 1999; Takahashi et al., 1999).

The mitogenic signaling pathways that regulate the proliferationof normal ECs have been intensively studied and are reviewedelsewhere (Carmeliet and Jain, 2011). However, relatively fewinvestigations have focused on the molecular mechanisms that

promote unregulated proliferation and transformation of ECsduring the development of vascular neoplasia. This is in part dueto the rarity of EC-derived malignancies as well as the focus ofmost investigations on end-stage disease, which precludes studyof the molecular events involved in the initiation and progressionof disease. Consequently, little progress has been made in thedevelopment of novel therapeutics for the treatment of endothelialmalignancies over the past few decades. By describing themolecular features of vascular neoplasias, as well as the cell linesand in vivo models that are available to model EC transformation,the aim of this review is to inform and motivate preclinical studiesof new therapeutic approaches to the treatment of specific ECneoplasias.

Clinical and molecular features of EC-derivedneoplasiasHemangiomaHemangioma is the most common tumor of infancy, affecting ~10%of Caucasian neonates (Table 1) (Drolet et al., 1999). This neoplasiadevelops as a rapidly growing disorganized mass of ECs. Mosthemangiomas appear within the first few weeks of birth and arecharacterized by an initial period of rapid tumor growth that isdriven by the proliferation of immature ECs. This is followed by a‘rest phase’, in which there is little change in the appearance of thetumor. A subsequent slow involuting phase associated with ECdifferentiation and apoptosis marks regression of the tumor inalmost all cases. The involuted tumor is replaced by fibrous andfatty tissue. Although histologically benign, non-invasive andunlikely to progress to a malignancy, visceral hemangiomas canresult in substantial morbidity and mortality by causing obstruction,hemorrhage or by diverting blood flow. Currently availabletreatments for life-threatening or otherwise complicated cases ofhemangioma have limited efficacy and commonly result intreatment-associated toxicity.

REVIEW Disease Models & Mechanisms 6, 1066-1079 (2013) doi:10.1242/dmm.012674

1Cancer Cell Development Group, Children’s Cancer Institute Australia for MedicalResearch, Lowy Cancer Research Centre, University of New South Wales, Randwick,NSW, Australia*Author for correspondence ([email protected])

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distributionand reproduction in any medium provided that the original work is properly attributed.

Endothelial cell (EC)-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, whichresponds poorly to current treatments and has a very high mortality rate. The development of treatments that aremore effective for these disorders will be expedited by insight into the processes that promote abnormal proliferationand malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by therarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a centralrole in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This reviewdescribes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes thatunderpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through theintroduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed inrelation to their relevance as models of vascular neoplasia.

Modeling human endothelial cell transformation invascular neoplasiasVictoria W. Wen1 and Karen L. MacKenzie1,*

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Modeling endothelial cell neoplasia REVIEW

Hemangiomas are composed of morphologically normal,immature ECs that are clonally derived and exhibit enhancedproliferation and migration (Mulliken et al., 1982; Boye etal., 2001; Dadras et al., 2004). Evidence has recently emergedto suggest that the excessive proliferation of ECs inhemangiomas is driven by an imbalance in angiogenicsignaling factors and activation of nuclear factor-κB (NFκB)(Jinnin et al., 2008; Greenberger et al., 2010). Notably, arecent study of genetic risk factors for hemangioma identifiedgermline mutations in the genes encoding vascularendothelial growth factor (VEGF) receptor 2 (VEGFR2) andanthrax toxin receptor 1 (also known as tumor endothelialmarker 8) that perturb VEGF signaling and contribute todisorganized angiogenesis (Jinnin et al., 2008). Othermolecular events resulting from the cytogeneticabnormalities that have been identified in hemangiomascould also play a role in this disorder. Chromosomalaberrations that have been observed in hemangioma includeloss of heterozygosity at chromosome regions 5q, 13q14 and17p13, as well as loss of the Y chromosome and amplificationof the cyclin D1 gene on chromosome 11 (Berg et al., 2001;Domfeh et al., 2006; Mohamed et al., 2009). Insights into themolecular regulation of different phases of hemangioma willbe valuable for the development of improved therapies forthese disorders.

Intermediate grade vascular malignanciesHemangioendotheliomas and Kaposi’s sarcoma (KS) areintermediate-grade endothelial tumors that are rankedbetween benign hemangioma and aggressive angiosarcomain terms of malignancy (Table 1; Fig. 1) (Weiss and Enzinger,1986; Salahuddin et al., 1988; Mentzel et al., 1997).Hemangioendotheliomas are rare tumors of variable andunpredictable malignancy that have been diagnosed in adultsof most age groups. These neoplasms most often present aslow-grade tumors in the trunk or limbs (Weiss and Enzinger,1986; Mentzel et al., 1997). The lesions arise from bloodvessels and are composed of cells that have beenphenotypically identified as vascular ECs (Mentzel et al.,1997; Pyakurel et al., 2006). Approximately 15% of patientsdie of disease associated with distant metastases (Mentzel et al., 1997). The molecular biology ofhemangioendothelioma is largely uncharacterized, althoughVEGF expression and abnormalities in the p53 tumorsuppressor pathway have been shown to be associated withless-differentiated (defined by a lack of endothelial-specificstorage granules known as Weibel-Palade bodies) tumorregions in a patient with metastatic disease (Theurillat et al.,2003). Chromosomal instability has been reported inmalignant hemangioendothelioma (Theurillat et al., 2003;Tsarouha et al., 2006).

