The Role of the Tumor Microenvironment in Regulating ...

The Role of the Tumor Microenvironmentin Regulating Angiogenesis

Randolph S. Watnick

Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115

Correspondence: [email protected]

The tumor-associated stroma has been shown to play a significant role in cancer formation.Paracrine signaling interactions between epithelial tumor cells and stromal cells are a keycomponent in the transformation and proliferation of tumors in several organs. Whereasthe intracellular signaling pathways regulating the expression of several pro- and antiangio-genic proteins have been well characterized in human cancer cells, the intercellular signalingthat takesplacebetween tumorcells and the surrounding tumor-associated stroma has not beenas extensively studied with regard to the regulation of angiogenesis. In this chapter we definethe key players in the regulation of angiogenesis and examine how their expression is regulatedin the tumor-associated stroma. The resulting analysis is often seemingly paradoxical, under-scoring the complexity of intercellular signaling within tumors and the need to better under-stand the environmental context underlying these signaling mechanisms.

In the earliest stages of cancer, epithelialtumors, carcinomas, are physically confined

within the region of the tissue from whencethey arise. These early lesions (carcinomas insitu) are separated from the tissue parenchymaby the basement membrane (Hanahan andWeinberg 2000). Opposite the basement mem-brane are a myriad of cells consisting of fibro-blasts, myofibroblasts, immune/inflammatorycells, and endothelial cells (Ronnov-Jessenet al. 1996). In addition to these cell types arethe extracellular matrix proteins which theysecrete and to which they, and tumor cells,attach (Ronnov-Jessen et al. 1996).

To progress to a clinically relevant andpotentially lethal disease, tumor cells mustalso acquire the ability to escape the confinesof the epithelial compartment and thus invade

locally and disseminate systemically. To makethis escape, tumor cells must degrade the base-ment membrane separating the epithelial com-partment from the tissue parenchyma. Theprocess of invading the tissue parenchyma, orbeing invaded by cells from the tissue paren-chyma, initiates a new phase of tumor progres-sion in which tumor growth becomes partiallyregulated by non-cell-autonomous processesregulated by paracrine and juxtacrine inter-actions with the tumor microenvironment(Chung and Davies 1996; Henshall et al. 2001;Tuxhorn et al. 2001). The tumor-associatedstroma provides oxygen and nutrients via thevasculature as well as soluble and matrix-boundgrowth factors and enzymes that promotetumor proliferation and progression (Hanahanand Folkman 1996). The evidence for stromal

Editors: Michael Klagsbrun and Patricia D’Amore

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Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a006676

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cells in tumor progression suggests that theyplay a key role in matrix remodeling, tumorinvasion, and metastatic spread (Picard et al.1986; Grey et al. 1989; Camps et al. 1990).More importantly, epithelial cancer cells areable to alter their surrounding stromal fibro-blasts to enhance tumor growth (Olumi et al.1999). Specifically, paracrine signaling interac-tions between epithelial tumor cells and stromalcells have been shown to be a key component inthe transformation and proliferation of tumorsin several organs (Cunha et al. 1985; Donjacourand Cunha 1991; Hom et al. 1998). It has beenshown, for instance, that stromal fibroblasts iso-lated from a prostate tumor induce tumor for-mation of immortal but nontransformedprostate epithelial cells when the mixture isinjected orthotopically into nude mice (Olumiet al. 1999).

One process in tumor progression enhancedby tumor–stromal signaling is the induction ofangiogenesis. As the endothelium does notcross the basement membrane in normal tissuearchitecture, in order for tumors to gain accessto blood vessels they must first invade the sur-rounding stroma. Once the tumor cells haveinvaded the tissue parenchyma they must beable to transmit paracrine signals to the stromalcells to induce a proangiogenic environment.Although a myriad of pro- and antiangiogenicfactors have been discovered and studied, themajor effort in understanding their regulationhas been in a cell-autonomous fashion. Todate, the tumor cell-autonomous regulation ofvascular endothelial growth factor (VEGF)(Rak et al. 1995; Damert et al. 1997; Wojtaet al. 1999; Xiong et al. 2001; Akiyama et al.2002) and Thrombospondin-1 (Tsp-1) (Raket al. 2000; Watnick et al. 2003), two of themajor positive and negative regulators of angio-genesis, have been described in detailed bio-chemical fashion.

The regulation of angiogenesis via signalingbetween epithelial tumor cells and stromalfibroblasts and endothelial cells may also beimportant in the establishment and prolifera-tion of metastases. It has been well documentedthat tumors from various organs have distinctmetastatic profiles (Chambers et al. 2002). For

example, prostate cancer metastasizes preferen-tially to bone and liver, whereas breast cancermetastasizes to bone and lung, although in xen-ograft models it is possible to isolate variants oftumor cell lines that metastasize preferentiallyto lymph nodes (Pettaway et al. 1996). The abil-ity of a tumor cell to survive and proliferate in ametastatic environment may ultimately rely onits ability to manipulate the angiogenicity ofthe stroma in this new environment.

The tumor-associated stroma, or tumormicroenvironment, can grossly be categorizedinto two types of cells: (1) cells that are presentin the normal tissue parenchyma before tumordevelopment; and (2) cells that are recruitedto the tumor-associated stroma from distal sites(i.e., the circulation or bone marrow). The firsttype is largely comprised of fibroblasts andendothelial cells, whereas the second type ofcells is largely comprised of immune/inflam-matory cells, including T- and B-cells, macro-phages, neutrophils, mast cells, and otherbone marrow–derived cells. In this work, thedifferent cell types, as well as the extracellularmatrix, and their contribution to tumor pro-gression will be detailed and explained.

FIBROBLASTS

The tissue parenchyma of most organs is largelycomprised of fibroblasts that, along with theextracellular matrix they secrete, make up thestructural scaffolding of organs (Fig. 1). Thereare two distinct types of fibroblasts in normaltissue: fibroblasts and myofibroblasts. Thesetwo types of fibroblasts are distinguished by,among other markers, the expression of smoothmuscle actin, which is expressed by myofibro-blasts but not normal fibroblasts. Fibroblasts,although morphologically distinct, are poorlydefined molecularly and were originally charac-terized more for what they are not: vascular,inflammatory, or epithelial cells (Tarin andCroft 1969). Fibroblasts are responsible for thesynthesis and deposition of the extracellularmatrix (ECM), the regulation of epithelial celldifferentiation, and the regulation of inflamma-tory response to tissue insults (Parsonage et al.2005; Tomasek et al. 2005). Fibroblasts are also

R.S. Watnick

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key mediators of wound repair, where theyinvade the wound, synthesize and secreteECM to anchor other cells recruited to the siteof the wound, and facilitate healing wound con-tractions via the contractile properties of colla-gen (Gabbiani 2003). Fibroblasts in woundrepair attain what has been termed an activatedphenotype (Castor et al. 1979).

