Seminars in Cell & Developmental Biology 38 (2015) 117130

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Seminars in Cell & Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Review

Antilymphangiogenic therapy to promote transplant survival and to

reduce cancer metastasis: What can we learn from the eye?
Deniz Hos a,1 , Simona L. Schlereth a,1 , Felix Bock a,b , Ludwig M. Heindl a , Claus Cursiefen a,
a
Department of Ophthalmology, University of Cologne, Cologne, Germany
b
Cologne Ophthalmological Reading and Image Analysis Center (CORIC), University of Cologne, Cologne, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The lymphatic vasculature is amongst other tasks essentially involved in inammation,
Available online 20 November 2014 (auto)immunity, graft rejection and cancer metastasis. The eye is mainly devoid of lymphatic vessels
except for its adnexa, the conjunctiva and the limbus. However, several pathologic conditions can result
Keywords: in the secondary ingrowth of lymphatic vessels into physiologically alymphatic parts of the eye such
Lymphangiogenesis as the cornea or the inner eye. Therefore, the cornea has served as an excellent in vivo model sys-
Cornea
tem to study lymphangiogenesis, and ndings from such studies have substantially contributed to the
Transplantation
understanding of central principles of lymphangiogenesis also with relevance outside the eye. Graft-
Ocular malignancies
Tumor-associated lymphangiogenesis
ing experiments at the cornea have been extensively used to analyze the role of lymphangiogenesis in
Antilymphangiogenic therapy transplant immunology. In this regard, we recently demonstrated the crucial role of lymphatic vessels in
mediating corneal allograft rejection and could show that antilymphangiogenic therapy increases graft
survival. In the eld of cancer research, we recently detected tumor-associated lymphangiogenesis in the
most common malignant tumors of the eye, such as conjunctival carcinoma and melanoma, and cilio-
choroidal melanoma with extraocular extension. These neolymphatics correlate with an increased risk
of local recurrence, metastasis and tumor related death, and may offer potential therapeutic targets for
the treatment of these tumors. This review will focus on corneal and tumor-associated ocular lymphan-
giogenesis. First, we will describe common experimentally used corneal lymphangiogenesis models and
will recapitulate recent ndings regarding the involvement of lymphatic vessels in corneal diseases and
transplant immunology. The second part of this article will summarize ndings about the participation of
tumor-associated lymphangiogenesis in ocular malignancies and their implications for the development
of future therapeutic strategies.
2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
2. The lymphatic vascular anatomy of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3. Corneal (lymph)angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.1. Molecular mechanisms of corneal avascularity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.2. Experimental abrogation of corneal avascularity: common corneal (lymph)angiogenesis models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.2.1. The corneal micropocket assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
3.2.2. The suture induced corneal neovascularization assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3.2.3. Imaging and quantication of corneal (lymph)angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3.3. The contribution of lymphangiogenesis to corneal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.3.1. Dry eye disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Corresponding author at: Department of Ophthalmology, University of Cologne, KerpenerStrasse 62, 50924 Cologne, Germany. Tel.: +49 221 478 4300;
fax: +49 221 478 5094.
E-mail address: claus.cursiefen@uk-koeln.de (C. Cursiefen).
URL: http://www.augenklinik.uk-koeln.de (C. Cursiefen).
1
Both authors contributed equally and should be considered as rst authors.

http://dx.doi.org/10.1016/j.semcdb.2014.11.003
1084-9521/ 2014 Elsevier Ltd. All rights reserved.
118 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130

3.3.2. Ocular allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

3.3.3. Herpetic keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.3.4. Corneal graft rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.4. Therapeutic inhibition of corneal lymphangiogenesis to promote transplant survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.4.1. Anti(lymph)angiogenic treatment options for immature, actively outgrowing blood and lymphatic vessels . . . . . . . . . . . . . . . . . . . 122
3.4.2. Clinically available anti(lymph)angiogenic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
3.4.3. Regression of mature corneal lymphatic vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4. Tumor-associated lymphangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.1. The eye as a model to study lymphangiogenesis in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.2. Ocular malignancies and their association to lymphatic vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.3. The role of lymphangiogenesis in the tumor microenvironment for cancer cell metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.4. Potential treatment options to prevent or treat ocular cancer metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5. Concluding remarks and further directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