KS encompasses a group of neoplasms in which thelesions are comprised of proliferating ‘spindle cells’ ofendothelial origin (Salahuddin et al., 1988; Kaaya et al., 1995;Ganem, 2010). Classic KS occurs predominantly in elderlymen, although endemic subtypes are observed amongyounger adults and children in specific geographical regionsof Africa (Szajerka and Jablecki, 2007). Classic KS is aT

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Modeling endothelial cell neoplasiaREVIEW

relatively indolent disease that is rarely life-threatening and is oftenleft untreated. Indeed, spontaneous remissions have been reported(Brooks, 1986). Cases of KS that do progress usually involve localinvasion, whereas distant dissemination is extremely rare. KS alsomanifests in association with immune suppression in individualswith AIDS and organ-transplant recipients (Gnepp et al., 1984). Incontrast to classic KS, AIDS-associated KS presents as a morewidespread, multifocal disorder where life-threateningcomplications can result from visceral involvement.

KS arises in individuals infected with human herpes virus 8 [alsoknown as KS-associated herpes virus (KSHV)] (Chang et al., 1994).Since the discovery of KSHV, there has been considerable

investigation of the genetics and oncogenic potential of this virus(reviewed by Ganem, 2010). The KSHV genome encodes viralhomologs of cellular genes that promote the cell cycle and cellularimmortalization, inhibit apoptosis, stimulate angiogenesis, andenable infected cells to evade the immune system (Boshoff et al.,1995; Bais et al., 1998; Knight et al., 2001; Bais et al., 2003; Sodhiet al., 2006; Chang et al., 2009). Consequently, activation of cellularoncogenes and mutation of tumor suppressor genes might play aless substantial role in the initiation of KS relative to other sarcomas.Although TP53 (encoding p53) mutations seem to be rare in KS,p53 protein function can be suppressed through direct interactionwith the KSHV-encoded latency-associated nuclear antigen

Classic KS

AIDS- and transplant-associatedKS

Angiosarcoma VEGF pathway dysfunction p53 mutation p14ARF inactivation Mdm-2 overexpression p16INK4a silencing Ras mutation ALT Complex or unstable karyotype

Malignant hemangioendothelioma

VEGF pathway dysfunction p53 dysfunction Unstable karyotype

Spontaneous regression

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invasion VEGF pathway dysfunction NFκB pathway dysfunction Cyclin D1 amplification Simple karyotype

Hemangioma

Hemangioendothelioma Wild-type p53 Simple karyotype

VEGF pathway dysfunction p53 dysfunction (LANA inactivated) mTOR activation (KSHV activated) Telomerase activation (KSHV activated) Simple or complex karyotype p53-p21Cip1 activation

p16INK4a -pRb activation Aneuploidy

Senescent ECs

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Fig. 1. Molecular alterations underlying vascular neoplasias. (A)Normal ECs (illustrated in the center) have a finite replicative lifespan that is dictated by theshortening of telomeres with each cell division. Critical shortening of telomeres leads to activation of p53 and pRb pathways, and the onset of senescence. (B-D)Abnormal proliferation of ECs is a common feature of vascular neoplasias. Abnormalities in the VEGF pathway are common across the spectrum of vascularneoplasias. Other mechanisms implicated in oncogenesis are listed, and chromosomal characteristics are described in blue. A simple karyotype is characterizedby a near-diploid chromosome number and two or fewer karyotypic abnormalities. A complex karyotype is defined as five or more chromosomal aberrations.(B)Hemangioma is a benign condition in which hyperproliferation of immature ECs is driven by constitutive activation of NFκB and VEGF signaling pathways.(B,C)Infection by KSHV gives rise to classic Kaposi’s sarcoma (KS), which is relatively indolent and can either undergo spontaneous regression or can becomelocally invasive. p53 is inactivated, whereas mTOR and telomerase are activated by the KSHV-encoded protein LANA and the G-protein coupled receptor,respectively. (C)In immune-compromised individuals, KSHV often progresses to a multifocal tumor. (C,D)Hemangioendothelioma also presents in variousgrades, with defects in the p53 pathway found in malignant hemangioendothelioma, but not in intermediate-grade hemangioendothelioma. Chromosomalabnormalities are observed in intermediate-grade and malignant EC tumors. (D)In addition to p53 dysfunction, malignant hemangioendothelioma andangiosarcoma often exhibit chromosomal instability. Activation of oncogenes such as Ras is also common in angiosarcoma. EC, endothelial cell; NFκB, nuclearfactor-κB; ALT, alternate telomere lengthening; KS, Kaposi’s sarcoma; KSHV, Kaposi’s sarcoma-associated herpes virus [also known as HHV-8 (human herpes virustype-8)]; VEGF, vascular endothelial growth factor; LANA, latency-associated nuclear antigen; AIDS, acquired immune deficiency syndrome; pRb, retinoblastomatumor suppressor protein; mTOR, mammalian target of rapamycin.

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(LANA) (Friborg et al., 1999). Insights into the role of KSHV inthe pathogenesis of KS have provided a platform for several earlyphase clinical trials of new therapeutic strategies, which pave theway for rapid progress towards the more effective treatment ofAIDS- and transplant-related KS. Promising novel therapeuticsinclude inhibitors of tyrosine kinase signaling and compounds thattarget the mTOR (mammalian target of rapamycin) pathway, whichare activated by the KSHV G-protein coupled receptor (Koon etal., 2005; Sodhi et al., 2006).

AngiosarcomaAngiosarcoma is a rare malignancy that makes up less than 1% ofall sarcomas. In contrast to other EC-derived neoplasias,angiosarcomas are highly malignant (Table 1; Fig. 1) (Young et al.,2010). This malignancy occurs with a peak incidence in the seventhdecade of life and tends to arise in the viscera, skin and soft tissue.Angiosarcomas have a propensity to recur locally and spread widely,exhibiting a high rate of lymph node and systemic metastases. Thedisease is associated with a high mortality rate as a result of poorresponse to treatment (Mark et al., 1996; Lezama-del Valle et al.,1998). With a reported 5-year survival rate of ~20% and very littlechange in treatment options over the past decades, innovativetherapies are urgently needed. Tumor resection with adjuvantradiotherapy is currently the main treatment approach toangiosarcoma, although a few groups have recently begun topursue biological and molecular targeted approaches (Koontz etal., 2008; George et al., 2009; Maki et al., 2009; Young et al., 2010).A more detailed understanding of the molecular pathways involvedin malignant transformation of ECs and more extensive preclinicalstudies will be invaluable for the validation of these therapies andthe optimization of effective therapeutic strategies.