Activated fibroblasts proliferate at a fasterrate, produce greater amounts of ECM and, asstated above, express a-smooth-muscle actin(Gabbiani 2003). Fibroblasts can be activatedby growth factors released from damaged epi-thelial cells, such as TGF-b and basic fibroblastgrowth factor (bFGF), or via direct cell–cellcontacts with leukocytes at the sites of wounds(Clayton et al. 1998; Choi and Tseng 2001).To accommodate the production and secretionof large amounts of ECM proteins, activatedfibroblasts contain an oval-shaped euchromaticnucleus, rough endoplasmic reticulum, andprominent Golgi apparatus (Castor et al.1979). Activated fibroblasts also produce pro-teases, such as matrix metalloproteases (MMPs)that degrade the ECM. These proteases aid inthe turnover and reorganization of the ECMand secrete growth factors like hepatocytegrowth factor (HGF) and bFGF (Rodemannand Muller 1991). Interestingly, once the wound

is repaired, the number of activated fibroblastsis greatly decreased, although the overall num-ber of fibroblasts in the area is not significantlychanged (Gabbiani 2003). Thus, it is not easilydiscernible whether the decrease in the numberof activated fibroblasts is caused by cell death orapoptosis and subsequent repopulation of nor-mal fibroblasts from neighboring tissue or ifthe activated fibroblasts revert back to normalfibroblasts. However, the general consensus isthat the activation is transient and once woundrepair is complete, the fibroblasts revert back toa quiescent phenotype.

CARCINOMA-ASSOCIATED FIBROBLASTS

Tumor progression from the in situ stage tometastatic disease has been shown to be pro-moted by fibroblasts present in the tumormicroenvironment (Elenbaas and Weinberg2001). These fibroblasts found in the tumormicroenvironment have been termed carci-noma-associated fibroblasts (CAFs) (Olumiet al. 1999). The molecular and genetic charac-terization of CAFs indicates that they aresimilar, if not identical, to activated fibroblastsfound in the stroma of tissues undergoingwound repair, described above (Durning et al.1984; Tsukada et al. 1987; Schor et al.

EndotheliumFibroblast

Immune cell

Stroma

Basementmembrane

ECM

Epithelial cells

Carcinoma cells

Figure 1. Schematic of tumor microenvironment.

Tumor Microenvironment in Regulating Angiogenesis

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1988a,b). Specifically, they express smoothmuscle actin, EGF (Normanno et al. 1995; Pan-ico et al. 1996), HGF (Montesano et al. 1991;Seslar et al. 1993; Jin et al. 1997; Vande Woudeet al. 1997; To and Tsao 1998), IGF-1 and -2(Yee et al. 1989; Cullen et al. 1992; Ellis et al.1994), and matrix remodeling enzymes suchas MMPs (Basset et al. 1990; Wolf et al. 1993;Basset et al. 1994; Engel et al. 1994; Newellet al. 1994; Heppner et al. 1996; Chambersand Matrisian 1997; Lochter et al. 1998; Massonet al. 1998; Noel et al. 1998; McCawley andMatrisian 2000).

Furthermore, in some cancers �80% of thefibroblasts within the tumor stroma are thoughtto become activated (Sappino et al. 1988). Thesignaling mechanism by which CAFs are gener-ated has not been conclusively demonstrated;however, in vitro studies have shown thatTGF-b can induce CAF-like properties in nor-mal fibroblasts (Ronnov-Jessen and Petersen1993). Moreover, studies suggest that humancarcinoma cells can convert normal fibroblastsinto CAFs in a mouse xenograft model (Orimoand Weinberg 2006). Once fibroblasts becomeCAFs, they can be cultured in the absence of car-cinoma cells and retain their CAF phenotype inculture until they undergo senescence (Orimoet al. 2005). It is interesting to note that chickensinfected with Rous sarcoma virus develop inva-sive carcinomas when wounded, showing the

oncogenecity of tumor stroma (Dolberg et al.1985). These studies show the importance oftumor stroma, and CAFs in particular, to theprocess of tumor progression.

When carcinomas progress to the invasivestate the basement membrane is degraded andstromal cells, including CAFs, inflammatoryresponse cells, and newly formed capillaries,come into contact with the tumor cells (Hana-han and Weinberg 2000). CAFs in the stromaof invasive carcinoma continue depositing largeamounts of ECM, including tenascin C in somecases (Chiquet-Ehrismann et al. 1986; Inagumaet al. 1988). It has been shown that in breast andbladder carcinomas expression of tenascin Ccorrelates with increased tumor invasiveness(Mackie et al. 1987; Brunner et al. 2004). Theaccumulation of ECM in tumors contributesto increased interstitial fluid pressure whichhinders oxygen and nutrient diffusion (Nettiet al. 2000; Brown et al. 2004). Thus, CAF-mediated hypoxia could lead to the expressionof HIF-1a and the induction of VEGF, thusproviding a mechanism by which CAFs can pro-mote angiogenesis in tumors (Fig. 2).

As stated above, CAFs are associated withtumor cells at most stages of cancer progres-sion. Many studies have shown the ability offibroblasts to promote cancer. For example,patients genetically predisposed to breast cancercontained skin fibroblasts that proliferated

FibroblastsBlood vessels

Tumor

Figure 2. Tumor–stromal paracrine signaling. Tumors secrete factors into the microenvironment that act on thestromal cells to either promote or inhibit the growth of new blood vessels (angiogenesis).

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more rapidly in vitro (Kopelovich 1982). It hasalso been shown that CAFs can promote tumorgrowth in a mouse xenograft model whereasnormal fibroblasts cannot (Olumi et al. 1999).Olumi et al. (1999) demonstrated that massivetumors grew in the grafts containing CAFs,whereas no tumors grew in grafts containingnormal fibroblasts. This shows how CAFs aidin the formation of tumors, probably throughinduction of tumorigenic changes in epithelialcells.