1. Introduction to correlate with an increased risk of local recurrence, metastasis

and tumor related death, probably offering a potential therapeutic
Lymphangiogenesis, the development and growth of lymphatic target to treat ocular tumors [2527].
vessels, is critically involved in uid and lipid homeostasis, inam- This article will mainly give an overview of two aspects of lym-
mation, blood pressure regulation, immune surveillance, graft phangiogenesis research at the eye: First, we will focus on the
rejection and cancer metastasis [1,2]. However, lymphangiogen- cornea. We will briey describe mechanisms of corneal avascu-
esis research has been neglected for a long time because lymphatic larity and how this angiogenic privilege can experimentally be
endothelial specic markers were not available, but with recent disrupted to study lymphangiogenesis. Furthermore, we will reca-
discoveries of key lymphatic specic markers such as prox-1, pitulate recent ndings regarding the involvement of lymphatic
podoplanin, VEGFR-3, and LYVE-1 our knowledge in this eld is vessels in corneal diseases and transplant immunology. Second, we
now rapidly growing. will focus on the role of lymphatic vessels in ocular tumor biology
The eye is mainly devoid of lymphatic vessels except for its and the participation of tumor-associated lymphangiogenesis in
adnexa, the conjunctiva and the limbus. However, ocular lymphan- cancer progression and metastasis.
giogenesis can occur under certain pathological conditions: the
cornea, the transparent and avascular windscreen of the eye can 2. The lymphatic vascular anatomy of the eye
secondarily be invaded by both vessel types in response to severe
inammation (Fig. 1) [35]. Furthermore, corneal lymphangiogen- In the eye, differentially lymphvascularized areas are in close
esis can experimentally be induced by several well-characterized proximity to each other and are strictly regulated by different
procedures and can easily be imaged due to the easy accessibil- local tissue factors. Furthermore, under certain circumstances, lym-
ity and transparent nature of the cornea [6]. Thus, the cornea has phatic vessels are able to grow into areas that are physiologically
served as an excellent model to study lymphangiogenesis, and nd- devoid of lymphatic vessels, thereby providing great models to
ings from experiments at the cornea have substantially contributed track and analyze lymphangiogenesis. At the eye, lymphatic ves-
to the unraveling of fundamental principles of lymphangiogenesis, sels are only found in the lacrimal gland, the extraocular muscles,
which also have relevance outside the eye [711]. the conjunctiva and the limbus, which is the transition zone where
Corneal transplantation, the most common and most successful the opaque sclera and the conjunctiva fade into the cornea [4]. The
form of transplantation, differs from other tissue and solid organ limbal lymphatic vasculature consists of a ring shaped network
grafting as the avascular cornea is immunologically privileged connected to the conjunctival lymphatic vessels, with generally
[4,12,13]. This results in excellent graft survival rates even without one main circumferential lymphatic vessel with small extensions
previous HLA-matching when corneal transplantation is performed directed toward the central cornea [28]. This main circumfer-
because of non-inammatory and non-vascular diseases (normal- ential lymphatic vessel may show gaps in its limbal circle and
risk keratoplasty). However, when corneal grafting is performed also shows remarkable plasticity, during both inammation as well
in severely inamed and prevascularized recipient beds, survival as aging [28,29]. However, inammatory stimuli can lead to an
rates signicantly drop (high-risk keratoplasty) [1416]. This increase in limbal lymphatic extensions toward the cornea, and
huge difference in graft survival between avascular and prevas- severe inammation can even result in a pathological growth of
cularized recipient corneas provides an excellent model system lymphatic vessels from the limbal lymphatic vasculature into the
to study the role of the lymphatic vascular system in transplant cornea (Fig. 1) [5,30]. During aging, the limbal lymphatic vascul-
immunology, and the majority of our knowledge regarding this ature regresses: gaps in the main limbal lymphatic vessel become
subject is indeed deduced from grafting experiments at the cornea. more frequent and the number of lymphatic extensions toward the
Using the mouse model of allogenic corneal transplantation, the cornea declines [28]. Thus, the ability of the limbal lymphatic vas-
important role of lymphatic vessels in mediating graft rejection culature to respond to inammatory and lymphangiogenic stimuli
could recently be demonstrated [7,17]. In this context, we could decreases with age [28].
show that anti(lymph)angiogenic treatment signicantly increases Similar to the cornea, the sclera and the inner eye are also
corneal graft survival [17], and current ndings outside the eye also physiologically devoid of lymphatic vessels, although in direct
indicate that lymphatic vessels have similar functions in solid organ neighborhood to lymphvascularized tissues [4,31,32]. Lymphatic
grafting [1820]. vessels accompany the extraocular muscles, which insert into the
Recently, the role of lymphatic vessels in ocular tumor biology sclera, but lymphatic vessel growth abruptly stops when approa-
could also be specied in more detail. Tumor-associated lym- ching the sclera. The most outer layer of the sclera, the episclera,
phangiogenesis could be detected in the most common malignant contains a tight network of blood vessels, which is surrounded
tumors of the ocular surface [2124]. These lymphatic vessels seem by LYVE1+ macrophages [32]. The fetal sclera is also alymphatic
 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
Fig. 1. Breakdown of the corneal (lymph)angiogenic privilege by severe inammation. (A and B) Corneal and limbal vascular anatomy; (A) the limbal vasculature consists of
a circumferential network with small extensions directed toward the avascular cornea. (B) Severe inammation can lead to a breakdown of the corneal (lymph)angiogenic
privilege and result in the secondary ingrowth of blood (red) and lymphatic (green) vessels from the limbus into the cornea.
but, in contrast to the adult sclera, contains only few macrophages
[33]. The exact function of these macrophages is not clear yet. Also
the sclera can secondarily be invaded by lymphatic vessels, e.g. by
perforating trauma or inltrating malignomas [21,26,34].
3. Corneal (lymph)angiogenesis
3.1. Molecular mechanisms of corneal avascularity
Corneal avascularity, also termed the corneal (lymph)
angiogenic privilege, is essential for its transparency and thereby
visual acuity [4]. The corneal (lymph)angiogenic privilege is estab-
lished early in embryonic development [35], is evolutionary
highly conserved, and is actively maintained by several redun-
dantly organized antiangiogenic mechanisms. In this context, it
was recently demonstrated that the corneal epithelium expresses
soluble forms of the three major vascular endothelial growth fac-
tor (VEGF) receptors (sVEGFR-1, sVEGFR-2, sVEGFR-3), which all
act as decoy receptors for the key hem- and lymphangiogenic
growth factors VEGF-A, VEGF-C and VEGF-D, and (experimen-
tal) inactivation of these endogenous VEGF traps abolishes the
corneal (lymph)angiogenic privilege [7,36,37]. Furthermore, we
could previously show that the corneal epithelium ectopically
expresses membrane bound VEGFR-3, which is also able to trap
lymphangiogenic ligands and thereby maintain corneal avascu-
larity [38]. Further potent antiangiogenic molecules expressed in
the cornea are angiostatin, endostatin, thrombospondin-1 (TSP-
1), thrombospondin-2 (TSP-2), pigment epithelium-derived factor,
and inhibitory PAS domain protein (IPAS), which negatively regu-
lates hypoxia-induced upregulation of (lymph)angiogenic growth
factors [3941]. Amongst these factors, especially TSP-1 seems to
be a key molecule critical for the maintenance of corneal lymphatic
avascularity. We could recently demonstrate that TSP-1 reduces
macrophage derived expression of lymphangiogenic VEGF-C and
inhibits accumulation of macrophages in the cornea. Accordingly,
mice defective in TSP-1 show increased VEGF-C expression and
higher numbers of corneal macrophages, resulting in spontaneous
and isolated corneal lymphangiogenesis in aged mice [9].
In fact, the avascular and alymphatic nature of the cornea allows
to use differences in limbal lymphatic vascular patterns and in the
lymphangiogenic response between mice from different genetic
backgrounds to identify novel endogenous regulators of lymphan-
giogenesis [42]. This powerful approach has already been used to
identify TSP-1 [9] and other novel regulators (Regenfuss et al., sub-
mitted).
119
Due to its anti(lymph)angiogenic mechanisms, the cornea
does not respond with lymphangiogenesis to minor inammatory
stimuli (to which it is constantly exposed due to its anatomical
position). However, these anti(lymph)angiogenic mechanisms are
not invincible. Severe and eye-threatening inammation can lead
to a breakdown of the corneal (lymph)angiogenic privilege and
result in corneal hem- and lymphangiogenesis (Fig. 1), sacricing
the functionality of the cornea for the safety of the whole eye [4].
Unlike blood vessels, lymphatic vessels do not lead to a signicant
reduction of corneal transparency per se, but have been shown to
substantially contribute to the development and maintenance of
several pathological conditions (described in Section 3.3).
3.2. Experimental abrogation of corneal avascularity: common
corneal (lymph)angiogenesis models
It is possible to experimentally abolish the corneal
(lymph)angiogenic privilege, and several corneal neovasculariza-
tion assays that result in the growth of blood and/or lymphatic
vessels into the cornea have been established. The great advantage
of these corneal neovascularization assays is that any vessel that
grows into the corneal stroma is newly formed and can easily
be detected. Furthermore, parameters such as the vascularized
area, vessel length, invasion distance, vascular density, and vessel
diameter are precisely quantiable [43,44]. Therefore, the cornea
is one of the most commonly used in vivo model systems to study
mechanisms of hem- and lymphangiogenesis. The easy accessibil-
ity and transparency of the cornea which allows simple ex vivo and
in vivo imaging has further contributed to the popularity of this
system [6]. Findings from corneal (lymph)angiogenesis models
have substantially contributed to the discovery of general princi-
ples of hem- and lymphangiogenesis, which also have relevance
outside the eye [711]. In addition, corneal (lymph)angiogenesis
models are widely used to analyze the impact of newly developed
drugs on blood and lymphatic vessels [4548].
Several commonly used corneal (lymph)angiogenesis models
exist. Amongst these, the corneal micropocket assay and the suture-
induced corneal neovascularization assay are the most frequently
used models (Fig. 2).
3.2.1. The corneal micropocket assay
In this assay, a small stromal pocket is surgically prepared in
the corneal stroma of an experimental animal (e.g. rabbits, rats or
mice). Then, a slow-release pellet containing a potentially angio-
genic agent is placed into the corneal micropocket (Fig. 2B). The
120 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130