Most cases of angiosarcoma arise sporadically; however, previousirradiation, exposure to toxic chemicals and chronic lymphedemaare known risk factors (Mark et al., 1996; Lezama-del Valle et al.,1998). Activation of the oncogene K-ras through acquisition ofspecific point mutations is a common event in angiosarcoma,reportedly occurring in 29% of hepatic angiosarcomas (7 out of 24)and 60% of cardiac angiosarcomas (3 out of 5) (Przygodzki et al.,1997; Boivin-Angèle et al., 2000; Garcia et al., 2000). The incidenceof K-ras mutations seems to be highest among angiosarcomasassociated with exposure to vinyl chloride, where mutations havebeen detected in ~80% of cases (5 out of 6 cases tested) (Boivin-Angèle et al., 2000). Ras mutations are likely to contribute to thehighly metastatic nature of angiosarcomas, given that they aregenerally not found in non-malignant vascular neoplasias (Table 1).The malignant growth of angiosarcoma is driven by theproliferation of pleomorphic cells that exhibit varying degrees ofEC differentiation and give rise to a chaotic tissue architecture(Young et al., 2010). VEGF is expressed at high levels inangiosarcoma and the expression of VEGF receptors is associatedwith a more differentiated tumor phenotype and favorableprognosis (Zietz et al., 1998; Itakura et al., 2008). Consistent withthose observations, inhibition of VEGF receptor 1 (VEGFR1) hasbeen shown to increase the proliferation of canine angiosarcomacells in vitro (Tamburini et al., 2009).

Abnormalities in the TP53 pathway – including p53 mutations,Mdm-2 overexpression and inactivation of p14ARF – are commonmolecular aberrations in angiosarcoma (Hollstein et al., 1994; Zietz

et al., 1998; Weihrauch et al., 2002). For example, mutations in theTP53 gene were detected in ~30% of liver angiosarcomas(Przygodzki et al., 1997; Weihrauch et al., 2002). Consistent withthe high frequency of p53 dysfunction, complex karyotypes andchromosomal instability are often observed in angiosarcomas.Chromosomal aberrations reported in angiosarcomas includedeletions within the CDKN2A locus at chromosome region 9p21,which encodes tumor suppressors p16INK4a, p15INK4b and p14ARF.Inactivation of p16INK4a by promoter methylation is anotherfrequent event in liver angiosarcoma (Tannapfel et al., 2001;Weihrauch et al., 2002). Studies of liver angiosarcoma showed thatp16INK4a was repressed by promoter methylation in 63% (12 out of19) of tumor samples, whereas 5% (1 out of 19) exhibitedhomozygous deletion and 10% (2 of 19) exhibited loss ofheterozygosity at the CDKN2A locus. Overall, inactivation ofp16INK4a was detected in 74% (14 out of 19) of angiosarcomas. Theimportance of p16INK4a repression in angiosarcoma was also evidentfrom canine studies that showed sustained expression of p16INK4a

in 100% of benign hemangiomas (a total of ten were examined),and p16INK4a suppression in 82% (32 out of 39) angiosarcomas(Yonemaru et al., 2007). Together, these studies implicate Rasactivation plus aberrations in the p53 pathway and silencing ofp16INK4a as key events in the pathogenesis of advanced, but notbenign, vascular tumors (Table 1, Fig. 1).

Immortalization and tumorigenic conversion of human ECsThe limited replicative potential of normal human ECsA fundamental step in the development of endothelial neoplasiasis the deregulation of cell proliferation. Normal human cells havea finite replicative ability, yet cancer cells are capable of unlimitedreplication, i.e. they are immortal. Cellular immortality isconsidered to be a requirement for malignant transformation andis a defining property of the subset of tumor cells that drive tumorgrowth and recurrence (Zhou et al., 2009). Previous studies haverevealed that the in vitro replicative potential of normal humancells, including ECs, is restricted by both intrinsically andextrinsically initiated stresses that induce senescence (e.g. telomereshortening and an oxidative environment, respectively) (Hayflickand Moorhead, 1961; Johnson and Longenecker, 1982; Yuan et al.,2008). Senescent ECs typically adopt a flattened morphology anddemonstrate cytoplasmic spreading, increased granularity,vacuolization and multi-nucleation. They are resistant to mitogenicstimulation and exhibit pH-dependent β-galactosidase activity(Johnson and Longenecker, 1982; Dimri et al., 1995).

It is well established that the shortening of chromosomal-endstructures, referred to as telomeres, plays a central role in the onsetof senescence (Artandi and DePinho, 2010). Telomeres normallyfunction to prevent chromosomal end-to-end fusions and tomaintain genomic integrity during cell division. However, telomericDNA is eroded during consecutive cell divisions as a consequenceof the inability of DNA polymerases to synthesize the 5� terminusof linear DNA. Exposure to oxidizing agents accelerates telomereshortening and the onset of EC senescence; conversely, free radicalscavengers reduce the rate of telomere shortening (von Zglinickiet al., 1995; Xu et al., 2000; Kurz et al., 2004; Napier et al., 2010).The progressive shortening of telomeres has been demonstratedin serially passaged cultures of human ECs derived from the

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umbilical vein, iliac arteries and veins, abdominal aorta, and bonemarrow (Chang and Harley, 1995; Aviv et al., 2001; MacKenzie etal., 2002). Telomere shortening has also been demonstrated inarterial ECs in vivo in association with increasing age (Okuda etal., 2000).