Tumor progression is also mediated byCAFs. It has recently been shown that mixinghuman breast carcinoma cells with CAFs in amouse xenograft gave rise to tumors that werelarger and more angiogenic than when mixedwith normal fibroblasts (Orimo et al. 2005).Furthermore, it was shown that the increase intumor cell proliferation was mediated by stro-mal cell–derived factor 1 (SDF1) secreted bythe CAFs binding to the CXCR4 receptor ontumor cells. Additionally, in this model it wasshown that secreted SDF1 stimulated angiogen-esis by recruitment of endothelial progenitorcells (EPCs) into the tumor. It has been previ-ously shown that EPCs are recruited duringtumor angiogenesis and differentiate into vas-cular endothelial cells (Lyden et al. 2001).

Another study showed the ability of CAFs toinduce invasiveness in vivo with rat colon carci-noma cells that were not invasive in vitro(Dimanche-Boitrel et al. 1994). CAFs alsosecrete MMPs that help degrade the basementmembrane and promote tumor invasion. Forexample, MMP3 secreted by CAFs can promotetumor cell invasiveness (Lochter et al. 1997).This is accomplished by MMP3-mediatedcleavage of the extracellular domain of the adhe-sive protein E-cadherin on the surface of mam-mary epithelial cells. Cleavage of E-cadherincauses mammary epithelial cells to disperseand undergo epithelial-to-mesenchymal transi-tion, which promotes tumor cell invasiveness.

CAFs have also been implicated in tumormetastasis, by promoting the proliferation oftumor cells at the metastatic site. For example,a hepatic metastatic cell line was shown tosecrete factors that activate fibroblasts in vitro(Olaso et al. 1997). These activated fibroblasts

were shown to be within the tumor stroma ofthe metastasis, and quiescent fibroblasts takenfrom the liver of mice were activated when cul-tured with conditioned media (CM) from themelanoma metastasis. The tumor CM inducedfibroblast migration, proliferation, and produc-tion of MMP2. This suggests that CAFs help tocreate a niche for tumor cells at metastatic sites(Olaso and Vidal-Vanaclocha 2003).

Yet another study showed that mice defi-cient for the Mts1 protein, which stimulatestumor metastasis, failed to grow metastaseswhen highly metastatic mammary carcinomacells were grafted onto these mice (Grum-Schwensen et al. 2005). Furthermore there wasa significant delay in tumor uptake as well adecrease in tumor incidence as compared towild-type mice injected with the carcinomacells. When the tumor cells were mixed withMts1 competent fibroblasts and injected intothe mts1 knockout mice, the ability of thesetumors to metastasize was partially restored.Additionally, it has been shown that condi-tioned media from metastatic human breastand prostate carcinoma cell lines are able torepress the expression of Tsp-1 in fibroblastsfrom tissues where the carcinoma is known tometastasize (Kang et al. 2009). This shows theability of tumors to prime metastatic sites forangiogenesis by decreasing the levels of anendogenous angiogenesis inhibitor.

It is clear that angiogenesis is an essentialstep in the progression and metastasis oftumors. Fibroblasts play an important role inpromoting not only tumor progression butangiogenesis as well. CAFs produce growth fac-tors like VEGF which aid in the recruitment andactivation of endothelial cells within the tumorstroma. During tumor invasion, CAFs producenot only angiogenic growth factors, but alsoproduce proteases which break down not onlythe basement membrane of the tumor-associated tissue but can break down the base-ment membrane of stromal blood vessels, anessential step in angiogenesis. Finally, duringtumor metastasis CAFs are able to create per-missive environments for tumor growth andangiogenesis at metastatic sites. Studies fromKalas et al. (2005) and Kang et al. (2009) have



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shown that tumors secrete factors that are ableto repress the expression of Tsp-1 in fibroblasts.These studies underscore the importance ofTsp-1 in the induction of angiogenesis. In orderfor tumor cells to induce angiogenesis, the bal-ance of angiogenic stimulators and inhibitorsmust be shifted toward induction of angiogenicstimulators in the tumor stroma.

CELL SIGNALING MECHANISMS ANDFACTORS INFLUENCING STROMALANGIOGENESIS

Vascular Endothelial Growth Factor

One of the most potent proangiogenic proteins,VEGF induces endothelial cell migration andproliferation as well as vascular permeability(Senger et al. 1983; Leung et al. 1989). The reg-ulation of VEGF in carcinoma cells has beenextensively studied. Signal transduction cas-cades emanating from Ras and PI3 kinase leadto the increased transcription of VEGF and con-sequently to its secretion into the extracellularspace (Rak et al. 1995). It has also been discov-ered that carcinoma cells secrete proteins intothe extracellular space that modulate VEGF pro-duction and secretion by stromal fibroblasts inthe tumor-associated stroma, such as TGF-b,platelet-derived growth factor (PDGF), andbFGF (Brogi et al. 1994; Tsai et al. 1995).

Stromal VEGF expression was first shown tobe regulated by carcinoma cells using a trans-genic mouse model in which GFP expressionwas driven by the VEGF promoter (Fukumuraet al. 1998). In this model, the activation ofthe VEGF promoter downstream from a signaltransduction cascade would result in the expres-sion of GFP. Examination of tumor xenograftsin these VEGF-GFP mice by fluorescencemicroscopy revealed that the fibroblasts infil-trating and immediately surrounding the carci-noma cells were bright green, indicating that theVEGF promoter had been activated. In theabsence of tumors, however, the fibroblasts innormal tissues in these mice did not expressGFP. These results indicated that tumorssecreted a protein into the extracellular spacethat stimulated VEGF expression in the stroma.