Fig. 2. Commonly used corneal (lymph)angiogenesis models. Left panels: suture induced corneal neovascularization assay; upper left: in vivo appearance after suture
placement; lower left: corneal whole mount stained with the lymphendothelial marker LYVE-1; right panels: corneal micropocket assay; upper right: in vivo appearance
after stromal implantation of a pellet containing recombinant VEGF-C; lower right: corneal whole mount stained with LYVE-1; dashed lines in the lower panels indicate the
border between the limbus and the cornea.

developing vessels can then be analyzed in vivo or in corneal
whole mounts (Fig. 2D). This assay is mainly used to investigate
the specic effect of certain cytokines or growth factors on hem-
and lymphangiogenesis, but also cells or tumor fragments can be
placed into the pocket [45,49,50]. Using this corneal micropocket
assay, the angiogenic potential and modulatory role of a plethora
of molecules and cell types could be identied [8,11,46,51,52]. The
main advantage of the corneal micropocket assay is that the specic
effects of one single growth factor can reliably be assessed and that
this model is considered as a low inammatory model. However,
a disadvantage of this model is that the amount of growth factor
in the slow-release pellet usually is considerably higher and might
therefore lead to non-physiological vascular responses.
3.2.2. The suture induced corneal neovascularization assay
In this model, 13 sutures are placed into the corneal stroma
(Fig. 2A), leading to a rapid and strong inammatory response
[15,53,54]. Already two days after suture placement, blood and
lymphatic vessels begin to grow from the limbal region and
invade the cornea [29]. Hem- and lymphangiogenesis progress and
reach their maximum density approximately 2 weeks after suture
placement. After suture removal (usually after 2 weeks), the inam-
matory response decreases within the next 12 weeks, and both
blood and lymphatic vessels slowly regress [29]. The regression of
lymphatic vessels starts earlier and is also more pronounced than
that of blood vessels. Whereas (partly) perfused blood vessels can
still be observed after 68 months, there are usually no lymphatic
vessels detectable beyond 6 months after suture placement [29].
The advantage of the corneal suture model is that this procedure
results in very robust and reliable corneal hem- and lymphangio-
genesis (Fig. 2C). However, when this model is used in small animals
such as mice, the surgical procedure can be challenging and time
consuming. Furthermore, recent studies indicate that the area of
suture placement has to be carefully selected and should not vary
between manipulated corneas, as the vascular response might dif-
fer between e.g. the nasal and the temporal parts of the cornea
[55].
Using the suture induced corneal neovascularization assay, it
was possible to decipher important regulatory mechanisms of
lymphangiogenesis that also have signicant implications for non-
ocular tissues [7,10]. Furthermore, the corneal suture model is
frequently used to induce corneal blood and lymphatic vessels prior
to corneal transplantation in order to analyze the role of blood
and/or lymphatic vessels in transplant immunology [56] (see Sec-
tion 3.4).
3.2.3. Imaging and quantication of corneal (lymph)angiogenesis
The above described corneal hem- and lymphangiogenesis
models are frequently used to analyze the molecular mecha-
nisms which underlie these processes or to test novel drugs
[7,8,10,11,45,48,57]. In these models, corneal hem- and/or lym-
phangiogenesis can be additionally stimulated or inhibited by
systemic or local treatment. Furthermore, the transparency of
the cornea permits excellent visualization of sprouting vessels
with low background. Usually, corneal hem- and lymphangio-
genesis are quantied by specic staining for blood vessels (e.g.
CD31) and lymphatic vessels (e.g. LYVE-1). The accuracy of vessel
detection and quantication is a critical step to determine e.g. the
 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
effectiveness of anti(lymph)angiogenic therapies. Therefore, we
recently established a novel, semiautomatic, gray value based
analysis method where only the areas which are in fact covered by
the vessel network are measured [43]. Furthermore, this method
also enables a fast and precise measurement of single vessels or
even the detection of all sprouts in a region of interest at once [43].
Starting with preclinical studies on histological whole mount spec-
imens of vascularized murine corneas, we further developed this
method for its application in clinical studies, where it is possible to
follow up changes of the corneal vessel network during the study
and to analyze vascular alterations of any magnitude [5860].
3.3. The contribution of lymphangiogenesis to corneal diseases
Several studies demonstrate the occurrence of pathological
lymphangiogenesis in corneal diseases, both experimentally and
clinically [3,30,61]. Mostly, corneal lymphatic vessels grow in par-
allel to corneal blood vessels, and it seems that the degree of corneal
lymphangiogenesis also correlates with the degree of corneal
hemangiogenesis. Whereas it is well accepted that pathological
corneal blood vessels directly cause a loss of corneal transparency
and also lead to secondary effects such as uid and lipid deposi-
tion through immature capillaries, the contribution of clinically
invisible corneal lymphatic vessels to disease development and
progression was less apparent [5]. However, recently, several
experimental studies provided evidence for the substantial con-
tribution of corneal lymphatic vessels to ocular pathologies, which
will be recapitulated in the following subchapters [17,30,62,63].
Furthermore, several corneal diseases have recently been shown
to result in a selected ingrowth of lymphatic, but not blood ves-
sels into the cornea [62], providing suitable model systems to
analyze the specic contribution of lymphangiogenesis to these
diseases.
3.3.1. Dry eye disease
Dry eye is one of the most frequent diseases in ophthalmology.
