Myeloid Cells and Lymphangiogenesis - CSHL Pperspectivesinmedicine.cshlp.org/content/2/6/a006494...Myeloid Cells and Lymphangiogenesis Adrian Zumsteg and Gerhard Christofori Institute

Myeloid Cells and Lymphangiogenesis

Adrian Zumsteg and Gerhard Christofori

Institute of Biochemistry and Genetics, Department of Biomedicine, University of Basel,CH-4058 Basel, Switzerland

Correspondence: [email protected]

The lymphatic vascular system and the hematopoietic system are intimately connected inontogeny and in physiology. During embryonic development, mammalian species derivea first lymphatic vascular plexus from the previously formed anterior cardinal vein,whereas birds and amphibians have a lymphatic vascular system of dual origin, composedof lymphatic endothelial cells (LECs) of venous origin combined with LECs derived frommesenchymal lymphangioblasts. The contribution of hematopoietic cells as buildingblocks of nascent lymphatic structures in mammals is still under debate. In contrast, theimportance of myeloid cells to direct lymphatic vessel growth and function postnatally hasbeen experimentally shown. For example, myeloid cells communicate with LECs viaparacrine factors or cell–cell contacts, and they also can acquire lymphatic endothelial mor-phology and marker gene expression, a process reminiscent of developmental vasculogen-esis. Here, we present an overview of the current understanding of how lymphatic vesselsand the hematopoietic system, in particular myeloid cells, interact during embryonic devel-opment, in normal organ physiology, and in disease.

THE INTERRELATIONSHIP OF LYMPHATICENDOTHELIUM AND HEMATOPOIETICCELLS DURING DEVELOPMENT

The current understanding of the origin of thelymphatic vascular system is that it fully

(mammals) or partially (birds, amphibians)originates from venous blood endothelial cells(Oliver 2004). The endothelial system is also,at an earlier stage, giving rise to hematopoieticstem cells, though mainly from arterial struc-tures within the embryo proper and also fromextraembryonic structures like the yolk sac andthe placenta (Cumano and Godin 2007). Theontological relationship between the hemato-poietic and the endothelial system is still under

intensive investigation. In mice, definitive he-matopoietic stem cells are derived from a so-called hemogenic endothelium. Lineage tracingstudies have shown that most leukocytes origi-nate from cells expressing endothelial markers(found on blood vessel endothelial cells [BECs]during development and in the adult), in-cluding vascular endothelial growth factorreceptor-2 (VEGFR-2), vascular endothelial cad-herin (VE-Cadherin) or Tie-2/TEK (Motoikeet al. 2003; Li et al. 2006; Chen et al. 2009).Hence, a hematopoietic system cannot be estab-lished in the absence of a primitive endothelialsystem.

The question arises whether, in the conversedirection, the hematopoietic system also has an

Editors: Michael Klagsbrun and Patricia D’Amore

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influence on the development of the vascularsystem. Analysis of mice deficient for certainhematopoietic compartments suggests that inthe presence of sufficient erythropoiesis andvessel flow most leukocytes are dispensable forthe generation of a functional blood andlymphatic vascular system. Several differentleukocyte-deficient mouse lines have beenestablished, including lymphocyte-deficientRag knockout mice (Mombaerts et al. 1992;Shinkai et al. 1992), macrophage-deficientop/op mice (Yoshida et al. 1990) and mast cell–deficient KitW-sh/W-sh-mice (Grimbaldestonet al. 2005); they all display normal blood vesseland lymphatic vessel development. Even in micedeficient for the transcription factor Runx1,which show severely impaired hematopoiesisduring development and die in utero, primitivelymph sacs develop normally (Srinivasan et al.2007). Reports describing mesenchymal cellswith macrophage and LEC characteristicswithin and around developing murine lym-phatic vessels nourish the speculation that alsoin mammals, there may be a dual origin of thelymphatic vascular system (Buttler et al. 2006,2008). However, functional inactivation of theProx1 transcription factor in cells derivedfrom the venous endothelium severely impairslymph sac development, suggesting that Prox1-expressing venous endothelial cells are the mainsource of LECs during lymphatic vasculaturedevelopment in the mouse (Srinivasan et al.2007). In addition, lineage-tracing studies usingthe Vav gene promoter driving Cre-mediatedinheritable expression of yellow fluorescent pro-tein (YFP) in definitive hematopoietic cells hasnot revealed any direct contribution of hemato-poietic cells to the formation of lymphatic ves-sels (Bertozzi et al. 2010).

However, the complete separation of lym-phatic sacs from the cardinal veins seems tocritically depend on the interaction of theendothelial and the hematopoietic systems.Failure in this separation is apparent by theoccurrence of cutaneous hemorrhage duringembryonic development and blood-filledlymphatic vessels. This phenotype has beenobserved in several murine models deficientfor genes important for LEC differentiation

and identity. For example, the glycoproteinpodoplanin expressed on LECs binds the recep-tor CLEC-2 on platelets to induce platelet ag-gregation via the activation of a downstreamcascade in platelets involving the signalingmolecules Syk, Slp-76 and phospholipase-Cg2.Activation of this signaling pathway appears tobe crucial for the establishment of separatedvenous and lymphatic endothelial systems by athus far unknown mechanism (Abtahian et al.2003; Bertozzi et al. 2010; Uhrin et al. 2010).However, as podoplanin deficiency reveals amuch less severe phenotype than Syk deficiencyin mice, there must be additional effects of Sykexpression on lymphatic development. Indeed,lineage tracing experiments have identified aSyk-expressing, prolymphangiogenic myeloidpopulation in the skin of murine embryos thatwas dramatically increased on Syk depletion,leading to lymphatic vessel hyperplasia andblood-lymphatic vessel shunts (Bohmer et al.2010). These results study suggest that Syk func-tions in leukocytes to repress the recruitment ofprolymphangiogenic myeloid cells to the skinand thus prevents overshooting lymphatic ves-sel sprouting. In contrast, the lack of macro-phages (as seen in op/op mice) in skin has noor only a very moderate effect on developmentallymphangiogenesis.

In conclusion, the lymphatic endothelialsystem in mammals seems to be exclusivelyderived from the blood endothelial compart-ment, and the contribution of hematopoieticcells integrating into lymphatic structures iseither very small or absent. In contrast, plateletaggregation seems to be indispensable for a cor-rect separation of the lymphatic and the venousendothelial systems.

LEUKOCYTE-MEDIATED REMODELINGAND EXPANSION OF LYMPHATICENDOTHELIUM

During embryonic development, the lympha-tic vascular system emerges independent ofthe hematopoietic system with the exceptionof the platelet-triggered separation of bloodand lymphatic vascular structures. However,studies in mice have shown the influence of

A. Zumsteg and G. Christofori

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macrophages on postnatal lymphatic vesseldevelopment. Analysis of osteopetrotic op/opmice, which are deficient for macrophagecolony stimulatory factor (M-CSF) and displaymarkedly reduced osteoclast and macrophagenumbers, has revealed that lymphatic vesselbranching is reduced in 15-d-old mice bythe absence of macrophages in the trachea andthe skin and that tissue fluid drainage isimpaired in limbs and ears of these mice. Inter-estingly, lymphatic vessels appear normal in3-mo-old op/op mice, indicating a role of mac-rophages in early lymphatic vessel patterningbut not in the physiological functions of estab-lished lymphatic vessels (Baluk et al. 2005).Accordingly, inhibition of the M-CSF receptorc-fms in adult mice has no measurable effecton the lymphatic vascular system. However,when studying the role of leukocytes duringvascular remodeling, one always has to considerthat some myeloid cells have been shown toexpress not only prolymphangiogenic factors,such as VEGF-A, -C, and -D, but also theirreceptors VEGFR-1 and VEGFR-3. These over-lapping expression patterns of receptors andligands renders the interpretation of experi-mental results in mouse models difficult, as itwill sometimes not be possible to assign thecausative cell type.