The significance of the stromal expression ofVEGF in tumor growth and progression was notimmediately obvious from these results. It wasclear that VEGF expression was being stimu-lated; however, it was not clear whether thisstimulation was required for tumor growth,supportive of tumor growth, or merely aby-product of tumor growth. The evidencethat stromally produced VEGF was importantfor tumor growth came from experiments test-ing the efficacy of the human specific anti-VEGF antibody bevacizumab (Avastin). In theseexperiments human tumor cells were implantedinto immunocompromised mice and the result-ing tumors were treated with the human-specific VEGF antibody (Kim et al. 1993).Whereas the antibody was effective at signifi-cantly slowing tumor growth, it was not 100%effective. It was postulated that perhaps a resid-ual angiogenic stimulus was being provided bythe production of murine VEGF, whose activitywas not inhibited by the human-specific anti-body. To test this hypothesis, human tumorxenografts were treated with human-specificVEGF antibodies as well as mFlt(1-3)-IgG,which inhibits both human and murine VEGFby acting as a decoy receptor (Gerber et al.2000). Treatment with both VEGF therapiesresulted in the complete blockade of tumorgrowth, thus demonstrating the requirementsfor tumor- and stromal-produced VEGF.

BASIC FIBROBLAST GROWTH FACTOR

Basic fibroblast growth factor (bFGF, FGF2), isanother extremely potent proangiogenic growthfactor (Shing et al. 1984; Klagsbrun et al. 1986;Folkman and Klagsbrun 1987). Interestingly,despite the presence of high-affinity cell surfacereceptors (Dionne et al. 1990) and the fact thatbFGF stimulates endothelial cell proliferationand angiogenesis both in vivo and in vitro, itlacks a signal sequence that would otherwisedirect its secretion (Abraham et al. 1986).Although the role of bFGF as a stimulator ofangiogenesis is unequivocal, its paracrine regu-lation in stromal fibroblasts and subsequenteffect on tumor angiogenesis is clouded by thefact that that it also potently stimulates tumor

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cell proliferation via FGFR signaling via bothautocrine and paracrine signaling (Rogelj et al.1988, 1989; Gleave et al. 1991). Nevertheless, ithas been shown that bFGF expression in thestroma of lung adenocarcinoma inversely corre-lates with disease progression and patient sur-vival (Guddo et al. 1999). In addition to itsparacrine regulation in fibroblasts, it has beenshown that stem cell factor (SCF) and TGF-bare potent stimulators of bFGF expression ininflammatory cells, including macrophages,mast cells, and neutrophils (Qu et al. 1998).The role of these cells in tumor angiogenesiswill be detailed later.

Thrombospondin-1

Tsp-1 is an endogenous inhibitor of angiogene-sis which functions via a bimodal approach: itbinds to CD36 on the endothelial cell surfaceand renders the cell insensitive to both VEGFand bFGF, via an as yet undetermined mecha-nism. Tsp-1 also binds to and functionally inac-tivates MMP-9, a MMP shown to liberate VEGFfrom the ECM (Bergers et al. 2000; Rodriguez-Manzaneque et al. 2001). One signal transduc-tion pathway that has been shown to inducethe repression of Tsp-1 leads from PI3-kinaseto the Rho GTPase to ROCK to Myc, whichrepresses Tsp-1 in a phosphorylation-depend-ent manner (Watnick et al. 2003). This pathwayhas been shown to be active in several humanbreast cancer cell lines in which Tsp-1 expres-sion was virtually silenced (Watnick et al.2003). Furthermore, in a majority of the sur-veyed breast cancer cell lines the pathwaypreviously described (Watnick et al. 2003) wasshown to be responsible for the silencing.Thus, this pathway represents the first biochem-ical elucidation of a cell-autonomous “angio-genic switch.”

Although the expression of VEGF in thetumor-associated stroma is widely accepted tohave a positive correlation with tumor progres-sion (Fukumura et al. 1998; Brown et al. 1999;Mueller and Fusenig 2002), the role of Tsp-1expression in the tumor-associated stroma isunclear. Tsp-1 expression by epithelial tumorcells is observed infrequently and ectopic

expression of Tsp-1 is inhibitory to tumorgrowth (Streit et al. 1999; Watnick et al. 2003).Stromal Tsp-1, meanwhile, has been correlatedwith a desmoplastic response and increasedinvasiveness in a subset of breast cancers(Wong et al. 1992; Bertin et al. 1997; Brownet al. 1999), whereas it has been shown to beinhibitory to early-stage breast cancers (Clezar-din et al. 1993). Expression of Tsp-1 by stromalfibroblasts has been shown to be inhibitory totumor formation and growth (Filleur et al.2001). Intriguingly, the same report showedthat tumors that arose in an environment highin Tsp-1 eventually overcame the inhibitoryeffects of this protein by increasing their pro-duction of VEGF. Thus, the complex interrela-tionship between these two proteins and theirrelative expression levels in the tumor-associated stroma can play a key role in theinduction and maintenance of the angiogenicphenotype in human tumors.

The work described above shows that VEGFexpression in the stroma is a critical componentin tumor-mediated angiogenesis. Conversely,Tsp-1 expression in the tumor-associatedstroma can be a potent inhibitor of tumorangiogenesis and growth. The question thatarises, then, is how do tumors stimulate theexpression of VEGF in the stroma while con-comitantly repressing the expression of Tsp-1?

TGF-b

One of the most interesting paradoxes involv-ing tumor-derived growth factors involves theeffects of TGF-b. It had been shown thatTGF-b displays a potent proangiogenic activityin vivo (Roberts et al. 1986). In seemingly dia-metric opposition are the in vitro data thatdemonstrate the growth-inhibitory activity ofTGF-b on cultured endothelial cells (Bairdand Durkin 1986; Frater-Schroder et al. 1986).These diverse activities were resolved withthe discovery that TGF-b stimulated the expres-sion of VEGF in both fibroblasts and tumorcells, indicating that tumor-secreted TGF-bcould elicit proangiogenic effects via the induc-tion of VEGF in stromal fibroblasts (Brogi et al.1994; Pertovaara et al. 1994). Moreover, TGF-b



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has been shown to have profound effects on theexpression of bFGF in fibroblasts, stimulatingits expression by more than sixfold (Goldsmithet al. 1991). These results suggest that at lowerlevels tumor-secreted TGF-bmay act on tumor-associated fibroblasts to stimulate VEGF andbFGF production to stimulate angiogenesis.Conversely, when expressed at higher levels,TGF-b may act directly on endothelial cells toinhibit their proliferation and thus inhibitangiogenesis.