Recent studies have clearly demonstrated that dry eye disease is
an inammatory condition characterized by the inux and acti-
vation of inammatory cells and an increase in pro-inammatory
mediators, resulting in abnormal tear composition and subsequent
damage of the ocular surface [6467]. Furthermore, it has been
shown that adaptive immune responses are accountable for main-
taining ocular surface inammation in dry eye disease [68,69]. As
these autoreactive processes are mainly generated and maintained
in regional lymph nodes, corneal lymphatics seem to serve as con-
duits that enable accelerated autoantigen-transport and migration
of antigen-presenting cells (APC) from the ocular surface to the
lymph nodes. In this context, our group demonstrated that in TSP-
1 decient mice, which develop Sjgrens syndrome-like dry eye
disease, an isolated ingrowth of lymphatic vessels into the cornea
can be observed, which may facilitate APC trafcking [9]. Further-
more, in a murine desiccation-stress model of dry eye disease, Goyal
et al. also demonstrated selective corneal lymphangiogenesis with-
out concurrent corneal hemangiogenesis [62]. Moreover, the same
group could also show that inhibition of VEGF-C in this model sig-
nicantly reduced dry eye-associated corneal lymphangiogenesis
and signicantly improved the clinical course of the disease [70].
3.3.2. Ocular allergy
The contribution of lymphangiogenesis to extraocular allergic
diseases has recently received considerable attention [71]. At the
ocular surface, antigen and APCs are drained to the regional lymph
nodes and may induce allergic responses [72]. Preventing the lym-
phatic migration of APCs, accomplished by CCR7 blockade, reduces
allergic signs and systemic IgE levels [73]. However, whether the
ocular surface also responds with the induction of neo-lymphatics
121
in response to allergic eye disease had so far not been determined.
Unpublished results indicate that in mice, severe forms of allergic
eye disease also result in the signicant development of corneal
lymphatic, but not blood vessels (Saban et al., submitted). Fur-
ther analyses revealed that these newly formed lymphatics enable
egress of antigen-loaded dendritic cells from the cornea, suggesting
a role in the migration of these cells to the regional lymph nodes and
a consequent contribution to a type-2 T cell (Th-2) response. In line
with this, blockade of corneal lymphangiogenesis in allergic mice
signicantly decreased Th-2 responses and ameliorated clinical dis-
ease (Saban et al., submitted). Therefore, lymphangiogenesis seems
to play also a crucial role in the development of ocular allergy and
offers a new and exciting therapeutic target to treat this frequently
occurring disease.
3.3.3. Herpetic keratitis
Herpes simplex virus 1 (HSV-1) is one of the most frequent
infections of the eye. Viral infection is permanent within the
trigeminal nerve and is further characterized by recurrent episodes
of reactivation. One frequent manifestation of ocular HSV-1 infec-
tion is herpetic keratitis, and severe forms of herpetic keratitis
can even lead to blindness. Although it is generally accepted that
herpetic keratitis can lead to corneal hemangiogenesis, it was
unknown whether lymphangiogenesis also occurs in this disease.
In a rst study that analyzed lymphangiogenesis during viral infec-
tion in vivo, Wuest and Carr could demonstrate that HSV-1 induces
corneal lymphangiogenesis in mice by expression of VEGF-A in
infected corneal epithelial cells [63]. Interestingly, HSV-1 asso-
ciated corneal lymphangiogenesis was strictly dependent on the
VEGF-A/VEGFR-2 axis but not on the VEGF-C/-D/VEGFR-3 axis
[63]. Furthermore, herpetic keratitis associated corneal lymphan-
giogenesis was specic for HSV-1 and not other viruses and was
in addition independent of macrophage recruitment, which is in
contrast to previously described models of inammatory corneal
lymphangiogenesis [10,74]. HSV-1 induced corneal lymphatic ves-
sels persisted after resolution of infection and might therefore
contribute to chronic inammation or even reactivation. However,
to our knowledge, there was so far no study investigating the effect
of interference with corneal lymphangiogenesis in herpetic kerati-
tis.
3.3.4. Corneal graft rejection
As previously mentioned (see Section 1), corneal transplanta-
tion usually results in excellent graft survival rates when grafting
is performed because of non-inammatory and non-vascular dis-
eases (normal-risk keratoplasty). However, if donor corneas
are grafted into inamed and prevascularized recipients, sur-
vival rates signicantly decrease even under aggressive systemic
immunosuppression (high-risk keratoplasty) [14,53]. Corneal
neovascularization is therefore considered as the most important
risk factor for the risk of immune reactions after transplantation
[16]. The reason for the decreased transplant survival in prevascu-
larized corneas is the accelerated induction of allogenic immune
responses, as donor and antigen-loaded recipient dendritic cells
from the graft have immediate access to the lymphatic vasculature
and thereby to the regional lymph nodes, where increased allosen-
sitization occurs [5,56,75,76]. Subsequently, effector cells can easily
reach and enter the graft via preexisting corneal blood vessels and
reject the graft [5].
As corneal lymphatic vessels therefore ease the connection
between the graft and the regional lymph nodes and provide impor-
tant paths for dendritic cell trafcking to the secondary lymphatic
organs, interference with this afferent arm of the immune reex
arch seems a very reasonable strategy to avoid graft rejection and
improve graft survival after corneal transplantation [5,30]. Indeed,
several experimental studies demonstrated a benecial effect of
122 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
Fig. 3. Absence of lymphatic vessels in the high-risk recipient cornea prior to transplantation signicantly improves graft survival in mice. (a) KaplanMeier survival curve;
(bd) corneal whole mounts stained with CD31 (blood vessel; green) and LYVE-1 (lymphatic vessels, red) in different recipient beds (modied from Dietrich et al. [17]). This