When telomeres reach a critically short length, they are proneto fusion and promote chromosome rearrangements. Hence,primary ECs that are maintained in culture for long periods of timefrequently harbor aberrant chromosomes (Wagner et al., 2001). Inaddition to structural rearrangements, changes in wholechromosome number (aneuploidy) tend to occur as cultured ECsage and approach senescence in vitro (Nichols et al., 1987; Johnsonet al., 1992; Zhang et al., 2000; Aviv et al., 2001). In normal humancells, chromosome fusions and rearrangements initiate a DNAdamage response that culminates in activation of tumor suppressorp53 and transcriptional upregulation of p21Cip1/Waf1, which haltscell cycle progression and initiates senescence (Stein et al., 1999).Tumor suppressor p16INK4a is subsequently upregulated, resultingin hypo-phosphorylation of the retinoblastoma tumor suppressorprotein (pRb) and a sustained growth arrest (Munro et al., 1999;Wagner et al., 2001; Freedman, 2005). The activation of tumorsuppressor pathways and induction of senescence plays a crucialrole in preventing replication of aged endothelial cells harboringcytogenetic aberrations. Disruption of these mechanisms enablescells to continue to proliferate and initiate neoplastic growth, asoutlined below.

Lifespan extension, immortality and genetic evolution ofECsDuring neoplastic transformation, the replicative lifespan of humancells is extended by either the activation of a telomere maintenancemechanism (described below) or inactivation of a tumor suppressorpathway (reviewed by Reddel, 2000). Inactivation of tumorsuppressor pathways enables cells to progress through the cell cyclewith critically short telomeres. However, short, dysfunctionaltelomeres that are present in proliferating cells promotechromosomal fusions and genomic instability (Ray et al., 1990;Counter et al., 1992). Telomere dysfunction in pre-malignant andcancerous cells is typically evidenced by dicentric chromosomes,unbalanced translocations, gene amplifications, deletions and ringchromosomes (Ducray et al., 1999; Gisselsson et al., 2001; O’Haganet al., 2002). These types of chromosomal aberrations are frequentlyobserved in KS and angiosarcoma, and have also been identifiedin immortalized EC lines, particularly those with defective tumorsuppressor pathways (Schuborg et al., 1998; Wong et al., 2001; Wenet al., 2006). Chromosomal instability accelerates the rate andaccumulation of molecular changes that facilitate malignanttransformation and tumor progression (Rudolph et al., 2001),including activation of a telomere maintenance mechanism,oncogene activation or silencing of tumor suppressor genes.

Under standard culture conditions, ECs that bypass senescenceusually succumb to a proliferative ‘crisis’ that is characterized byan increased rate of cell death and reduced cell expansion(MacKenzie et al., 2002; Gu et al., 2003; Nisato et al., 2004; Wenet al., 2006). Crisis is thought to be initiated by telomeredysfunction, genetic catastrophe and/or fatal oxidative damage.Cells that are driven into crisis by the deactivation of tumorsuppressor pathways resume proliferation if they undergo a

spontaneous molecular event that activates a telomere maintenancemechanism: either activation of the enzyme telomerase or analternate telomere lengthening mechanism (ALT) (Counter et al.,1992; Bryan et al., 1995). Telomere maintenance stabilizeschromosomes and thereby enables both escape from crisis andunlimited proliferation.

Extension of replicative lifespan and immortalization ofECs following transfection with viral antigensThe extended replicative lifespan that results from the inactivationof tumor suppressor pathways is thought to represent a pre-malignant stage of the multistep process of cancer development.Several investigations have demonstrated that the replicativelifespan of normal human ECs derived from bone marrowmicrovasculature, the umbilical vein and the iliac vein is extendedby transfection with Simian virus 40 (SV40) T antigen or humanpapilloma virus (HPV)-encoded E6 and E7 oncogenes (Table 2).The large T antigen of SV40 and the HPV E6 and E7 oncogenesfunctionally inactivate the p53 and pRb tumor suppressor pathwaysand thereby enable transfected cells to bypass senescence (Girardiet al., 1965; Werness et al., 1990; Shay et al., 1991). Viral-antigen-transfected ECs have been shown to replicate for up to 42 passagesbeyond senescence (Table 2) (Gimbrone and Fareed, 1976; Schwartzet al., 1991; Fickling et al., 1992; Hohenwarter et al., 1992b; Lassalleet al., 1992; Bailey et al., 1994; Rhim et al., 1998; Krump-Konvalinkova et al., 2001; O’Hare et al., 2001; MacKenzie et al.,2002).

A number of studies also reported that viral transformation leadsto immortalization of ECs (Ades et al., 1992; Fontijn et al., 1995;Burger et al., 1998; Rhim et al., 1998; Rood et al., 2000). However, agrowth crisis prior to immortalization was noted in several studies(Ades et al., 1992; Fontijn et al., 1995; Rood et al., 2000; MacKenzieet al., 2002) and two studies showed that telomerase wasspontaneously activated in the immortalized ECs (Rhim et al., 1998;Gagnon et al., 2002). Ectopic activation of telomerase was shown tocooperate with SV40 transformation to overcome crisis andefficiently promote immortalization of bone-marrow- andmammary-derived ECs (O’Hare et al., 2001; MacKenzie et al., 2002).Together, these studies suggest that inactivation of tumor suppressorpathways and activation of a telomere maintenance mechanism isnecessary and sufficient for in vitro immortalization of human ECs.

Immortalization of ECs by transfection with viral oncogenes andactivation of telomerase is not sufficient for malignant conversionof ECs (Burger et al., 1998; Rhim et al., 1998; MacKenzie et al.,2002). Nevertheless, viral-antigen-transfected ECs do exhibit somecharacteristics of transformed cells, including the ability toproliferate independently of exogenous VEGF, and reducedexpression of proteins that define the endothelial phenotype,including VEGFR2, platelet endothelial cell adhesion molecular-1(CD31; also known as PECAM-1), VE cadherin and von Willebrandfactor (vWF) (Table 2) (Gimbrone and Fareed, 1976; Hohenwarteret al., 1992b; Rood et al., 2000; MacKenzie et al., 2002; Shao andGuo, 2004). These features parallel the abnormal expression ofVEGF receptors and tendency for downregulation of vWF andCD31 in late-stage hemangiomas (Takahashi et al., 1994). Viral-oncogene-transformed ECs also accumulate genomicrearrangements that are similar to those observed in ECmalignancies (Gimbrone and Fareed, 1976; MacKenzie et al., 2002).