Adding to the paradox of TGF-b’s role intumor angiogenesis is the fact that TGF-b stim-ulates the expression of Tsp-1 and, in turn,is activated by Tsp-1 (Penttinen et al. 1988;Murphy-Ullrich et al. 1992; Schultz-Cherryand Murphy-Ullrich 1993; Schultz-Cherryet al. 1994a,b). TGF-b is produced and secretedby cells in an inactive, latent form that cova-lently attached to the latent associated peptide.TGF-b can be activated by proteases that cleavethe latent form or by undergoing a conforma-tional change, induced by Tsp-1 binding thatexposes the receptor-binding region. TGF-bexpression in fibroblasts has also been shownto be induced by hypoxia (Falanga et al.1991). Thus, the Tsp-1-mediated inhibition ofangiogenesis would result in a state of hypoxia,which would then trigger the expression ofTGF-b by the tumor stroma. The latentTGF-b would subsequently be activated byTsp-1 and induce the expression of VEGF,whose expression would also be stimulated bythe Tsp-1-induced hypoxia (Brogi et al. 1994).In this way a tumor could overcome the effectsof Tsp-1 and induce angiogenesis. However, ifthe levels of Tsp-1 were so high as to inhibitthe excess VEGF produced, then the tumorwould remain in a state of dormancy until thebalance of angiogenic activity was tipped deci-sively toward the positive.

Intriguingly, although TGF-b induces theexpression of both VEGF and Tsp-1, it accom-plishes these diverse activities through the acti-vation of different transcription factors. TGF-bbinding to its receptors TGFbR1 and R2 acti-vates a signal transduction cascade which cul-minates in the activation of the smad family oftranscription factors (Hoodless et al. 1996; Liu

et al. 1996). The TGF-b-mediated stimulationof Tsp-1 expression is achieved via activationof Smad2, whereas stimulation of VEGF ismediated via activation of Smad3 (Nakagawaet al. 2004). Thus, depending on the contex-tual environment within the tumor-associatedstroma, TGF-b can stimulate the expression ofVEGF, Tsp-1, or both proteins.

Platelet-Derived Growth Factor

Another growth factor that seemingly defiescharacterization as a pro- or antiangiogenicprotein is PDGF. It was shown by Goldsmithet al. (1991) that PDGF was a potent stimulatorof bFGF. Specifically, treatment of lung fibro-blasts with recombinant PDGF resulted in atwofold increase in bFGF expression (Gold-smith et al. 1991). In response to the observa-tions described above that the contribution ofstromal VEGF needed to be inhibited to effec-tively block tumor growth, it was determinedthat stromal VEGF expression was stimulatedby tumor-derived PDGF (Dong et al. 2004).Furthermore, inhibition of PDGF activity viaadministration of soluble PDGFR abrogatedthe stimulation of VEGF in the tumor-associated stroma and inhibited tumor angio-genesis. Moreover, a second member of thePDGF family, PDGF B, had previously beenshown to up-regulate VEGF expression invascular smooth muscle cells (Brogi et al.1994). These data indicated that PDGF expres-sion by tumor cells was a potent inducer ofVEGF expression in the tumor-associatedstroma.

One conclusion that could be drawn fromthe above study was that PDGF expression bytumor cells promotes angiogenesis via stromalVEGF induction. As with TGF-b, however, theactivities of this growth factor are not as straightforward as these results would indicate. In addi-tion to its ability to stimulate VEGF and bFGFexpression to stimulate angiogenesis, PDGFhas also been shown to stimulate Tsp-1 expres-sion (Majack et al. 1987). The stimulation ofTsp-1 by PDGF was shown to be via the Raf-MAPK pathway in an analogous fashion to thestimulation of Tsp-1 by serum (Majack et al.

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1985). Intriguingly, unlike the differentialactivation of transcription factors by TGF-b,the PDGF-mediated stimulation of VEGF isalso via the Raf-MAPK pathway (Chang et al.2006). Thus, its effect on angiogenesis mediatedby tumor-associated fibroblasts is most likelymediated by the activation of different recep-tor/signal transduction pathways.

Hormones and Nuclear Receptors

The results presented above indicate that two ofthe most potent inducers of stromal VEGF and,in turn, angiogenesis also possess the seeminglycounterproductive ability to stimulate the ex-pression of Tsp-1. These seemingly diverseevents downstream from TGF-b and PDGFligation to its receptor indicate that tumor-derived TGF-b and PDGF expression shouldhave no net angiogenic activity, because theiractivity stimulates the expression of both pro-and antiangiogenic proteins. However, inhibi-tion of PDGF activity has been shown toinhibit tumor angiogenesis despite its effectson Tsp-1 (Dong et al. 2004). Similarly, TGF-bhas been shown to stimulate angiogenesis,again, despite its stimulatory effect on Tsp-1.One explanation for the observed proangio-genic activities of these two proteins is that theexpression of Tsp-1 in the tumor-associatedstroma is somehow suppressed by an indepen-dent signaling mechanism. The suppression ofTsp-1 would result in the net stimulation ofonly the proangiogenic growth factors VEGFand bFGF by these two growth factors andwould account for their observed proangiogenicactivity.

Two candidates for such a Tsp-1-repressingfactor are the hormones estrogen and androgen.Estrogen has, in fact, been shown to repress theexpression of Tsp-1 (Sengupta et al. 2004). Sim-ilarly, androgen has been shown to repress Tsp-1expression (Colombel et al. 2005). Althoughthese two hormones have similar effects onTsp-1 expression, the mechanisms by whichthey repress Tsp-1 are different. Estrogen inhib-ition of Tsp-1 is dependent on both ERK1/2and JNK activity (Sengupta et al. 2004). Fur-thermore, the repression of Tsp-1 expression

by estrogen appears to be mediated through acombination of transcriptional repression andinhibition of protein secretion. Conversely,androgen-mediated suppression of Tsp-1 ex-pression appears to be solely via transcriptionalrepression, as an androgen-responsive elementhas been identified in the Tsp-1 promoter(Colombel et al. 2005).

Whereas hormone-mediated effects ontumor growth have been largely studiedthrough their actions on hormone-responsivetumor cells, it has recently been shown thatestrogen can have a systemic proangiogeniceffect (Gupta et al. 2007). This study showedthat estrogen receptor (ER)-positive stromalcells stimulate angiogenesis and promote tumorgrowth in response to estrogen even for ER-neg-ative tumor cells. Although the regulation ofVEGF and Tsp-1 was not the focus of that study,it is not unreasonable to assume that the stimu-lation of estrogen-mediated stimulation ofangiogenesis observed could be partially medi-ated by a proangiogenic activity.