Infection of the upper respiratory tract inmice by Mycoplasma pulmonis is associatedwith dramatic architectural changes in the wallsof the airways and in the vasculature they con-tain (Baluk et al. 2005). Within 2 wk, blood ves-sels and lymphatic vessel undergo a dramaticangiogenic expansion. Blood vessel remodelingseems to be driven by the inflammatory func-tion of tumor necrosis factor-a (TNF-a) stim-ulating its receptor TNFR1, independent ofVEGF-A (Baluk et al. 2009). TNFR1 expressionis mainly detected on BECs but not on LECs,and the expansion of the lymphatic vasculaturein this model has been shown to depend onVEGF-C and/or VEGF-D, classical lymphan-giogenic factors (Baluk et al. 2005). In thesame model, an unexpected role for adaptiveimmunity has been discovered: Upon infectionof lymphocyte-deficient Rag1 – / – mice, theexpansion of the blood and lymphatic system

was severely compromised as compared to im-muno-competent mice. Deposition of immu-noglobulin G (IgG) produced by B cells in aT-cell-dependent manner appears to be essen-tial for vascular remodeling in this model.Notably, IgG deposited at the site of infectionmay have a role in recruiting and activatinginfiltrating neutrophils and monocytes (Auroraet al. 2005).

Lymphatic vessel expansion is also criticalduring infection and inflammation in theskin and their subsequent resolution. Follow-ing an inflammatory reaction, the local lym-phatic vessels may be less functional due totissue edema and high leukocyte infiltration.However, within days, afferent lymphaticvessels connecting to draining lymph nodeswill expand, thereby acquiring a higher capacityto transport antigens and antigen-presentingcells (APCs) to the lymph nodes. In several dif-ferent mouse models, it is VEGF-A, the potentblood endothelial mitogenic factor, ratherthan the lymphangiogenic VEGF-C or D thatinduces inflammation-mediated lymphangio-genesis. In parallel to the peripheral afferentlymphatic vessels, also within the draininglymph nodes lymphatic vessels proliferate(sinusoidal hyperproliferation). B cells havebeen shown to be crucially involved in lymphnode lymphangiogenesis, because after immu-nization with keyhole limpet hemocyanin themitogenic response of lymphatic vessels in thelymph node is diminished in B cell–deficientmice. In fact, B cells have been identified as aprominent source of VEGF-A (Angeli et al.2006). In contrast, different inflammatory stim-uli (e.g., inducing a delayed type hypersensitiv-ity reaction in ears by oxazolone application)rather identify the inflamed tissue as theprimary source of VEGF-A, and lymph nodelymphangiogenesis seems a consequence ofdrainage of VEGF-A to the lymph nodes, inde-pendent of B cells (Halin et al. 2007). However,the main source of VEGF is under debate. Forexample, it has been shown that VEGF-A orVEGF-C expressed as transgenes under the ker-atinocyte-specific K14 promoter induces tumorlymphangiogenesis and lymph node lymphan-giogenesis and promote metastasis to the lymph


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node in chemical carcinogenesis models (Hira-kawa et al. 2005, 2006). Conversely, CD11bþ/Gr1þ macrophages infiltrating the skin onapplication of bacterial polysaccharides arealso an important source of VEGF-A, C, andD. Macrophage depletion by treatment withliposome-encapsulated clodronate (Clodrolip)attenuates inflammation-associated cutaneousand lymph node lymphangiogenesis, as well aslymph flow and inflammatory cell transport tothe draining lymph nodes (Kataru et al. 2009).A critical role of VEGFR1 tyrosine kinaseactivity in VEGF-A-mediated macrophagerecruitment has been shown by the transplanta-tion of VEGFR1 tyrosine kinase–deficient(Vegfr1 tk – / – ) bone marrow cells into miceoverexpressing VEGF-A in the ear skin. Macro-phage recruitment, lymphangiogenesis, andblood vessel angiogenesis were significantlydecreased in mice transplanted with bone mar-row cells from Vegfr1 tk – / – mice, as comparedto mice transplanted with wild-type bone mar-row (Fig. 1) (Murakami et al. 2008). Theseexperiments suggest that VEGF-A expressionhas different consequences: Locally, it inducesangiogenesis, lymphangiogenesis, and vesselpermeability. In parallel, it induces recruitmentand facilitates extravasation of leukocytes,which are themselves prominent producers ofangiogenic factors like VEGF-A, -C, and -Dand also deliver inflammatory cytokines likeTNF-a, which can further increase the localproduction of VEGF-C and change the expres-sion of endothelial cell adhesion molecules crit-ical for leukocyte adhesion.

In the peritoneum of mice, LECs are alsoimportant early players in orchestrating theinflammatory response: Via Toll-like receptor4 (TLR4) they can sense bacterial lipopolysac-charide (LPS) and through activation of theNF-kB pathway express leukocyte chemoattrac-tants, such as CCL2, CCL5, and CX3CL1, topromote macrophage homing to the draininglymphatic vessels (Kang et al. 2009). Theinfiltrating macrophages provide VEGF-C and-D to induce lymphangiogenesis in the perito-neal and the pleural side lymphatic vessels ofthe diaphragm, as well as in the central tendonand the lymph nodes. Depletion of these

macrophages using Clodrolip or trapping ofthe lymphangiogenic VEGF-C and -D by solu-ble VEGFR-3 represses this process. Moreover,LPS or TNF-a can induce the expression ofthe LEC-specific transcription factor Prox-1,followed by Prox-1/NF-kB synergistic promo-tion of VEGFR-3 expression. Higher levels ofVEGFR-3 will render LECs more susceptibleto stimulation by suboptimal concentrationsof VEGF-C and -D (Flister et al. 2010). Impor-tantly, intraperitoneally injected LPS causes anextremely prominent inflammatory infiltratein the diaphragm, with increased fibrosis andreduced lymphatic drainage capacity withinthe lymphatic vessels and to the lymph nodes(Kim et al. 2009). Hence, acute inflammation,even though leading to active lymphangio-genesis, can reduce lymphatic vessel drainagecapacity, probably through fibrotic depositsperturbing lymphatic vessel function.

A further challenge for a sole passive role ofthe lymphatic vascular system in simply offeringa transport route for APCs to draining lymphnodes comes from the finding that ICAM-1on LECs binds to CD11b on dendritic cells(DCs), thereby attenuating DC maturation andthe DC’s ability to stimulate T-cell proliferation.Importantly, the suppressive effect of the lym-phatic endothelium on DCs has been observedonly in the absence of pathogen-derived signals(Podgrabinska et al. 2009). This implies that ina steady-state flow of DCs to the lymph nodes,in the absence of a pathogenic signal, the lym-phatic endothelium ensures an even-temperedstate of the immune system. Conversely, de-tection of pathogens via Toll-like receptors ex-pressed on LECs ablates their suppressivefunction on APCs. Also, D6, a scavenger recep-tor for CC chemokines mainly expressed onLECs, has been shown to modulate the localchemokine microenvironment and by thismechanism control local inflammatory cellinfiltration (Nibbs et al. 2007).