work clearly demonstrates the key role of lymphatic vessels in mediating immune responses after (corneal) transplantation.
this approach. Surgical excision of draining regional lymph nodes
(cervical lymphadenectomy) in mice resulted in 100% graft sur-
vival after normal-risk and in 90% graft survival after high-risk
keratoplasty in mice [77,78]. Since then, we and other groups ana-
lyzed the feasibility of a molecular lymphadenectomy, namely a
pharmacological blockade of pathological corneal lymphangiogen-
esis to improve graft survival after corneal grafting. Using various
pharmacological (lymph)angiogenesis modulators at the cornea,
we had previously shown that it is possible to inhibit both hem-
and or lymphangiogenesis [10] or to solely block lymphangiogen-
esis without affecting hemangiogenesis [79,80]. By application of
these inhibitors after performing the suture induced corneal neo-
vascularization assay, we were able to compare different recipient
beds prior to corneal grafting: (1) healthy, avascular beds (normal-
risk); (2) hem- and lymphvascularized beds (high-risk); and
(3) only hem- but not lymphvascularized beds after treatment
with selective lymphangiogenesis inhibitors (alymphatic high-
risk) (Fig. 3). After allogenic corneal transplantation, we observed
that grafts placed into alymphatic but hemvascularized recipient
beds had similar graft survival rates when compared to grafts
placed into completely avascular, normal-risk recipient beds [17].
Contrary, graft survival was signicantly lower when lymphatic
vessels were present in the high-risk setting (Fig. 3). Therefore,
we could demonstrate that lymphatic vessels, but not blood ves-
sels, primarily mediate immune reactions after keratoplasty and
dene the high-risk status of prevascularized hosts [17]. Similar
results could be obtained by the administration of soluble VEGFR-
2, which selectively traps prolymphangiogenic VEGF-C and inhibits
lymphangiogenesis without affecting hemangiogenesis. Treatment
with soluble VEGFR-2 markedly enhanced corneal allograft survival
[7].
Taken together, these experiments demonstrate that lymphatic
vessels are crucial for corneal graft rejection and play a superior
role in mediating allogenic immune responses when compared to
blood vessels. Furthermore, specic antilymphangiogenic therapy
markedly increases corneal allograft survival. These results may
have important implications also in solid organ grafting, as these
organs much more depend on the intact blood vasculature to main-
tain organ perfusion and function.
3.4. Therapeutic inhibition of corneal lymphangiogenesis to
promote transplant survival
As described above, we could demonstrate that lymphatic
vessels play a crucial role in the mediation of corneal trans-
plant alloimmunity and that corneal transplant survival in mice
can signicantly be improved by the blockade of lymphatic ves-
sels (Fig. 3) [17]. Therefore, anti(lymph)angiogenic treatment is
a feasible approach to promote transplant survival. A variety of
(lymph)angiogenesis inhibitors have already been developed and
are currently tested and will be summarized in this section. Further-
more, as this eld of research is rapidly growing, more inhibitors
will surely be available in the near future.
3.4.1. Anti(lymph)angiogenic treatment options for immature,
actively outgrowing blood and lymphatic vessels
Several scenarios of anti(lymph)angiogenic therapy in the con-
text of corneal transplantation exist to promote graft survival.
Actively outgrowing blood and lymphatic vessels depend on proin-
ammatory and angiogenic stimuli. Therefore, antiinammatory
strategies as well as antibodies targeting VEGF are in clinical use
already [8184].
Furthermore, there are several preclinically tested
anti(lymph)angiogenic drugs such as integrin blocking pep-
tides, anti-VEGFR3 antibodies, VEGFR-tyrosine kinase inhibitors
and soluble VEGFRs [7,37,48,79,80,85]. Also endogenous factors
such as thrombospondin-1 (TSP-1) have been shown to inhibit
lymphangiogenesis [9]. Eye drops containing TSP-1 could signif-
icantly block progressive corneal neovascularization in animal
models of corneal neovascularization [9].
In addition to the already known prolymphangiogenic growth
factors VEGF-C and -D the prohemangiogenic growth factor VEGF-
A could also be established as a prolymphangiogenic factor, and
 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
we could show that the blockade of VEGF-A also blocks corneal
lymphangiogenesis in preclinical models [10,47,86].
3.4.2. Clinically available anti(lymph)angiogenic agents
In the clinical setting, it is so far not possible to reliably
detect corneal lymphatics in vivo without obtaining specimens,
e.g. for histological analyses. Therefore, patient studies usually
assess clinically visible blood vessels. Nevertheless, we know from
experimental studies that several clinically used antihemangio-
genic agents at the cornea also are potent inhibitors of corneal
lymphangiogenesis.
Today, topical corticosteroid therapy is still the standard of
antiangiogenic therapy at the cornea. Besides its known anti-
hemangiogenic effects it was recently shown in experimental
studies that corticosteroids also inhibit lymphangiogenesis [54].
Our group has demonstrated that this effect is not only due
to direct inhibitory effects on lymphatic endothelium, but also
due to reduced recruitment of prolymphangiogenic inammatory
cells such as macrophages [54]. The antilymphangiogenic potency
of corticosteroids varies with prednisolone having the strongest
effects. Nonetheless do corticosteroids alone not achieve complete
inhibition e.g. of postkeratoplasty corneal neovascularization [87],
and may cause several side effects such as elevated intraocular
pressure or cataract.
Antibodies directed against VEGF have recently been shown
to also block lymphangiogenesis: Bevacizumab and Ranibizumab
are potent inhibitors of both corneal hem- and lymphangiogenesis
[47,86]. Both can penetrate well into the cornea and reach sufcient