It has also been recently shown that anothernuclear receptor family, the peroxisomeproliferator-activator receptors (PPAR), canregulate the expression of VEGF and Tsp-1. Spe-cifically, it has been shown that tumor cellsinjected into PPARa2/2 mice failed to growbeyond a microscopic size (Kaipainen et al.2007). It was shown that the dormant state ofthese tumors was the result of increased Tsp-1expression in the host stroma. Surprisingly, itwas later determined that two ligands ofPPARa, fenofibrate and WY14643, also stimu-lated the expression of Tsp-1 (Panigrahy et al.2008). These seemingly discordant observa-tions with respect to Tsp-1 expression couldindicate that, in the absence of PPARa, anothermember of the PPAR family—perhaps PPARg,which has been shown to stimulate expressionof CD36 (Han et al. 2000), a receptor forTsp-1—may compensate and stimulate theexpression of Tsp-1. In keeping with theseobservations, it was shown that the PPARgagonists rosiglitazone and pioglitazone inhibitbFGF and VEGF-mediated angiogenesis in thechick chorioallantoic membrane assay (CAM)(Aljada et al. 2008).



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Nonprotein Mediators of Angiogenesis

The vast majority of paracrine signaling studiesfocus on the roles of cytokines and growthfactors. However, one largely understudied sig-naling mechanism, mediated by lipids andphospholipids, has recently been investigatedwith respect to the regulation of Tsp-1 expres-sion. Two independent studies revealed thatgeneration of phospholipids and the resultantdownstream signal transduction cascades couldpotently repress the expression of Tsp-1 in stro-mal fibroblasts. The first showed that generationof phospholipids, specifically sphingosine 1phosphate (S1P), mediated by platelets couldrepress the expression of Tsp-1 in dermal fibro-blasts via activation of Gi-protein-coupled S1Preceptors (Kalas et al. 2005). It has been widelynoted that platelets are commonly trapped intumors, most likely caused by the presence ofhigh levels of heparin-sulfates (Sack et al.1977). Thus, platelet-mediated repression ofTsp-1, and increased angiogenesis and tumorgrowth, may be a by-product of this seeminglyrandom event.

The second study investigating the role ofS1P in repression of Tsp-1 showed that Ras-transformed cells secrete a low molecularweight (,3 kD) molecule that represses Tsp-1in dermal fibroblasts via an S1P-dependentmechanism (Kalas et al. 2005). These resultssuggest that angiogenic tumor cells secrete fac-tors that actively repress Tsp-1 in the surround-ing tumor-associated stroma. As with theactivity of estrogen and androgen noted above,the secretion of factor(s) by tumor cells thatsuppress the expression of Tsp-1 in the tumor-associated stroma may be a critical process inthe escape from dormancy.

Matrix Metalloproteases

Local invasion across the basement membraneand within the tissue microenvironment oftumors is critical for tumor growth and ulti-mately progression to metastasis. One criticalstep in tumor migration and invasion is thedegradation of the ECM and the resultantremodeling. A key driver of matrix degradationand remodeling is the MMPs. For example,

it has been shown that co-injection of MCF7breast cancer cells with fibroblasts significantlyaccelerated tumor growth (Noel et al. 1993).When the fibroblasts were engineered toexpress TIMP-2 (tissue inhibitor of metallopro-tease 2), an inhibitor of MMP activity, thetumor-stimulating activity was lost (Noel et al.1998). Analogously, when a general inhibitorof MMP activity, batimastat, was administeredto mice that were co-injected with MCF7 cellsand fibroblasts the tumors grew at the samerate as MCF7 cells alone (Noel et al. 1998).

In addition to allowing tumor cells tomigrate and invade, MMP-mediated matrixremodeling also allows endothelial cells tomigrate and form the leading edge of new bloodvessels. Additionally, MMPs may function toliberate angiogenic growth factors like VEGFand bFGF that would otherwise be sequesteredby the ECM. Evidence for the role of MMPs inangiogenesis was generated by crossing tumor-prone RIP-TAG2 mice with different matrixprotease knockout mice (Bergers et al. 2000).In this model mice develop pancreatic islet celltumors driven by the expression of the SV40Large T antigen from the rat insulin promoter(Hanahan 1985). Crossing the RIP-TAG micewith MMP2 knockout mice impaired tumorgrowth but had no effect on angiogenesis(Bergers et al. 2000). Crossing the RIP-TAGmice with urokinase knockout mice had noeffect on tumor growth. However, crossing RIP-TAG mice with MMP9 knockout mice inhibitedtumor growth and angiogenesis (Bergers et al.2000). In addition to cleaving matrix proteins,it has also been shown that MMP9 can cleaveTGF-b, which is normally secreted as a pro-protein covalently bound to the latent associ-ated peptide and is thus inactive. The ability ofMMP9 to convert TGF-b from the latent toactive form has been shown to stimulate growthof mammary tumor model (Yu and Stamen-kovic 2000).

BONE MARROW–DERIVED CELLS

In addition to fibroblasts, the tumor microen-vironment is made up several other types ofcells that were present before tumorigenesis or

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migrated as a result. Most prominent amongthese cells are bone marrow–derived cells: mes-enchymal stem cells (MSCs), macrophages,neutrophils, mast cells, and T cells. These cellsmigrate in response to the growing tumormass, often interpreted as inflammation, andby the secretion of discrete growth factors andchemokines produced by the tumor cells.

Mesenchymal Stem Cells

MSCs are bone marrow–derived cells that havebeen characterized by the ability to differentiateinto a myriad of mesenchymal cells: fibroblasts,osteoblasts, chondrocytes, adipocytes, peri-cytes, and muscle cells. MSCs are an exceedinglyrare cell type within the bone marrow, compris-ing between 0.01% and 0.001% of the mononu-clear cells (Civin et al. 1996; Pittenger et al.1999). Human MSCs are defined by the expres-sion of cell surface markers: CD44 adhesionmolecule (HCAM), CD73, CD90, CD105(endoglin), CD106 (VCAM-1), and STRO-1(Dennis and Charbord 2002).