Impaired wound healing is a common com-plication of diabetes, and it has been shown thatlymphangiogenesis can be a rate-limiting stepduring wound healing. Promotion of lymphan-giogenesis by delivery of lymphangiogenicgrowth factors can accelerate wound healing in



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an excision skin-wounding model in db/db dia-betic mice (Saaristo et al. 2006). The investiga-tors have identified an increased recruitmentof VEGFR-3-positive macrophages on VEGF-Cexpression and speculate that the recruitedinflammatory cells support wound healing.Other work has subsequently revealed thatdb/db mice have a reduced capacity for macro-phage mobilization and that these macrophagesare in a hyporeactive state, probably caused by

hyperglycemia of the diabetic mice. Activationof hyporeactive macrophages isolated fromdb/db using IL-1b restores their expression ofVEGFR-3 and VEGF-C in vitro. When theseIL-1b-activated macrophages are applied onexcision wounds in db/db mice, an accelerationof wound closure is observed (Maruyama et al.2007).

An additional role of macrophages in main-taining skin homeostasis via control of the

(2) Macrophage recruitment

(1) Direct stimulation

(3) Transdifferentiation

(1) Direct stimulation

Infection/ inflammation High-salt diet

VEGF-R2

VEGF-A/-C/-D

Cancerous/inflamed tissue

Blood vessel

VEGF-R1/R3

VEGF-R2/R3

Lymphatic vessel

VEGF-R3

FGFRTLR

VEGF-CVEGF-C/-D

NF-κB TonEBP

Figure 1. Macrophage involvement in adult pathological lymphangiogenesis. Macrophages contribute inthree complementary ways to lymphangiogenesis in pathological situations. (1) Direct stimulation: Inflam-mation, infection, or high-salt diet can activate VEGF-C and -D expression in tissue-resident macrophagesvia NF-kB and TonEBP transcription factors. (2) Immune amplification by angiogenesis-promoting factors.Tissue-derived VEGF-A, -C, and -D can recruit peripheral monocytes via VEGFR-1 and -3. VEGFs alsodirectly stimulate angiogenesis, lymphangiogenesis, and vessel permeability. Recruited monocytes eventuallydifferentiate into macrophages and function as in (1). (3) Peripheral monocytes of unknown polarizationcan intravasate from the bloodstream into sites of ongoing lymphangiogenesis and integrate into growinglymphatic vessels. In vitro, this conversion is dependent on signaling from Toll-like receptor (TLR) andfibroblast growth factor receptors (FGFR). Monocytes/macrophages are indicated in blue with ruggedcell shapes.



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lymphatic vasculature is the regulation of bloodpressure. Experimentally induced hypertonicaccumulation of Naþ in the skin of rats fed ahigh-salt diet (HSD) leads to hypertension.On an HSD, the mononuclear phagocyte system(MPS), including macrophages and DCs, isorchestrating the expansion of the lymphaticcapillary network in the skin (Machnik et al.2009). Lymphatic capillary density in the skinincreases twofold on an HSD, an increase thatis paralleled by higher MPS cell density. Impor-tantly, MPS cell depletion using Clodrolip abro-gates lymphatic vessel hyperplasia and furtherincreases hypertension. The MPS system there-fore regulates a buffering system that attenuatesHSD-triggered hypertension. Molecularly, thelymphangiogenic growth factor VEGF-C isinduced in MPS cells by the tonicity-responsiveenhancer binding protein (TonEBP), which onelevated Naþ levels acts as a transcriptional acti-vator. VEGF-C then directly induces lymphaticvessel hyperplasia and the expression of endo-thelial nitric oxide synthase (eNOS). Mechanis-tically, increased lymph drainage capacity andeNOS-mediated vasodilation are thought tocounteract hypertension (Machnik et al. 2009).

LEUKOCYTES, TUMORLYMPHANGIOGENESIS, ANDLYMPH NODE METASTASIS

The role of leukocytes in cancer progression hasbeen extensively investigated, and experimentalstudies in mice and rats as well as histologicalcorrelation analysis in patients have firmlyestablished a protumorigenic, proangiogenicbranch of the hematopoietic system (Allavenaet al. 2008; Zumsteg and Christofori 2009).For example, high levels of tumor-associatedmacrophages (TAM) in human epithelialtumors usually correlate with poor prognosis,with few exceptions (Bingle et al. 2002). Therole of leukocytes, especially of myeloid cells,on tumor lymphangiogenesis has been lessintensively studied. Above, we have illustratedthe importance of myeloid cells and B cell in dif-ferent models of inflammatory lymphangio-genesis. The pathologist Rudolf Virchow,already in the 19th century, regarded cancer as

“wounds that never heal,” anticipating inflam-mation as an integral part of tumorigenesis.Therefore, many concepts described for inflam-mation also hold true for the interaction ofinflammatory cells and lymphangiogenesis dur-ing carcinogenesis.

In human cancer patients, metastatic spread-ing of tumor cells to regional lymph nodes (sen-tinel lymph nodes) is considered an importantindicator of the likelihood of metastasis toother organs. Correlation studies in humancancers, including breast, head and neck, andmelanoma, link the expression of VEGF-Cand/or VEGF-D to peritumoral lymphangio-genesis, intralymphatic tumor cell clusters,and lymph node metastasis (Kinoshita et al.2001; Stacker et al. 2002; Dadras et al. 2005;Siriwardena et al. 2008). Other correlative stud-ies imply inflammatory cells and processesinvolved in regulation of VEGF-C expressionin lymphangiogenesis and lymph node metas-tasis. For example, VEGF-C expression corre-lates with COX-2 expression in breast cancer(Zhang et al. 2008), and VEGF-C expression isstimulated by the inflammatory mediatorsinterleukin-1b, TNF-a, and COX-2 and itsinduced prostaglandins (Ristimaki et al. 1998;Su et al. 2004). In head and neck cancer, theactivity of inducible nitric oxide synthase(iNOS) is found to correlate with VEGF-C ex-pression, lymphangiogenesis, and lymph nodemetastasis, and the human epidermoid carci-noma cell line A431 up-regulates VEGF-C onnitric oxide (NO) treatment (Franchi et al.2006). iNOS can be produced by tumor or stro-mal cells, including macrophages, and is in-duced by inflammatory cytokines or LPS.Interestingly, iNOS is found to activate COX-2by S-nitrosylation, thereby enhancing its activ-ity (Kim et al. 2005). As high NO productionis a feature of M1 polarized macrophages, whichare antimicrobial and tumoricidal (Allavenaet al. 2008), it may be a critical integrator of pro-tective inflammation and subsequent resolu-tion, which also includes lymphangiogenesis.

Inflammatory cells are also a source of lym-phangiogenic factors in tumors: In human cer-vical cancer, a fraction of TAM expressesVEGF-C and D, and the levels of TAM correlate



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with peritumoral lymphatic vessel density.Moreover, naıve CD14 monocytes isolatedfrom healthy donors express VEGF-C onTNF-a or LPS stimulation in vitro (Schopp-mann et al. 2002). Notably, TAM also expressVEGFR-3, and murine peritoneal macrophagesare chemoattracted by VEGF-C in transwellassays, attributing VEGF-C also an immuno-modulatory function (Skobe et al. 2001).