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Telomerase-mediated immortalization of human ECsThe active core of the telomerase holoenzyme is a ribonuclearprotein complex that includes a reverse transcriptase (hTERT), anon-coding RNA that functions as a template for telomere synthesis(hTR) and an RNA-binding and -modifying protein, dyskerin, whichstabilizes the RNA component (Feng et al., 1995; Nakamura et al.,1997; Cohen et al., 2007). Although telomerase is not expressed inmature somatic cells, its activation accounts for telomeremaintenance in ~85% of all human cancers and immortal cell lines.

Telomerase activity is also readily detected in human germ cells,stem cells and normal endothelial progenitors (Kim et al., 1994;Wright et al., 1996). Telomerase is downregulated duringdevelopment, differentiation and with in vitro passage (Hsiao etal., 1997; Ingram et al., 2005). Consequently, it is not usuallydetectable in differentiated ECs.

Ectopic expression of hTERT reactivates telomerase in matureECs and is further bolstered by co-expression of hTR (Yang et al.,1999; MacKenzie et al., 2002; Gu et al., 2003; Nisato et al., 2004;

Table 2. Viral-antigen-transformed EC lines

Exogenous genes

Tissue source

Cell line

name

SV40 ER

HPV E6

HPV E7

Other

viral

gene Lifespan CD31 VEGFR1 vWF ICAM-1 Karyotypeb References

Bone marrow BMSVT,

BMEC-1 + – – – Extended + +/− +/− + Unstable Candal et al., 1996;

MacKenzie et al., 2002

Mammary

microvascular HMME + – – – Extended NE NE NE NE NE O’Hare et al., 2001

Dermal

microvascular HMEC-1 + – – – Immortalized + +/− +/− + NE Ades et al., 1992;

Ribeiro et al., 1995;

Unger et al., 2002a; Shao and Guo, 2004

Umbilical vein CS2, Q2, IB10 + – – – Extended NE NE +/− NE Abnormal Hohenwarter et al.,

1992b; Hohenwarter

et al., 1994; Hohenwarter et al.,

1997

Umbilical vein SVHEC-D + – – – Extended NE NE – NE Unstable Gimbrone and

Fareed, 1976

Umbilical vein EC-pSV1 + – – – Extended NE NE +/− + NE Lassalle et al., 1992

Umbilical vein SGHEC-7 + – – – Extended NE NE +/− + Abnormal Fickling et al., 1992

Umbilical vein Clone 1,

Clone 6 + – – – Extended NE NE + + NE Bailey et al., 1994

Umbilical vein EVLC2 + – – – NE – + – + NE van Leeuwen et al.,

2000; Unger et al.,

2002a

Umbilical vein Clone 1, Clone 2

+ – – – Extended NE NE +/− NE NE Schwartz et al., 1991

Umbilical vein E7 HUVEC – – + – Extended NE NE NE NE NE Burger et al., 1998;

Rhim et al., 1998

Umbilical vein E6 HUVEC – + – – Extended NE NE NE NE NE Burger et al., 1998; Rhim et al., 1998

Pulmonary HPMEC + – – – Extended + NE + + NE Krump-Konvalinkova

et al., 2001

Bone marrow HBMEC-27,

HBMEC-28, HBMEC −33,

HBMEC-60

– + + – Immortalized NE NE + + NE Rood et al., 2000

Umbilical vein EC-RF7, 24 – + + – 2/24 clones

immortalizedc + NE + + NE Fontijn et al., 1995

Umbilical vein PymT-EC – – – + Extended + NE + NE NE Primo et al., 2000 aCharacteristics of the EC phenotype, i.e. the direction of the expression change for each of the molecular markers, is explained in the text. A positive sign indicates correspondence

with the EC phenotype; a negative sign indicates deviation from the EC phenotype. bAn unstable karyotype was defined by a rapidly evolving karyotype with a high frequency of

random aberrations and numerous non-clonal marker and ring chromosomes. An abnormal karyotype denotes unspecified chromosomal abnormalities or aneuploidy.

cImmortalized via growth crisis.

NE, not evaluated.

EC phenotypea

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Napier et al., 2010). Reactivation of telomerase activity has beenshown to extend the replicative lifespan of normal human ECs, butis not sufficient for immortalization. A number of studies haveshown that ECs that overexpress hTERT are subject to a growthcrisis following an extended period of replication (Krump-Konvalinkova et al., 2001; O’Hare et al., 2001; MacKenzie et al.,2002; Gu et al., 2003; Nisato et al., 2004; Wen et al., 2006; Takanoet al., 2008) (Table 3). The occurrence of crisis in hTERT-transducedEC cultures highlights a requirement for additional molecularevents to cooperate with telomerase in the immortalization process.The efficient immortalization of ECs co-transfected with hTERTand viral oncogenes is consistent with the inactivation of tumorsuppressor genes being a crucial requirement (Table 3) (O’Hare etal., 2001; MacKenzie et al., 2002). This is further supported by

studies that showed that hTERT-transduced ECs frequently silencedtumor suppressor p16INK4a, which functions as a major regulatorof the RB pathway during hTERT-driven immortalization (Wen etal., 2006). Furthermore, abnormalities in the TP53 pathway havealso been reported in hTERT-immortalized EC lines (Wen et al.,2006). This inactivation of tumor suppressor pathways is likely tobe required for telomerase-transduced ECs to overcome cellularstress (e.g. oxidative stress) during immortalization.