MSCs have been shown to be recruited tosites of wounding or inflammation, as well asto tumors (Hall et al. 2007). MSCs are recruitedto tumors by multiple different growth factorsand cytokines, including VEGF, bFGF, IL-8,EGF, HGF, and PDGF as well as CCL2, CCL7,and CXCL12 (SDF-1) (Schichor et al. 2006;Birnbaum et al. 2007; Dwyer et al. 2007; Kiddet al. 2008; Spaeth et al. 2008). In melanoma,a correlation has been demonstrated betweenMSCs and angiogenesis (Sun et al. 2005). Fur-thermore, following recruitment to the tumor,MSCs have been shown to secrete VEGF tostimulate angiogenesis (Coffelt et al. 2009).

In addition to correlation and expressionstudies examining the role of MSCs in angio-genesis, they have also been shown to berecruited and stimulate angiogenesis in vitroas well as in murine pancreatic xenografts(Beckermann et al. 2008). Tumors in miceinjected with wild-type, vector control MSCshad twice as many blood vessels as normaltumors. Conversely, tumors in mice injectedwith MSCs in which VEGF had been silencedby lentiviral VEGF shRNA had comparable

numbers of blood vessels to normal tumors(Beckermann et al. 2008). Thus, the ability ofMSCs to home to, produce, and secrete VEGFcan contribute to tumor growth via enhancedangiogenesis.

Macrophages

By far the most prevalent immune/inflamma-tory cell type present in tumors is the tumorassociate macrophage (TAM; Balkwill andMantovani 2001). Activated macrophages (i.e.,those that have been recruited to sites of inflam-mation) are generally categorized into twotypes, M1 and M2, depending on the type ofinflammation (Sher et al. 2003; Mantovaniet al. 2004; Balkwill et al. 2005). M1 macro-phages are effector cells that are able to potentlykill microorganisms as well as tumor cellsand secrete high levels of proinflammatorycytokines (Balkwill et al. 2005). M2 macro-phages can have different response phenotypesbased on the type of signals present in theinflammation. Thus, they are able to scavengedebris and stimulate angiogenesis, as well as tis-sue remodeling and repair (Goerdt and Orfanos1999; Mantovani et al. 2002; Gordon 2003;Mosser 2003; Balkwill et al. 2005). TAMs, tothe extent they have been studied, are most sim-ilar to, and share many properties with, M2macrophages.

TAMs have been shown to display a growth-promoting activity for both human and exper-imental tumor models (Crowther et al. 2001).TAMs are preferentially recruited to sites ofhypoxia which, in nontumorous tissue gener-ally, is symptomatic of wounded or infected tis-sue (Crowther et al. 2001). Hypoxia stimulatesthe activity of the transcription factor HIF-1which, in turn, stimulates the expression ofthe proangiogenic growth factors VEGF, bFGF,TNFa, and CXCL8 (Crowther et al. 2001).VEGF and bFGF directly stimulate endothelialmigration and proliferation leading to newblood vessel growth into the hypoxic region ofthe tumor. Additionally, hypoxia stimulatesthe secretion of CXCL12 (SDF1), which poten-tiates the activity of VEGF and bFGF on endo-thelial cells (Salcedo et al. 1999; Schioppa



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et al. 2003). In addition, SDF1 has been shownto induce angiogenesis by recruiting bone mar-row–derived endothelial precursor cells totumors (Orimo et al. 2005).

Although for most of the time the net resultof macrophage activity is growth promotingon tumors, macrophages can also inhibit thegrowth of tumors. Specifically, it has beenshown that CSF stimulates macrophage produc-tion and secretion of metalloelastase (Donget al. 1997, 1998). Metalloelastase is an extra-cellular protease which, among other substrates,cleaves plasminogen into multiple fragments,one of which is the antiangiogenic proteinangiostatin (O’Reilly et al. 1994). Thus, macro-phage activity with respect to tumor growth ishighly context dependent and can, in certaincircumstances, inhibit angiogenesis and tumorgrowth—in keeping with the macrophage’sgeneral role of protecting the body from disease.

Mast Cells

Mast cells are multifunctional secretory cells,characterized by numerous large electron-densegranules composed of proteoglycans, of whichheparin is the major component (Norrby2002). Mast cells descend from pluripotentbone marrow progenitor cells that express thecell surface markers CD34, c-kit, and CD13(Kirshenbaum et al. 1999). Mast cells in circula-tion are progenitor-like cells that differentiate/mature after being recruited to a given tissue.One consistent characteristic of precursormast cells is the ability to produce MMP9,which is essential for migration into differenttissue types (Tanaka et al. 1999). Mast cellsexpress a variety of proteases including chy-mases, tryptases, and MMPs, which are storedin secretory granules (Norrby 2002). These pro-teases, especially the MMPs, specifically MMP2and 9, are vital to mast cells’ ability to promotetissue remodeling and repair (Matrisian 1990).In addition to proteases, the secretory granulesof mast cells are also depots for cytokines andgrowth factors, including TNF-a, GM-CSF,SCF, bFGF, EGF, PDGF, VEGF, and IFN-g,IL-3, -4, -5, -6, -8, -10, -13, -14, and chemo-kines, such as (MIP)-1-a, I-309, (MCP)-1,

and lymphotactin (Norrby 2002). The releaseof proteases, cytokines, and growth factorsstored in the secretory granules of macrophagescan be triggered by multiple cytokines, includ-ing IL-1, IL-3, and GM-CSF, Platelet factor 4,IL-8, SCF, (MCP)-1, and MIP-1-a (Tazzymanet al. 2009). Moreover, mast cells also produceand secrete MMPs 2 and 9, which have beenshown to promote angiogenesis by liberatingVEGF and bFGF from the ECM (Coussenset al. 1999, 2000). Interestingly, mast cells havebeen shown to be recruited to tumors by theproangiogenic proteins VEGF, bFGF, andTGF-b (Gruber et al. 1994, 1995). Thus, condi-tions within a tumor that necessitate the growthof new blood vessels recruit mast cells, which inturn further stimulate angiogenesis.