Experimental studies in mice have shown adirect connection between inflammatory cellsand tumor lymphangiogenesis. For example,tumor lymphangiogenesis and lymph nodemetastasis are repressed with the pharmacolog-ical inhibition of COX-2 in mouse models oforthotopic gastric carcinoma and carcinoma-tous peritonitis (Iwata et al. 2007). Inhibitionof COX-2 in macrophages and tumor cellsresults in a reduction of VEGF-C expressionmainly by macrophages. In a closely relatedmodel, intraperitoneal injection of human ovar-ian cancer cell lines in mice induced carcinoma-tosis and lymphangiogenesis in diaphragm andmesentery (Jeon et al. 2008). However, similarto the situation in LPS-induced peritoneal lym-phangiogenesis, this lymphangiogenesis is dys-functional as assessed by tracer injection intothe peritoneum (Kim et al. 2009). A high num-ber of inflammatory cells is observed at theperitoneal side of the diaphragm of thesemice, mainly CD11bþ/LYVE-1þ macrophagesexpressing VEGF-A, -C, and -D, and depletionof macrophages by Clodrolip reduced the extentof aberrant lymphangiogenesis.

Only few studies employed geneticallymodified mice to explore the interactions ofleukocytes and tumor lymphangiogenesis. In amurine transgenic breast cancer model, macro-phages have been shown to be important for theangiogenic switch as well as for promotion ofmetastasis, as these parameters were severelyimpaired in op/op mice (deficient for mostmyelomonocytic cell types) as compared towild-type mice (Lin et al. 2001, 2006). However,effects on tumor lymphangiogenesis have notbeen assessed. In a subcutaneous osteosar-coma model, inhibition of the M-CSF/c-fmsaxis by anti-c-fms antibody or by pharmacolog-ical inhibition severely reduced macrophage

infiltration, tumor angiogenesis, lymphangio-genesis, lymphatic tumor drainage, and meta-stasis (Kubota et al. 2009).

A critical role in mobilization and recruit-ment of inflammatory cells to primary tumorsand metastatic sites has been assigned toVEGFR-1, also by VEGFR-1-expressing in-flammatory cells playing a pivotal role in thedevelopment of resistance to antiangiogenictreatment and in preparing the “premetastaticsoil” (Kaplan et al. 2005; Bergers et al. 2008).VEGFR-1 is a receptor for the angiogenicgrowth factors VEGF-A, VEGF-B, and placentalgrowth factor (PlGF), and its function hasbeen implicated in the tumor recruitment ofmacrophages. In particular, recent studiesusing anti-PlGF antibodies illustrate thatPlGF can be the main factor attracting macro-phages to tumors, where they provide angio-genic and lymphangiogenic growth factors(Fischer et al. 2007; Van de Veire et al. 2010).However, the efficacy of anti-PlGF treatmentand the molecular mechanisms underlyingthe repression of tumor angiogenesis is stilldebated (Bais et al. 2010). For example, B16melanomas grow to the same extent in micecarrying a nonfunctional VEGFR-1 tyrosinekinase domain (Vegfr1 tk– / – ) as compared towild-type mice, a tumor model that was previ-ously shown to be sensitive to anti-PlGF treat-ment. Further studies are warranted to resolvethis issue.

In conclusion, tumor-associated or tumor-activated leukocytes can actively participate inpromoting lymphangiogenesis at the tumorsite and within draining lymph nodes, bothprocesses that may facilitate lymph node meta-stasis. Macrophages can be potent producers oflymphangiogenic growth factors and theirdepletion in many murine models severelyimpairs lymphatic vessel function. Macro-phages can also trigger the expression of lym-phangiogenic growth factors in stromal cellsor cancer cells via the secretion of inflamma-tory cytokines. Overshooting recruitment ofinflammatory cells, as seen in models of peri-toneal lymphangiogenesis, can result in de-creased lymph drainage capacity even in astate of lymphatic vessel hyperplasia. This is



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an important finding, because it uncoupleslymphatic vessel density and drainage functionat inflammatory sites.

MYELOID-ENDOTHELIAL PLASTICITYIN PATHOLOGICAL INFLAMMATIONAND CANCER

Recent findings have challenged the notion thata cell’s identity cannot be changed once differ-entiated, or can only change along a defined(tissue) stem cell–progenitor cell–differenti-ated cell axis (e.g., in the hematopoietic system).Experimentally, cells have been converted intodifferent lineages (e.g., somatic cells of variousorigins into induced pluripotent cells or acinarcells of the pancreas into insulin producingb-cells) by the forced expression of certain tran-scription factors (Takahashi et al. 2007; Zhouet al. 2008). Cellular conversions have alsobeen observed in the absence of experimentalmanipulation (e.g., in metaplasia in which cellsconvert from one cell type into another with adifferent morphology and function [Slack2007]). Also in cancer, epithelial–mesenchymaltransition (EMT) is observed, believed to resultfrom a combination of cell intrinsic events(oncogene activation, tumor suppressor func-tion loss) and cell extrinsic factors (e.g., TGF-b signaling) (Thiery and Sleeman 2006). Suchcell plasticity has also been observed in endo-thelial cells: BECs give rise to fibroblasts thatdeposit extracellular matrix proteins in a mousemodel of cardiac fibrosis in a process referred toas endothelial–mesenchymal transition (Zeis-berg et al. 2007). These examples illustrate thatconversions from one cell fate into another arelikely to be associated with inflammation, ascheme holding true also for the plasticityobserved between hematopoietic and (lymph)endothelial cells.

Contribution of hematopoietic cells duringembryonic development to the lymphatic endo-thelial vasculature through processes referred toas transdifferentiation, or transdeterminationhas not been observed, at least in mice in whichstringent lineage-tracing studies are feasible.However, an increasing number of reports showplasticity of myeloid cells in pathophysiological

processes. These cells can display a mixed mye-loid/endothelial phenotype, they are derivedfrom the bone marrow and they have been func-tionally and structurally implicated in inflam-matory lymphangiogenesis.

The idea that vasculogenesis occurs inadults and not exclusively during embryonicdevelopment has been prompted by the findingthat bone marrow–derived cells can integrateinto growing blood vessels, cells called bytheir marker profile rather than by their func-tionality endothelial progenitor cells (EPCs).The initial descriptions of EPCs have beenbased on the characterization of CD34þ (anendothelial marker) cells isolated from bloodand cultured under proangiogenic conditions(Asahara et al. 1997). EPC characteristicsare now generally thought to reside withinthe CD14þ/CD34low-monocytic fraction ofperipheral (human) blood (Romagnani et al.2005). The occurrence of lymphvasculogenesis,however, by bone marrow–derived progenitorcells and/or cellular conversion, has been lessintensely studied. The identification of aCD133þ/VEGFR-3þ subfraction of CD34þ

cells in human fetal liver (Salven et al. 2003)and of a CD14þ/VEGFR-3þ subfraction inhuman peripheral blood (Schoppmann et al.2002) suggested that (1) during development(and maybe also postnatally) lymphatic endo-thelial progenitor cells exist, and (2) monocytes(CD14þ) could be responsive to lymphan-giogenic growth factors via their VEGFR-3expression.