concentrations especially in case of an absent or altered corneal
epithelial barrier (as is the case in vascularized corneas) [88]. Both
are not approved by the FDA for the use at the ocular surface. How-
ever, they are frequently used off label in the clinical setting to
treat corneal neovascularization, and several groups have proven
the effectiveness and safety of Bevacizumab and Ranibizumab in
inhibiting corneal hemangiogenesis [81,82,84,89,90].
Insulin receptor substrate-1 (IRS-1) has recently been shown
to be an important intracellular signaling molecule in the angio-
genic cascade. Blockade of IRS-1 signaling inhibits angiogenesis
and as recently shown also lymphangiogenesis in mice [57,91].
Aganirsen, an antisense oligonucleotide against IRS-1, has now suc-
cessfully been introduced in the clinical setting and is currently
tested as eye drops in phase II and phase III trials [58,59]. The
phase II/III data show that Aganirsen eye drops are safe, well toler-
ated and effective to inhibit progressive corneal neovascularization
when applied topically [58,59]. Furthermore, Aganirsen eye drops
are able to reduce the need for transplantation in patients with
viral keratitis-associated central corneal neovascularization [59].
Aganirsen will be the rst therapeutic that will be approved for the
topical treatment of corneal neovascularization. Since Aganirsen
eye drops in primate models also reached sufcient levels at the
retina and choroidea, Aganirsen also holds great promise for novel
topical treatment options against AMD and retinal vascular dis-
eases [92].
3.4.3. Regression of mature corneal lymphatic vessels
Strategies to stop progressive corneal hem- and lymphangio-
genesis have been established in preclinical and clinical studies.
However, most patients show already preexisting and mature
blood and lymphatic vessels. Therefore, regression of both vessel
types is even more important to improve transplant survival after
corneal grafting. We could demonstrate that, both in the clinical and
experimental setting, corneal lymphatic vessels regress over time
and that graft survival is enhanced in recipient beds with partly
regressed inammatory neovascularization [3,29,93]. Current clin-
ically used angioregressive strategies are limited to the treatment
of clinically visible blood vessels by ne needle cauterization or
123
corneal photodynamic therapy (PDT, see Section 3.4.3.2) [94,95].
However, whether these treatment strategies affect lymphatic ves-
sels is not known so far.
3.4.3.1. Molecular regression strategies. Interrupting e.g. the
Angiopoetin/Tie-2 signal pathway might be an additional strategy
to force the regression of blood and lymphatic vessels. It is well
known that the Ang/Tie system plays a crucial role in blood and
lymphatic vessel development and in adult blood vessel mainte-
nance. A Tie2-Fc fusion protein can inhibit the Ang-1 mediated
outgrowth of lymphatic vessels in a corneal micropocket assay
[96]. Furthermore, the stabilizing function of the Ang/Tie system
on isolated human lymphatic endothelial cells is abolished by
a blocking anti-Tie2 antibody [97]. Therefore, it is possible that
anti-Ang/Tie therapies at the cornea could lead to forced regres-
sion of corneal blood and lymphatic vessels and thereby might
allow the restoration of a normal-risk situation prior to corneal
transplantation. Up to date, no clinical studies are available for
these approaches.
3.4.3.2. Photodynamic therapy of lymphatic vessels. An even more
direct method to induce lymphatic vessel regression would be
the specic destruction of lymphatics. In this regard, photo-
dynamic therapy after Verteporn application seems to be an
interesting approach. Verteporn, a benzoporphyrin derivative, is
a photosensitizer and produces, when stimulated by nonthermal
red light in the presence of oxygen, highly reactive short-lived
singlet oxygen and other reactive oxygen radicals, resulting in
local damage to the endothelium of actively proliferating vessels.
Verteporn is used for the treatment of subfoveal choroidal neo-
vascularization in patients with age-related macular degeneration,
pathologic myopia, choroidal haemangioma, polypoidal choroidal
vasculopathy and for patients with chronic or recurrent central
serous chorioretinopathy. As already mentioned, it was shown
that corneal blood vessels can be regressed by the use of systemi-
cally applied Verteporn and subsequent photodynamic therapy
[95,98,99].
Very recently this method was also transferred to the treat-
ment of lymphatic vessels in the skin. Here, Verteporn was not
injected intravenously (as usual), but intradermally into the tip of
mouse ears, which were subsequently treated by PDT. This resulted
in the specic destruction of lymphatic vessels [100]. Our own
group has shown similar effects on corneal lymphangiogenesis: we
performed corneal suture placement in mice (see Section 3.2.2)
to induce corneal hem- and lymphangiogenesis and afterwards
injected Verteporn intrastromally, followed by PDT. This resulted
in a selective regression of corneal lymphatic, but not blood vessels
(Fig. 4), arguably via the selective uptake of Verteporn by drain-
ing lymphatics [101]. Therefore, PDT after intrastromal injection
of Verteporn is a novel technique to specically induce regres-
sion of corneal lymphatics without affecting blood vessels, and may
provide a new model to further characterize the specic role of
lymphatic vessels in corneal graft rejection. PDT after intrastromal
injection of Verteporn could be a novel strategy to precondition
vascularized high-risk corneas by reducing lymphatic vessel den-
sity prior to corneal transplantation.
4. Tumor-associated lymphangiogenesis
The eye is predestined to study lymphangiogenesis also in
the tumor context, since tumor-associated lymphangiogenesis has
been recently detected in the most common malignant tumors
of the ocular surface, including conjunctival melanomas, conjunc-
tival carcinomas and ciliochoroidal melanomas with extraocular
extension, and has been correlated with an increased risk of local
124 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130