EC lines immortalized following hTERT transduction retainmany of the properties of normal human ECs, including continuedexpression of normal EC surface markers, including CD31 andICAM-1, and a requirement for VEGF signaling for continuedproliferation (Table 3) (Yang et al., 1999; Krump-Konvalinkova etal., 2001; MacKenzie et al., 2002; Nisato et al., 2004; Shao and Guo,

Table 3. hTERT-mediated immortalization of EC lines

Tissue source Cell line name Replicative

lifespan

EC phenotypea

Karyotypeb

TSG pathwayc

References CD31 vWF ICAM-1 p53-p21 p16INK4a- pRB

Aortic HAEC-TERT Immortalized + NE + + Simple NE +p Yang et al., 1999

Aortic HAEC Immortalized NE NE NE NE NE NE NE Matsushita et al., 2001

Bone marrow BMhTERT-1 Immortalizedd + + + + Simple + – MacKenzie et al., 2002; Wen et al.,

2006

Bone marrow BMhTERT-2 Immortalizedd + + NE + Complex + – MacKenzie et al., 2002; Wen et al., 2006

Bone marrow BMhTERT

clones (p16-

positive)

Immortalizedd + + NE + Simple + + Wen et al., 2006

Bone marrow BMhTERT

clones

(p16-negative)

Immortalizedd + + NE + Complex + – Wen et al., 2006

Bone marrow BM2A4 Immortalizedd + + NE + Unstable – – Wen et al., 2006

Dermal lymphatic TERT-HDLEC Extended + + NE NE NE NE NE Nisato et al., 2004

Dermal microvascular

TIME Immortalized + NE NE NE Normal NE NE Venetsanakos et al., 2002

Mammary

microvascular HMME Extended NE NE NE NE NE NE NE O’Hare et al., 2001

Microvascular HMVEC Immortalized + + NE NE NE NE NE Shao and Guo, 2004

Brain microvascular

TERT-HBEC Extended NE NE + NE NE NE NE Gu et al., 2003

Dermal

microvascular HDMEC-TERT Immortalized + NE + + Normal NE +p Yang et al., 1999

Umbilical vein i-HUVEC Immortalized NE NE NE NE Abnormal + NE Freedman and Folkman, 2004

Umbilical vein HUVEC Extended + + NE NE NE NE NE Takano et al., 2008

Umbilical vein HUVEC Extended NE NE NE NE NE NE NE Young et al., 2003

Pulmonary HPMEC Extended + NE + + NE NE NE Krump-Konvalinkova et al., 2001

Umbilical vein HUVEC-TERT Immortalized + NE + + Simple NE +p Yang et al., 1999

Coronary arteries HCAEC-TERT Immortalized + NE + + Simple NE +p Yang et al., 1999

Saphenous vein HSVEC-TERT Immortalized NE NE + NE Simple NE NE Yang et al., 1999 aCharacteristics of the EC phenotype, i.e. the direction of the expression change for each of the molecular markers, is explained in the text. A positive sign indicates correspondence

with the EC phenotype; a negative sign indicates deviation from the EC phenotype. bA simple karyotype is characterized by a near-diploid chromosome number and two or fewer

karyotypic abnormalities. A complex karyotype included five or more chromosomal aberrations. An unstable karyotype was defined by a rapidly evolving karyotype with a high

frequency of random aberrations and numerous non-clonal marker and ring chromosomes. An abnormal karyotype denotes unspecified chromosomal abnormalities or

aneuploidy. cAnalysis of TP53 and RB tumor suppressor pathways in the late passage EC cells; + denotes presence and – denotes dysfunction of p53-p21 or p16INK4a-pRB pathways; pNormal phosphorylation of pRB while p16INK4a status was not evaluated. dImmortalized via growth crisis.

EC, endothelial cell; NE, not evaluated; TSG, tumor suppressor gene.

VEGFR1

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2004; Takano et al., 2008; Dill et al., 2012). Interestingly, somehTERT-transduced EC lines were also shown to form functionalvessels in vivo and in vitro (Yang et al., 2001; MacKenzie et al., 2002;Freedman and Folkman, 2004). Thus, despite their deregulatedreplicative lifespan, hTERT-immortalized ECs generally seem toretain more accurately the properties of normal, untransformedECs than do viral-antigen-transformed ECs. Nevertheless, it isimportant to note that spontaneous molecular alterations that occurduring hTERT-driven immortalization can confer neoplasticproperties. For instance, our recent investigations revealed thatrepression of p16INK4a during immortalization of ECs is associatedwith cytoskeletal changes, altered motility and defects inmorphogenesis in vitro (Kan et al., 2012). These changes are likelyto reflect some of the phenotypic changes required for theacquisition of metastatic ability in vivo.

Repression of p16INK4a by promoter methylation in hTERT-immortalized ECs is a molecular event that has also been observedin angiosarcoma (Fig. 1) (Tannapfel et al., 2001; Weihrauch et al.,2002). In our study of hTERT-transduced bone marrow ECs(BMECs), the subset of clones that repressed p16INK4a all developedcomplex karyotypes (Wen et al., 2006). The cytogeneticheterogeneity and abnormalities detected in the p16INK4a-negativeBMECs were typical of those that have been documented in EC-derived tumors. Specific aberrations that recur in p16INK4a-repressed cell lines and are also observed in angiosarcoma includemonosomy 13, loss of the Y chromosome, rearrangements involving17p, and trisomies 20 and 11 (Mandahl et al., 1990; Kindblom etal., 1991; Gil-Benso et al., 1994; Cerilli et al., 1998; Schuborg et al.,1998; Wong et al., 2001; Zu et al., 2001; Baumhoer et al., 2005;Tsarouha et al., 2006; Wen et al., 2006). Repression of p16INK4a andthe concurrent development of karyotypic complexity is a feasibledepiction of the molecular alterations that permit the accumulationof genomic imbalances in EC neoplasias.