Particularly instructive have been studiesundertaken in the cornea and the conjunctivaof the murine eye, which under inflammationshow a marked influx of inflammatory cellswith robust hemangiogenesis and lymphangio-genesis. The cornea is usually avascular, butunder induced inflammatory conditions likecornea transplantation, suturing, electric cau-tery or angiogenic pellet placement, blood andlymphatic vessels can grow from the conjunc-tiva through the limbus into the cornea. DCsin the cornea and monocytic cells in the con-junctiva express VEGFR-3 (Hamrah et al.2003, 2004). Corneal inflammation, triggeredby electric cautery, induces a rapid influx of



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CD11cþDCs into the stroma of the cornea. Thedendritic cells then express VEGF-C and alsoreallocate VEGFR-3 from an intracellular tomembranous location (Hamrah et al. 2003).In a similar inflammation model, intrastromalcorneal suturing causes a rapid influx of inflam-matory Gr1þ neutrophils and F4/80þ macro-phages into the cornea, concomitant withactivation of hemangiogenesis and lymphan-giogenesis (Cursiefen et al. 2004). Depletionof VEGF-A using a receptor trap has diminishedrecruitment of inflammatory cells and angio-genic responses. Similarly, inhibition of inflam-matory cell infiltration, either of all bonemarrow–derived cells by irradiation orof monocytic cells by Clodrolip treatment,abolishes angiogenic responses, suggesting astringent requirement of inflammatory cellinfiltration for hemangiogenesis and lymphan-giogenesis. CD11bþ myeloid cells were shownto express VEGF-C and D 48 h after injury,thus inducing lymphangiogenesis.

Inflammatory cells themselves can also becomponents of the newly formed lymphaticvessels. In a cornea transplantation model,newly formed corneal LYVE-1þ lymphatic ves-sels originate from bone marrow–derivedCD11bþ macrophages and also express theLEC-specific transcription factor Prox-1 (Mar-uyama et al. 2005). Direct evidence for denovo lymphatic vessel formation is providedby the ex vivo culturing of previously unchal-lenged, isolated corneas (not containing anyendothelial cells but CD11bþ myeloid cells),which under IL1b stimulation developLYVE-1þ/CD31þ structures in absence of anyconnected endothelial structure. This studyand a second one, placing FGF2-loaded micro-pellets in corneas, also show integration of bonemarrow–derived cells into newly formed lym-phatic vessels in the cornea, illustrating therapid mobilization of bone marrow cells andrecruitment to the cornea on inflammation(Religa et al. 2005).

Also in humans, there is evidence for re-cruitment of lymphatic endothelial progenitorcells during lymphangiogenesis. An elegant ret-rospective study with sex-mismatched, rejectedkidney transplants shows the integration of

recipient-derived cells into lymphatic vessels,constituting about 4.5% of all LECs in therejected organ (Kerjaschki et al. 2006). Con-trary, in control organs of sex-mismatchedbone marrow transplantation recipients, nei-ther intestinal nor skin lymphatic vesselsshowed integration of bone marrow–derivedcells (Fig. 2). Along these lines, in biopsies ofhuman Onchocerca (a filarial nematode caus-ing skin disease and river blindness) nodules,LYVE-1-positive macrophages were identifiedintegrated into lymphatic vessels in the fibrouscapsule of the nodule (Attout et al. 2009).Finally, in human idiopathic pulmonary fibro-sis, alveolar lymphangiogenesis correlates withthe disease severity. CD11bþ macrophagesfrom bronchoalveolar lavage fluid from pulmo-nary fibrosis patients, but not from healthy sub-jects, are able to form tube like structures invitro expressing the LEC markers LYVE-1 andpodoplanin (El-Chemaly et al. 2009). Theseresults indicate that inflammation is a prerequi-site for the potential transdifferentiation ofmyeloid cells into LECs.

The contribution of bone marrow–derivedcells to tumor lymphangiogenesis is very con-troversial. In mice subcutaneously transplantedwith B16F1 melanoma and Lewis lung carci-noma and previously transplanted with geneti-cally labeled bone marrow, no integration ofbone marrow–derived cells into tumor-associ-ated lymphatic vessels has been observed (Heet al. 2004). In contrast, contribution of bonemarrow cells has been observed in subcutane-ous T241 fibrosarcoma tumors and in thegenetic ApcMin/þ model of intestinal adenoma(Religa et al. 2005; Jiang et al. 2008). In thetransgenic Rip1Tag2 mouse model of pancreaticb-cell carcinogenesis and in subcutaneouslygrown TRAMP-C1 prostate adenocarcinoma,a combination of adoptive bone marrow trans-fer and genetic lineage tracing experiments hasrevealed that bone marrow–derived cells ofthe myeloid lineage integrate into tumor-associated lymphatic vessels (Zumsteg et al.2009). Furthermore, in vitro differentiated mac-rophages are able to aggregate into lymphaticlike structures and, while losing the expressionof macrophage markers, to express LEC



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Bone marrow transplantationA

B

Irradiation

Bone marrowtransplantation(GFP labeled)

Lineage tracing

CAG-promoter

CD11b-promoter

LYV

E-1

/GF

P

Cre recombinaseMyeloid cell–specific recombination

and GFP expression in progeny

StopIoxP IoxP

EGFP

CAG-promoter EGFP

Integration intotumor lymphatics

Integration intotumor lymphatics

Figure 2. Experimental demonstration of monocyte-lymphatic endothelial plasticity. (A) Transplantation ofGFP-labeled bone marrow is a strategy to identify bone marrow–derived cells in diverse tissues of the recipient,including tumors. Alternatively, using Cre/Lox-mediated lineage-tracing strategy, myeloid cells expressing Crerecombinase under the myeloid specific CD11b promoter can activate a GFP reporter transgene and thus labelall cells that have been passed through a CD11b-expressing lineage. Bone marrow transplantation and lineagetracing can also be combined. (B) Transplantation of GFP-labeled bone marrow into double-transgenicRip1Tag2;Rip-VEGF-C mice that develop pancreatic b-cell tumors with a high extent of peritumoral lymphan-giogenesis and lymph node metastasis. A histological section has been stained with antibodies against the lym-phatic marker LYVE-1 (red) and GFP (green). DAPI staining visualizes nuclei (blue). Note a GFPþ, bonemarrow–derived cell (green) integrated in a tumor-surrounding LYVE-1þ lymphatic vessel (red), as indicatedby an arrowhead and shown in higher-magnification insets.



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markers, such as podoplanin, LYVE-1, Prox1,and FoxC2. The molecular link between in-flammation and the transdifferentiation poten-tial of macrophages, however, is only poorlyunderstood.

What could be the role of macrophagesacquiring lymphatic endothelial characteristicin inflammation, be it tissue wounding or incancer? One speculation is that macrophagescan de novo form lymphatic tubes in previouslyavascular organs, such as the cornea, to speedup immune reactions and overcome the veryslow ingrowth of distant lymphatic vessels bysprouting lymphangiogenesis. The presence ofmacrophages in basically all organs and theirfast mobilization and replenishment makesthese cells ideally suited for this task. Anotherfunction could be to support endothelialsprouting, as macrophages are a rich source ofproteinases that are needed to degrade theECM. This guidance might be more efficientwhen macrophages employ homotypic inter-actions with their followers, like the expressionof VE-cadherin. Normally, macrophages andother APCs are designed to transmigratethrough endothelial sheets, and only the acquis-ition of lymphatic traits may allow stable inte-gration of macrophages into a lymphaticendothelial cell collective.

In conclusion, this article indicates thatmacrophages may serve a dual role in lymphan-giogenesis, either acting directly as lymphaticendothelial progenitors or indirectly by provid-ing lymphangiogenic growth factors. Becausemonocytes/macrophages are sensitive to stim-uli directly promoting angiogenesis and lym-phangiogenesis, they can potentiate initiallyweak proangiogenic signaling cues and, thus,may offer an appropriate target for the designof antiangiogenic and antilymphangiogenictherapy.