Fig. 4. Selective regression of corneal lymphatic vessels by photodynamic therapy (PDT) after intrastromal injection of the photosensitizer Verteporn in mice. Representative
sections of corneal atmounts 10 days after photodynamic therapy (PDT) after intrastromal PBS (upper panels) or intrastromal Verteporn (lower panels) application. Prior
to PDT, corneal suture placement was performed to induce corneal hem- and lymphangiogenesis. The selective regression of lymphatic vessels is shown in the lower right
panel, and the dashed lines indicate the border between the limbus and the cornea (modied from Bucher at al. [101]).

recurrence, metastasis and tumor related death [2127]. There-
fore, the second part of this review focuses on ocular cancer to
study tumor-associated lymphangiogenesis, metastasis and poten-
tial therapeutic strategies.
4.1. The eye as a model to study lymphangiogenesis in cancer
The physiological inner eye is usually devoid of lymphatic ves-
sels [13,102] (see Section 2). Therefore, studying tumors in usually
lymphatic-free tissues of the eye, e.g. uveal melanoma, may serve
as models for lymphangiogenesis where the tumor overcomes nat-
ural suppressive antilymphangiogenic mechanisms. Studying this
in an early stage may help to understand how ocular lymphatic ves-
sels are generated. It will help to identify whether new lymphatic
vessels are growing out of pre-existing lymphatics from the vicin-
ity or are built de novo from e.g. macrophages or tumor stem cells.
It may also help to develop new therapeutic strategies to interfere
with early stages of tumor lymphangiogenesis.
Moreover, the eye is easy accessible and most structures are
uncomplicated and painless to visualize by microscopy, optical
coherence tomography, uorescence angiography, ultrasonogra-
phy or in vivo confocal microscopy. Furthermore, two photon
microscopy may help to further study tumor cell interactions with
blood and/or lymphatic vessels in vivo, which could already been
shown for immune cells at the cornea [6]. By administration of
topical eye drops, new anticancer treatments can be tested with
generally no or only little systemic side effects and the effect on
the tumor can easily be visualized and documented.
4.2. Ocular malignancies and their association to lymphatic
vessels
Tumor-associated lymphangiogenesis has been shown for dif-
ferent malignancies of the ocular surface, including the conjunctival
carcinoma and melanoma, as well as the ciliary body melanoma
with extraocular extension [2127].
Our group examined whether lymphatic vessels can invade
the physiologically lymphatic-free intraocular space in malignant
melanoma of the ciliary body with extraocular extension. Here,
intra- and extraocular lymphatic vessels have been detected (Fig. 5),
whereas the origin of the vessels is not quite clear yet. These
could have their origin in conjunctival lymphatic vessels, which
are attracted inwards the eye by following a VEGF-C or -D gradi-
ent secreted by the intraocular tumor. They could also originate
from proliferating melanoma lymphatic vessels or from prox1+ or
LYVE1+ CD68+ macrophages from the sclera [32] or the choroid
[31], which seem to have the ability to transform into lymphatic
vascular endothelium [74]. The intact sclera seems to serve as a
natural barrier against invasion of lymphatic vessels, but when
overcoming this barrier, for example in extraocular extension of the
intraocular ciliary body melanoma, it becomes clinically relevant.
Patients with intraocular or peritumoral lymphatic vessels in cil-
iary body melanoma with extraocular extension had an increased
mortality risk showing that intraocular tumor-associated lym-
phangiogenesis has a prognostic signicance [21,25]. Lymphatic
metastasis has also been documented occasionally in patients with
uveal melanoma and extraocular extension [103], although the
major route for metastasis in these tumor entities is hematogenous.
Similar to the ciliary body melanoma, the malignant melanoma
of the conjunctiva showed high amounts of tumor-associated lym-
phatic vessels surrounding and within the tumor (Fig. 6) [24].
In conjunctival melanoma the lymphangiogenic stimulus is initi-
ated very early [22], even before the invasive phenotype of the
tumor [27]. In conjunctival melanoma the main route for metas-
tasis is via lymphatic vessels, and therefore the main function of
these vessels seem to be the transport of tumor cells to draining
lymph nodes and then further lymphatic spread. Tumor cell clusters
could be identied within intratumoral lymphatics in conjunctival
melanoma (Fig. 7) [27]. When analyzing lymph node metastasis it
has been shown that lymphatic vessels are occluded with tumor
cells [104,105]. However, neither in conjunctival melanoma nor
in eyelid tumors, lymph node biopsies are standard procedures
yet, although recent publications lay emphasis on the importance
 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130 125