Relatively little is known about the mechanism(s) that supporttelomere maintenance in the various types of human vascularneoplasias in patients. However, one study demonstratedtelomerase activity in 22 out of 22 KS lesions (Chen et al., 2001),and others have shown that KSHV-encoded LANA directlyactivates Sp1-mediated transcription of hTERT (Knight et al., 2001;Verma et al., 2004). In contrast, an assessment of telomerase activityin canine angiosarcomas showed no detectable telomerase enzymeactivity in 6 out of 7 cases (Yazawa et al., 1999; Chandler et al.,2009). Telomerase activity was also undetectable in two single-clone-derived angiosarcoma cell lines, ISO-HAS.1 and AS-M.5(Krump-Konvalinkova et al., 2003). These studies suggest that ALTcould be a common mechanism of telomere length maintenancein angiosarcoma. However, this possibility remains to be confirmedby demonstration of the specific molecular characteristics of ALT-immortalized cells, such as very long heterogeneous telomerelengths, DNA c-circles and PML bodies in angiosarcoma tissue(Bryan et al., 1997; Henson et al., 2005; Fasching et al., 2007).

Tumorigenic conversion of human EC linesImmortalized EC lines established by co-expression of hTERT andSV40 T antigen exhibit a partially transformed phenotype (Table 4)(Salmon et al., 2000; O’Hare et al., 2001; MacKenzie et al., 2002;Krump-Konvalinkova et al., 2003). BMECs that co-expressed SV40T antigen and hTERT were shown to be capable of robust

anchorage-independent growth in soft-agarose, but were notovertly tumorigenic, because implantation in immune-compromised mice resulted in very small subcutaneous nodulesthat regressed within 3 weeks (MacKenzie et al., 2002). Pulmonaryand sinusoidal ECs transformed with hTERT and SV40 ER weresimilarly non-tumorigenic (Salmon et al., 2000; Krump-Konvalinkova et al., 2001). In addition to the downregulation ofphenotypic markers, ECs immortalized by SV40 T antigen grewindependently of exogenous VEGF (Tables 2, 4), which is consistentwith the constitutive activation of VEGF signaling andheterogeneous phenotype observed in human vascular neoplasias(Ohsawa et al., 1995; MacKenzie et al., 2002; Bais et al., 2003; Itakuraet al., 2008; Young et al., 2010).

Overexpression of oncogenic N-Ras mediated tumorigenicconversion of immortalized BMECs (MacKenzie et al., 2002).Upon subcutaneous injection into immunocompromised mice,BMECs that co-expressed hTERT, SV40 T antigen and N-Rasgenerated large, rapidly growing tumors that were organized intolumen-like structures and in some mice metastasized to lymphnodes (MacKenzie et al., 2002). When injected via the tail vein, theECs lodged within the lungs, where they formed tumors that causedmorbidity within 3 weeks of implantation. Similarly, humanumbilical vein endothelial cells (HUVECs) transfected with HPVE6 and E7 oncogenes and immortalized via spontaneous activationof telomerase were vulnerable to tumorigenic conversion bytransfection with activated K-ras (Rhim et al., 1998). Thedemonstration of K-ras mutations in angiosarcoma underscores therelevance of these models to human vascular neoplasias (Boivin-Angèle et al., 2000; Hong et al., 2000).

EC hybrid and vascular tumor-derived cell linesIn addition to genetically engineered ECs, immortalized EC lineshave been generated by fusion of HUVECs with human tumor celllines, such as the adenocarcinoma A549 cell line (Table 5) (Edgellet al., 1983; Hohenwarter et al., 1992a; Unger et al., 2002b). Hybridendothelial tumor cell lines have unlimited replicative potential andretain some, but not all, EC characteristics (Unger et al., 2002a).In addition, because the hybrid EC A549 cell line displays karyotypicchanges that have also been identified in A549 cells, it does notseem an ideal representation of the molecular biology of endothelialtumor cells (Edgell et al., 1983).

Authentic EC lines isolated directly from vascular tumors areclearly valuable for studies of the molecular biology of the disease.Cell lines established by ex vivo culture of human tumor tissuesinclude the angiosarcoma-derived cell lines ISO-HAS, AS-M andHAEND (Hoover et al., 1993; Masuzawa et al., 1999; Unger et al.,2002a; Krump-Konvalinkova et al., 2003). Aberrant p53 expression,a lack of telomerase activity and an ability to form tumors in vivo(evaluated in ISO-HAS and ISO-HAS.1) are some of thecharacteristics that angiosarcoma-derived clonal EC lines have incommon with primary angiosarcomas. At least four KS-derivedEC lines have been established from AIDS- and non-AIDS-associated KS patients (Table 5) (Salahuddin et al., 1988; Lunardi-Iskandar et al., 1995b; Popescu et al., 1996; Albini et al., 1997;Casalone et al., 2001). Most of the reported EC-tumor-derived celllines retained typical endothelial characteristics, includingexpression of CD31, VEGFR1, vWF and I-CAM. One exceptionwas the HAEND cell line, which suppressed expression of all of

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the defining EC markers that were examined in twoseparate reports (Hoover et al., 1993; Unger et al.,2002b).

It is also important to mention that two presumptiveEC lines, ECV304 and TEC61, which were broadly useduntil ~2000, were shown by DNA profile analysis to bemisidentified cell lines (MacLeod et al., 1999). ECV304was originally reported as an immortal EC line derivedfrom spontaneous transformation of HUVECs, whereasTEC61 was established from thyroid endothelium.However, ECV304 was subsequently found to begenetically identical to the T24 bladder carcinoma cellline and TEC61 was identified as the humanchoriocarcinoma cell line, JEG3 (http://www.atcc.org/en/Products/Cells_and_Microorganisms/Cell_Lines/Misidentified_Cell_Lines.aspx). These discoverieshighlight the importance of cell line authentication andcharacterization of endothelial phenotypes prior to theutilization of presumptive tumor-derived cell lines inresearch and drug development. It is also ofconsideration that tumor-derived cell lines representend-stage disease, and therefore will be most valuablewhen used in conjunction with well-characterized EClines that have been genetically manipulated torepresent earlier stages of EC transformation.