ACKNOWLEDGMENTS

We apologize to all colleagues whose importantwork we could not cite due to space restrictions.Research in the laboratory of the authors relatedto this review article has been supported bythe NCCR Molecular Oncology of the Swiss

National Science Foundation, the EU-FP6framework programme LYMPHANGIOGE-NOMICS LSHG-CT-2004-503573, and theSwiss National Science Foundation.

REFERENCES

Abtahian F, Guerriero A, Sebzda E, Lu MM, Zhou R, MocsaiA, Myers EE, Huang B, Jackson DG, Ferrari VA, et al.2003. Regulation of blood and lymphatic vascular separa-tion by signaling proteins SLP-76 and Syk. Science 299:247–251.

Allavena P, Sica A, Solinas G, Porta C, Mantovani A. 2008.The inflammatory micro-environment in tumor pro-gression: The role of tumor-associated macrophages.Crit Rev Oncol Hematol 66: 1–9.

Angeli V, Ginhoux F, Llodra J, Quemeneur L, Frenette PS,Skobe M, Jessberger R, Merad M, Randolph GJ. 2006. Bcell-driven lymphangiogenesis in inflamed lymph nodesenhances dendritic cell mobilization. Immunity 24:203–215.

Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R,Li T, Witzenbichler B, Schatteman G, Isner JM. 1997.Isolation of putative progenitor endothelial cells forangiogenesis. Science 275: 964–967.

Attout T, Hoerauf A, Denece G, Debrah AY, Marfo-Debrekyei Y, Boussinesq M, Wanji S, Martinez V, MandS, Adjei O, et al. 2009. Lymphatic vascularisation andinvolvement of Lyve-1þ macrophages in the humanonchocerca nodule. PLoS One 4: e8234.

Aurora AB, Baluk P, Zhang D, Sidhu SS, Dolganov GM,Basbaum C, McDonald DM, Killeen N. 2005. Immunecomplex-dependent remodeling of the airway vascula-ture in response to a chronic bacterial infection. J Immu-nol 175: 6319–6326.

Bais C, Wu X, Yao J, Yang S, Crawford Y, McCutcheon K, TanC, Kolumam G, Vernes JM, Eastham-Anderson J, et al.2010. PlGF blockade does not inhibit angiogenesisduring primary tumor growth. Cell 141: 166–177.

Baluk P, Tammela T, Ator E, Lyubynska N, Achen MG, Hick-lin DJ, Jeltsch M, Petrova TV, Pytowski B, Stacker SA, et al.2005. Pathogenesis of persistent lymphatic vessel hyper-plasia in chronic airway inflammation. J Clin Invest115: 247–257.

Baluk P, Yao LC, Feng J, Romano T, Jung SS, Schreiter JL, YanL, Shealy DJ, McDonald DM. 2009. TNF-a drives remod-eling of blood vessels and lymphatics in sustained airwayinflammation in mice. J Clin Invest 119: 2954–2964.

Bergers G, Hanahan D. 2008. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 8: 592–603.

Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,Chen M, Chen CY, Xu B, Lu MM, Zhou D, et al. 2010.Platelets regulate lymphatic vascular developmentthrough CLEC-2-SLP-76 signaling. Blood 116: 661–670.

Bingle L, Brown NJ, Lewis CE. 2002. The role of tumour-associated macrophages in tumour progression: Im-plications for new anticancer therapies. J Pathol 196:254–265.

Bohmer R, Neuhaus B, Buhren S, Zhang D, Stehling M,Bock B, Keifer F. 2010. Regulation of developmental



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



lymphangiogenesis by Sykþ leukocytes. Dev Cell 18:437–449.

Buttler K, Kreysing A, von Kaisenberg CS, Schweigerer L,Gale N, Papoutsi M, Wilting J. 2006. Mesenchymal cellswith leukocyte and lymphendothelial characteristics inmurine embryos. Dev Dyn 235: 1554–1562.

Buttler K, Ezaki T, Wilting J. 2008. Proliferating mesodermalcells in murine embryos exhibiting macrophage and lym-phendothelial characteristics. BMC Dev Biol 8: 43.

Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA.2009. Runx1 is required for the endothelial to haemato-poietic cell transition but not thereafter. Nature 457:887–891.

Cumano A, Godin I. 2007. Ontogeny of the hematopoieticsystem. Annu Rev Immunol 25: 745–785.

Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radzie-jewski C, D’Amore PA, Dana MR, Weigand SJ, StreileinJW. 2004. VEGF-A stimulates lymphangiogenesis andhemangiogenesis in inflammatory neovascularizationvia macrophage recruitment. J Clin Invest 113: 1040–1050.

Dadras SS, Lange-Asschenfeldt B, Velasco P, Nguyen L, VoraA, Muzikansky A, Jahnke K, Hauschild A, Hirakawa S,Mihm MC, et al. 2005. Tumor lymphangiogenesis pre-dicts melanoma metastasis to sentinel lymph nodes.Mod Pathol 18: 1232–1242.

El-Chemaly S, Malide D, Zudaire E, Ikeda Y, Weinberg BA,Pacheco-Rodriguez G, Rosas IO, Aparicio M, Ren P, Mac-Donald SD, et al. 2009. Abnormal lymphangiogenesis inidiopathic pulmonary fibrosis with insights into cellularand molecular mechanisms. Proc Natl Acad Sci 106:3958–3963.

Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S,Pattarini L, Chorianopoulos E, Liesenborghs L, KochM, De Mol M, et al. 2007. Anti-PlGF inhibits growth ofVEGF(R)-inhibitor-resistant tumors without affectinghealthy vessels. Cell 131: 463–475.

Flister MJ, Wilber A, Hall KL, Iwata C, Miyazono K, NisatoRE, Pepper MS, Zawieja DC, Ran S. 2010. Inflammationinduces lymphangiogenesis through up-regulation ofVEGFR-3 mediated by NF-kB and Prox1. Blood 115:418–429.

Franchi A, Massi D, Santucci M, Masini E, Degl’InnocentiDR, Magnelli L, Fanti E, Naldini A, Ardinghi C, CarraroF, et al. 2006. Inducible nitric oxide synthase activity cor-relates with lymphangiogenesis and vascular endothelialgrowth factor-C expression in head and neck squamouscell carcinoma. J Pathol 208: 439–445.

Grimbaldeston MA, Chen CC, Piliponsky AM, Tsai M, TamSY, Galli SJ. 2005. Mast cell–deficient W-sash c-kitmutant KitW-sh/W-sh mice as a model for investigatingmast cell biology in vivo. Am J Pathol 167: 835–848.

Halin C, Tobler NE, Vigl B, Brown LF, Detmar M. 2007.VEGF-A produced by chronically inflamed tissue induceslymphangiogenesis in draining lymph nodes. Blood 110:3158–3167.

Hamrah P, Chen L, Zhang Q, Dana MR. 2003. Novel expres-sion of vascular endothelial growth factor receptor(VEGFR)-3 and VEGF-C on corneal dendritic cells. AmJ Pathol 163: 57–68.

Hamrah P, Chen L, Cursiefen C, Zhang Q, Joyce NC, DanaMR. 2004. Expression of vascular endothelial growth

factor receptor-3 (VEGFR-3) on monocytic bonemarrow-derived cells in the conjunctiva. Exp Eye Res79: 553–561.