Experimental evidence for the functionalrole of mast cells in angiogenesis and tumorgrowth was provided by an elegant murinegenetic model in which Myc expression inb cells was driven via fusion to a mutant formof the ER (Soucek et al. 2007). In this modelit was shown that Myc activation by systemicadministration of 4-hydroxy tamoxifen, in-duced b-cell tumors characterized by bloodvessel infiltration accompanied by mast cellrecruitment. These findings indicated thatmast cells are required for angiogenesis at theonset of tumorigenesis and for maintenanceof angiogenesis during tumor growth andprogression.

Neutrophils

Whereas macrophages, TAMS, are the mostprevalent and common leukocyte present inthe tumor microenvironment, neutrophils arethe most abundant leukocyte in the circulation(Tazzyman et al. 2009). Leukocytes originate inthe bone marrow from hematopoietic pluripo-tent stem cells and differentiate via a processtermed myelopoiesis. Neutrophil recruitmentfrom the bone marrow is, in part, mediated byCXCL12 (SDF-1) and its cognate receptor,CXCR4, is expressed at high levels on the cellsurface of neutrophils (Suratt et al. 2004). Thereare two types of neutrophils present in thecirculation: circulating neutrophils which, as

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their name suggests, are freely circulating; andmarginated neutrophils, which are bound tothe endothelium of small blood vessels (Tazzy-man et al. 2009). The marginated pool can bemobilized into the circulating pool by the cyto-kines such as IL-6 (Steele et al. 1987; Suwa et al.2000). Neutrophils from the circulating pool arethose recruited to sites of inflammation andtumors (Kanwar and Cairo 1993; Friedman2002).

Elevated levels of neutrophils have beenobserved in multiple human tumors, includingcolon, lung, myxofibrosarcoma, gastric carci-noma, and melanoma (Nielsen et al. 1996;Bellocq et al. 1998; Mentzel et al. 2001;Mhawech-Fauceglia et al. 2006). In additionto CXCL12, one of the most potent chemo-attractants of neutrophils is CXCL8, which isexpressed by both tumor and stromal cells ofmany types of human tumors (Bellocq et al.1998; Xie 2001). Once recruited to tumors, neu-trophils are able to stimulate angiogenesis bydirectly secreting VEGF and by secretingMMPs, which can release angiogenic growthfactors such as VEGF and bFGF from theirsequestration by the ECM (Coussens andWerb 1996; Gaudry et al. 1997).

Experimentally, it has been shown that, in agenetic murine model of squamous cell skincarcinoma, the MMP9 produced and secretedby neutrophils is required for the angiogenicswitch (Coussens et al. 2000). In this model, itwas observed that the source of MMP9 in theskin tumors was not from the tumors them-selves but from neutrophils. These results havesince been recapitulated by anti-GR1-mediatedneutrophil ablation in the RIP-TAG2 islet celltumor model and in a human ovarian cancerxenograft model in MMP9 deficient mice(Huang et al. 2002; Nozawa et al. 2006). Thus,it is now firmly established that neutrophilsare important and, in some cases, required fortumor angiogenesis.

Like macrophages and mast cells, neutro-phils also possess antitumor activity. For exam-ple, as early as 1975 it was observed thatneutrophils could kill tumor cells (Clark andKlebanoff 1975). It was originally thought thatthe killing was mediated exclusively by myelo-

peroxidase. However, it has since been shownthat neutrophils can kill tumor cells by multipledifferent mediators, including the release ofproteases, membrane perforating agents, reac-tive oxygen species, and cytokines such asTNFa and IL-1b (Di Carlo et al. 2001). More-over, neutrophils can inhibit angiogenesis viatwo distinct mechanisms, both of which aremediated by the protease neutrophil elastase.First, neutrophil elastase can inhibit angio-genesis by degrading VEGF and bFGF (Aiet al. 2007). Second, neutrophil elastase cancleave plasminogen into angiostatin, whichinhibits VEGF- and bFGF-mediated angiogene-sis (Scapini et al. 2002). Thus, this is anotherexample of a cell type which can influenceangiogenesis in the opposite way dependingon the contextual signals within the tumormicroenvironment.

CONCLUSIONS

Angiogenesis is a complex process driven bymany different growth factors and cytokinesand inhibited by a diverse range of proteins.As such, the regulation of angiogenesis bythe tumor microenvironment is equally, ifnot more, complex. The signaling moleculessecreted by tumors that act on stromal cells ina paracrine fashion can often have differentactivities with respect to the production andsecretion of pro- and antiangiogenic proteins.As such, the composition of the tumor micro-environment as well as the stage of the tumorhave profound effects on determining whetherthe tumor microenvironment is proangiogenicand growth promoting or antiangiogenic andthus growth inhibitory. The complex signalingmechanisms provide a myriad of potential,and as yet largely untapped, targets for thera-peutic intervention to inhibit tumor growth inpatients. Ultimately, the strategy of targetingtumor–stromal signaling molecules may proveto be hugely successful as the accounts ofgenomic instability and mutation in the stromaare exceedingly rare. As such, stromal-basedantiangiogenic therapy may encounter lessacquired resistance than traditional therapeuticstrategies.



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Michael O'Hare and Joseph F. Arboleda-VelasquezPatterningThe Beauty and Complexity of Blood Vessel

Victoria L. Bautch and Yoh-suke MukouyamaMicroRNAs in Mechanical Homeostasis

Jeremy A. Herrera and Martin A. SchwartzLeukocyte Trafficking in Lymphatic Vessels

Aline Bauer, Hazal Tatliadim and Cornelia HalinHuman Endothelial Colony-Forming Cells

Juan M. Melero-Martin Job in Vascular Decision-MakingEndothelial Cell Fate Determination: A Top Notch

KitajewskiL.A. Naiche, Stephanie R. Villa and Jan K.

Lymphatics in Cardiovascular Physiology

MitchellDakshnapriya Balasubbramanian and Brett M. Vitro Modeling and In Vivo Regeneration

Lymphatic Tissue and Organ Engineering for In

Anna M. Kolarzyk, Gigi Wong and Esak Lee

InteractionsEndothelial Cell−Mechanisms Regulating T Cell

Pilar AlcaideSpecificationEndothelial Cell Differentiation and Hemogenic

Jordon W. Aragon and Karen K. Hirschi

Models of Vascular AgingBeyond Static Pipes: Mechanisms and In Vitro

Martin W. Hetzer and Simone Bersini

Lymphedema and ObesityChristopher L. Sudduth and Arin K. Greene

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