Animal models of vascular neoplasiasIn addition to cell lines, animal models are required forpreclinical testing of novel therapeutics. Models thathave been established for in vivo study of vascularneoplasias include mice and non-human primatestreated with chemical carcinogens or infected withKSHV. Whereas infection with purified KSHV providesa relevant model of KS (Dittmer et al., 1999; Mutlu etal., 2007; Chang et al., 2009), the high incidence of Rasmutations in chemically induced murine vascularneoplasia is consistent with human angiosarcoma(Hong et al., 1980; Sato et al., 1986). Other murinemodels include mice with a combined knock out ofTP53 and the gene encoding p18INK4c, which results inthe development of angiosarcomas as well as othercancers of soft tissue (Harvey et al., 1993; Zindy et al.,2003). Interestingly, Notch1 knockout mice were alsofound to develop hepatic angiosarcoma with a very highpenetrance (Dill et al., 2012). However, to date, Notch1defects have not been demonstrated in humanangiosarcoma.

Clinical trials of novel therapeutics are usuallypreceded by in vivo tests using patient-derived cell linesor genetically engineered cell lines xenografted inanimal models. Tumors that form in micexenoengrafted with human ECs co-expressing viralantigens that inactivate tumor suppressors p53 andpRb, together with exogenous expression of hTERT andoncogenic Ras, could be used to model angiosarcomaor malignant hemangioendothelioma (Table 4) (Rhimet al., 1998; MacKenzie et al., 2002). By contrast,cavernous endothelial tumors established by infectionT

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of neonatal mice with polyoma virus middle T antigen,which induces constitutive tyrosine kinase activity, mightbe a more relevant model of infantile hemangioma(Bautch et al., 1987). A benign neoplasia resemblinghemangioma was also modeled in mice xenografted withHUVECs expressing polyoma virus middle T antigen.The xenografted cells formed tumors that regressed after3 weeks, mirroring the typical course of hemangioma(Primo et al., 2000; Schaffhausen and Roberts, 2009).

Engraftment of patient-derived cells inimmunocompromised mice is a clinically relevantalternative to genetically engineered xenograft models.Similar to the polyoma middle T-antigen-drivenxenograft tumors, patient-derived hemangiomaprogenitor cells were shown to form transientsubcutaneous tumors when engrafted inimmunocompromised mice (Khan et al., 2008; Xu et al.,2011). Xenograft tumors have also been establishedfrom canine angiosarcoma (Akhtar et al., 2004; Kodamaet al., 2009). In addition to providing a useful model forpreclinical testing of novel therapeutics, xenograftspotentially provide a means for expansion of rare tumormaterial via serial transplantation (Khan et al., 2008).

Perspectives and conclusionsThus far, investigations of the molecular biology of EC-derived malignancies have been limited to molecular andcytogenetic case studies and investigations of specificoncogenes and tumor suppressors in small patientcohorts. The data accumulated to date implicateabnormalities in the VEGF pathway as a common eventacross the spectrum of benign and malignant vascularneoplasias (Fig. 1). Defects in the TP53 and p16INK4a-RBtumor suppressor pathways feature in intermediate andaggressive malignancies, and are often associated withthe development of chromosomal abnormalities. It isnotable that, aside from aberrant VEGF signaling, therehave been no reports of oncogenic changes that clearlydistinguish EC neoplasias from other cancers, such asepithelial cancers. Hence, genetic models of ECneoplasias have largely relied on combinations that havealso been shown to transform epithelial cells (Boehm andHahn, 2005).

Studies involving the genetic manipulation of humanECs have confirmed that perturbation of TP53 andp16INK4a-RB tumor suppressor pathways, together withthe activation of a telomere maintenance mechanism, arecrucial for EC immortalization. However, in contrast tocarcinomas, which predominantly rely on telomerase fortelomere maintenance, the limited literature availablesuggests that the activation of ALT might be relativelycommon in angiosarcoma. Nevertheless, ALT-immortalized EC lines are underrepresented among thecurrently available EC lines. In contrast to the well-documented approach to the reconstitution oftelomerase by overexpression of hTERT, there arecurrently no known means of directly activating ALT inprimary mammalian cells. However, studies that showedT

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spontaneous activation of ALT in SV40-transformed epithelial andfibroblastic cell lines allude to an indirect means of establishingALT-immortalized EC lines (Bryan et al., 1995).

The currently available genetic models might also be made moreclinically relevant by substituting viral oncogenes with short hairpinRNA (shRNA) targeting the specific tumor suppressor genesimplicated in EC malignancies, and by constitutive activation ofVEGF signaling. Conditional deletion of tumor suppressor genesspecifically within ECs would also provide a valuable alternativemurine model of endothelial neoplasia. Additional refinement ofgenetic models will then be contingent upon furthercharacterization of the specific molecular defects that underpin theinitiation and progression of specific vascular neoplasias. Althoughhampered by sample numbers, genome-wide mapping and next-generation sequencing might be applied toward this goal.Information from broad-platform technologies such as these willbe valuable for improving diagnosis, identifying therapeutic targets,and establishing new in vitro and in vivo models that accuratelydepict the molecular biology of specific EC-derived neoplasias.Such models will then provide an avenue for preclinical tests ofnew therapeutics and for ultimately improving outcomes forpatients with these disorders.COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

FUNDINGThe Children’s Cancer Institute Australia is affiliated with the University of NewSouth Wales and the Sydney Children’s Hospital Network. K.L.M. received researchgrants from National Health and Medical Research Council Career DevelopmentAward (510378), NSW Cancer Council and Cancer Institute NSW.

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