He Y, Rajantie I, Ilmonen M, Makinen T, Karkkainen MJ,Haiko P, Salven P, Alitalo K. 2004. Preexisting lymphaticendothelium but not endothelial progenitor cells areessential for tumor lymphangiogenesis and lymphaticmetastasis. Cancer Res 64: 3737–3740.

Hirakawa S, Kodama S, Kunstfeld R, Kajiya K, Brown LF,Detmar M. 2005. VEGF-A induces tumor and sentinellymph node lymphangiogenesis and promotes lymphaticmetastasis. J Exp Med 201: 1089–1099.

Hirakawa S, Brown LF, Kodama S, Paavonen K, Alitalo K,Detmar M. 2006. VEGF-C-induced lymphangiogenesisin sentinel lymph nodes promotes tumor metastasis todistant sites. Blood 109: 1010–1017.

Iwata C, Kano MR, Komuro A, Oka M, Kiyono K, JohanssonE, Morishita Y, Yashiro M, Hirakawa K, Kaminishi M,et al. 2007. Inhibition of cyclooxygenase-2 suppresseslymph node metastasis via reduction of lymphangiogen-esis. Cancer Res 67: 10181–10189.

Jeon BH, Jang C, Han J, Kataru RP, Piao L, Jung K, Cha HJ,Schwendener RA, Jang KY, Kim KS, et al. 2008. Profoundbut dysfunctional lymphangiogenesis via vascular endo-thelial growth factor ligands from CD11bþmacrophagesin advanced ovarian cancer. Cancer Res 68: 1100–1109.

Jiang S, Bailey AS, Goldman DC, Swain JR, Wong MH,Streeter PR, Fleming WH. 2008. Hematopoietic stemcells contribute to lymphatic endothelium. PLoS ONE3: e3812.

Kang S, Lee SP, Kim KE, Kim HZ, Memet S, Koh GY. 2009.Toll-like receptor 4 in lymphatic endothelial cells contrib-utes to LPS-induced lymphangiogenesis by chemotacticrecruitment of macrophages. Blood 113: 2605–2613.

Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L,Costa C, MacDonald DD, Jin DK, Shido K, Kerns SA,et al. 2005. VEGFR1-positive haematopoietic bone mar-row progenitors initiate the pre-metastatic niche. Nature438: 820–827.

Kataru RP, Jung K, Jang C, Yang H, Schwendener RA, BaikJE, Han SH, Alitalo K, Koh GY. 2009. Critical role ofCD11bþ macrophages and VEGF in inflammatory lym-phangiogenesis, antigen clearance, and inflammationresolution. Blood 113: 5650–5659.

Kerjaschki D, Huttary N, Raab I, Regele H, Bojarski-Nagy K,Bartel G, Krober SM, Greinix H, Rosenmaier A,Karlhofer F, et al. 2006. Lymphatic endothelial progenitorcells contribute to de novo lymphangiogenesis in humanrenal transplants. Nat Med 12: 230–234.

Kim SF, Huri DA, Snyder SH. 2005. Inducible nitric oxidesynthase binds, S-nitrosylates, and activates cyclooxyge-nase-2. Science 310: 1966–1970.

Kim KE, Koh YJ, Jeon BH, Jang C, Han J, Kataru RP,Schwendener RA, Kim JM, Koh GY. 2009. Role ofCD11bþ macrophages in intraperitoneal lipopolysac-charide-induced aberrant lymphangiogenesis and lym-phatic function in the diaphragm. Am J Pathol 175:1733–1745.

Kinoshita J, Kitamura K, Kabashima A, Saeki H, Tanaka S,Sugimachi K. 2001. Clinical significance of vascularendothelial growth factor-C (VEGF-C) in breast cancer.Breast Cancer Res Treat 66: 159–164.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Kubota Y, Takubo K, Shimizu T, Ohno H, Kishi K, ShibuyaM, Saya H, Suda T. 2009. M-CSF inhibition selectivelytargets pathological angiogenesis and lymphangiogene-sis. J Exp Med 206: 1089–1102.

Li Z, Chen MJ, Stacy T, Speck NA. 2006. Runx1 function inhematopoiesis is required in cells that express Tek. Blood107: 106–110.

Lin EY, Nguyen AV, Russell RG, Pollard JW. 2001. Colony-stimulating factor 1 promotes progression of mammarytumors to malignancy. J Exp Med 193: 727–740.

Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzeski DA, QianH, Xue XN, Pollard JW. 2006. Macrophages regulate theangiogenic switch in a mouse model of breast cancer.Cancer Res 66: 11238–11246.

Machnik A, Neuhofer W, Jantsch J, Dahlmann A, TammelaT, Machura K, Park JK, Beck FX, Muller DN, Deer W,et al. 2009. Macrophages regulate salt-dependent volumeand blood pressure by a vascular endothelial growthfactor-C-dependent buffering mechanism. Nat Med 15:545–552.

Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H,Tomita M, Van Rooijen N, Takenaka H, D’Amore PA,Stein-Streilein J, et al. 2005. Inflammation-induced lym-phangiogenesis in the cornea arises from CD11b-positivemacrophages. J Clin Invest 115: 2363–2372.

Maruyama K, Asai J, Ii M, Thorne T, Losordo DW, D’AmorePA. 2007. Decreased macrophage number and activationlead to reduced lymphatic vessel formation and contrib-ute to impaired diabetic wound healing. Am J Pathol 170:1178–1191.

Mombaerts P, Iacomini J, Johnson RS, Herrup K, TonegawaS, Papaioannou VE. 1992. RAG-1-deficient mice have nomature B and T lymphocytes. Cell 68: 869–877.

Motoike T, Markham DW, Rossant J, Sato TN. 2003. Evi-dence for novel fate of Flk1þ progenitor: Contributionto muscle lineage. Genesis 35: 153–159.

Murakami M, Zheng Y, Hirashima M, Suda T, Moriat Y,Ooehara J, Ema H, Fong GH, Shibuya M. 2008. VEGFR1tyrosine kinase signaling promotes lymphangiogenesis aswell as angiogenesis indirectly via macrophage recruit-ment. Arterioscler Thromb Vasc Biol 28: 658–664.

Nibbs RJ, Gilchrist DS, King V, Ferra A, Forrow S, HunterKD, Graham GJ. 2007. The atypical chemokine receptorD6 suppresses the development of chemically inducedskin tumors. J Clin Invest 117: 1884–1892.

Oliver G. 2004. Lymphatic vasculature development. NatRev Immunol 4: 35–45.

Podgrabinska S, Kamalu O, Mayer L, Shimaoka M, SnoeckH, Randolph GJ, Skobe M. 2009. Inflamed lymphaticendothelium suppresses dendritic cell maturation andfunction via Mac-1/ICAM-1-dependent mechanism.J Immunol 183: 1767–1779.

Religa P, Cao R, Bjorndahl M, Zhou Z, Zhu Z, Cao Y. 2005.Presence of bone marrow-derived circulating progenitorendothelial cells in the newly formed lymphatic vessels.Blood 106: 4184–4190.

Ristimaki A, Narko K, Enholm B, Joukov V, Alitalo K. 1998.Proinflammatory cytokines regulate expression of thelymphatic endothelial mitogen vascular endothelialgrowth factor-C. J Biol Chem 273: 8413–8418.

Romagnani P, Annunziato F, Liotta F, Lazzeri E, MazzinghiB, Frosali F, Cosmi L, Maggi L, Lasagni L, Scheffold A,et al. 2005. CD14þCD34 low cells with stem cell pheno-typic and functional features are the major source of cir-culating endothelial progenitors. Circ Res 97: 314–322.

Saaristo A, Tammela T, Farkkila A, Karkkainen M, Suomi-nen E, Yla-Hertuala S, Alitalo K. 2006. Vascular endothe-lial growth factor-C accelerates diabetic wound healing.Am J Pathol 169: 1080–1087.

Salven P, Mustjoki S, Alitalo R, Alitalo K, Rafii S. 2003.VEGFR-3 and CD133 identify a population of CD34þ

lymphatic/vascular endothelial precursor cells. Blood101: 168–172.

Schoppmann SF, Birner P, Stockl J, Kalt R, Ullrich R, CaucigC, Kriehuber E, Nagy K, Alitalo K, Kerjaschki D. 2002.Tumor-associated macrophages express lymphaticendothelial growth factors and are related to peritumorallymphangiogenesis. Am J Pathol 161: 947–956.

Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendel-sohn M, Charron J, Datta M, Young F, Stall AM, et al.1992. RAG-2-deficient mice lack mature lymphocytesowing to inability to initiate V(D)J rearrangement. Cell68: 855–867.

Siriwardena BS, Kudo Y, Ogawa I, Udagama MN, Tilakar-atne WM, Takata T. 2008. VEGF-C is associated withlymphatic status and invasion in oral cancer. J Clin Pathol61: 103–108.

Skobe M, Hamberg LM, Hawighorst T, Schirner M, WolfGL, Alitalo K, Detmar M. 2001. Concurrent inductionof lymphangiogenesis, angiogenesis, and macrophagerecruitment by vascular endothelial growth factor-C inmelanoma. Am J Pathol 159: 893–903.

Slack JM. 2007. Metaplasia and transdifferentiation: Frompure biology to the clinic. Nat Rev Mol Cell Biol 8:369–378.

Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, TsaiMJ, Samokhvalov IM, Oliver G. 2007. Lineage tracingdemonstrates the venous origin of the mammalian lym-phatic vasculature. Genes Dev 21: 2422–2432.

Stacker SA, Achen MG, Jussila L, Baldwin ME, Alitalo K.2002. Lymphangiogenesis and cancer metastasis. NatRev Cancer 2: 573–583.

Su JL, Shih JY, Yen ML, Jeng YM, Chang CC, Hsieh CY, WieLH, Yang PC, Kuo ML. 2004. Cyclooxygenase-2 inducesEP1- and HER-2/Neu-dependent vascular endothelialgrowth factor-C up-regulation: A novel mechanism oflymphangiogenesis in lung adenocarcinoma. CancerRes 64: 554–564.

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T,Tomoda K, Yamanaka S. 2007. Induction of pluripotentstem cells from adult human fibroblasts by defined fac-tors. Cell 131: 861–872.

Thiery JP, Sleeman JP. 2006. Complex networks orchestrateepithelial-mesenchymal transitions. Nat Rev Mol CellBiol 7: 131–142.

Uhrin P, Zaujec J, Breuss JM, Olcaydu D, Chrenek P, Stock-inger H, Fuertbauer E, Moser M, Haiko P, Fassler R, et al.2010. Novel function for blood platelets and podoplaninin developmental separation of blood and lymphatic cir-culation. Blood 115: 3997–4005.

Van de Veire S, Stalmans I, Heindryckx F, Oura H, Tijeras-Raballand A, Schmidt T, Loges S, Albrecht I, Jonckx B,



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Vinckier S, et al. 2010. Further pharmacological andgenetic evidence for the efficacy of PlGF inhibition incancer and eye disease. Cell 141: 178–190.

Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S,Okamura H, Sudo T, Shultz LD, Nishikawa S. 1990.The murine mutation osteopetrosis is in the codingregion of the macrophage colony stimulating factorgene. Nature 345: 442–444.

Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL,McMullen JR, Gustafsson E, Chandraker A, Yuan X, PuWT, Roberts AB, et al. 2007. Endothelial-to-mesenchymaltransition contributes to cardiac fibrosis. Nat Med 13:952–961.

Zhang XH, Huang DP, Guo GL, Chen GR, Zhang HX, WanL, Chen SY. 2008. Coexpression of VEGF-C and COX-2and its association with lymphangiogenesis in humanbreast cancer. BMC Cancer 8: 4.

Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. 2008.In vivo reprogramming of adult pancreatic exocrine cellsto b-cells. Nature 455: 627–632.

Zumsteg A, Christofori G. 2009. Corrupt policemen:Inflammatory cells promote tumor angiogenesis. CurrOpin Oncol 21: 60–70.

Zumsteg A, Baeriswyl V, Imaizumi N, Schwendener R,Ruegg C, Christofori G. 2009. Myeloid cells contributeto tumor lymphangiogenesis. PLoS One 4: e7067.



ww

w.p

ersp

ecti

vesi

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edic

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February 21, 20122012; doi: 10.1101/cshperspect.a006494 originally published onlineCold Spring Harb Perspect Med

Adrian Zumsteg and Gerhard Christofori Myeloid Cells and Lymphangiogenesis

Subject Collection Angiogenesis

miRNAs as Modulators of Angiogenesis

William C. SessaShira Landskroner-Eiger, Isabelle Moneke and Malformation Syndromes

Arteriovenous Malformations and Other Vascular

Y. LiKevin J. Whitehead, Matthew C.P. Smith and Dean

VEGF and Notch in Tip and Stalk Cell SelectionRaquel Blanco and Holger Gerhardt Development

Molecular Parallels between Neural and Vascular

Anne Eichmann and Jean-Léon Thomas

Regulating AngiogenesisThe Role of the Tumor Microenvironment in

Randolph S. WatnickResponses, Resistance, and the Path ForwardThe VEGF Pathway in Cancer and Disease:

Mark W. Kieran, Raghu Kalluri and Yoon-Jae Cho

DisordersAngiogenic Factors in Preeclampsia and Related

Ana Sofia Cerdeira and S. Ananth Karumanchi

Common Polymorphisms in AngiogenesisMichael S. Rogers and Robert J. D'Amato

Anti-VEGF Therapies in the ClinicKellen L. Meadows and Herbert I. Hurwitz Signaling in Physiology and Pathology

Endothelial Cell-to-Cell Junctions: Adhesion and

Maria Grazia Lampugnani

Tie Signaling Pathway−Angiopoietin The Complex Role of Angiopoietin-2 in the

Gavin Thurston and Christopher DalyCells to OrganismVEGF-Directed Blood Vessel Patterning: From

Victoria L. Bautch

Disease-Restricted ActivityPlGF: A Multitasking Cytokine with

Mieke Dewerchin and Peter CarmelietModels for Therapeutic TrialsVascular Anomalies: From Genetics toward

VikkulaMelanie Uebelhoer, Laurence M. Boon and Miikka

Human Endothelial Progenitor CellsMervin C. Yoder Growth Factor Receptors

Signal Transduction by Vascular Endothelial

Sina Koch and Lena Claesson-Welsh

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Myeloid Cells and Lymphangiogenesis - CSHL Pperspectivesinmedicine.cshlp.org/content/2/6/a006494...Myeloid Cells and Lymphangiogenesis Adrian Zumsteg and Gerhard Christofori Institute