Fig. 5. Intraocular tumor-associated lymphangiogenesis in a malignant melanoma of the ciliary body with extraocular extension. Ciliary body malignant melanoma (CB-MM)
with extraocular extension displacing the lens (L) and showing extraocular (AC) and intraocular (DF) podoplanin and Lyve-1 positive lymphatic vessels. The extraocular
conjunctiva (con) is endowed with peritumoral lymphatic vessels, similar to the intraocular peritumoral lymphatic vessels in the sclera (sc) and in the intraocular tumor
component (modied from Heindl et al. [21]). This is the rst evidence for secondary lymphangiogenesis into the physiologically alymphatic eye.

of sentinel lymph node biopsies [106,107]. A signicant relation
between tumor-associated lymphangiogenesis and mortality, local
recurrence and metastasis has also been shown for conjunctival
melanoma [27].
Higher recurrence rates in patients with intraepithelial and
invasive squamous cell carcinoma of the conjunctiva could be
related to actively proliferating tumor associated lymphangiogene-
sis. In this tumor entity, intra- and peritumoral lymphangiogenesis
has been detected [23]. Similar to the malignant melanoma of the
conjunctiva, lymphangiogenesis is already detectable in very early
stages, even before the tumor invades the deeper layers.
4.3. The role of lymphangiogenesis in the tumor
microenvironment for cancer cell metastasis
The general ability of tumor cells to metastasize depends on dif-
ferent factors: (i) the tumor microenvironment and (ii) the tumor
cell itself [108,109]. Fibroblasts, endothelial cells, mesenchymal
stem cells, tumor-associated macrophages (TAMs), and extracel-
lular matrix form the tumor microenvironment [110]. VEGF [111],
chemokines or colony stimulating factors (CSF) [112] induce the
recruitment of further cells like macrophages into the tumor. High
amounts of factors such as transforming growth factor beta (TGF-
) or CSF1 can modulate these TAMs into M2 macrophages [113].
These cells have reduced immune activating properties and secrete
immunomodulating factors including TGF- or IL-10, proangio-
genic factors such as VEGF-A and prolymphangiogenic factors
such as VEGF-C or -D [113115]. M2 macrophages have also been
detected in uveal melanoma, where most TAMs are CD68+ CD163+
M2 macrophages and patients with low amounts of CD68+ CD163+
macrophages had a better prognosis compared to patients with
high amounts of CD68+ CD163+ macrophages [116]. Studies in mice
revealed that depletion of macrophages during tumor induction
prevented tumor growth, whereas in non-depleted mice all ani-
mals developed ocular tumors [116118]. Next to TAMs in uveal
melanoma CD8+ T-cells and CD3+FoxP3+ regulatory T-cells (Tregs)
126 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130

Fig. 6. Proliferating lymphatic vessels in a malignant melanoma of the conjunctiva. Conjunctival melanoma (A, hematoxylineosin) showing intratumoral (B and C) and
peritumoral (D and E) lymphatic vessels which stain positive for LYVE-1 (brown)/Ki-67 (red) (B and D) and podoplanin (brown)/Ki-67 (red) (C and E) (modied from Heindl
et al. [27]).

have been detected [119]. Tregs modulate other immune cells
toward a tolerance state and high numbers of Tregs are associated
with a poor prognosis [120].
In the microenvironment of tumors, high amounts of angio-
genic and lymphangiogenic factors are secreted and thereby induce
hemangiogenesis and lymphangiogenesis within and around the
tumor [8,121124]. Increased blood vessel growth in tumor tis-
sue for nutrition and oxygen supply as well as for evacuation of
metabolic waste seems obvious [125], whereby the function of
tumor-associated lymphatic vessels in tumor growth is still less
clear. Obviously, it is the initial step in lymphogenic metastasis,
as this has been shown for several malignancies outside the eye
[126128]. Lymphatic vessels lack tight junctions and a continuous
basal membrane, such as seen in blood vessels. Therefore invasion
into lymphatic vessels might be easier for tumor cells than invasion
into blood vessels [127]. Moreover, some tumor cells express CCR7
[129131] which is important for homing of lymphoid cells to the
draining lymph nodes, and might further support the invasion of
tumor cells into lymphatic vessels.
4.4. Potential treatment options to prevent or treat ocular cancer
metastasis
To date, physicians can barely offer effective curative therapies
for patients with metastatic diseases of primarily ocular tumors.
A broad spectrum of common ocular malignancies including the
conjunctival melanoma, the ciliochoroidal melanoma with extraoc-
ular extension or the conjunctival carcinoma show a high tendency

Fig. 7. Tumor-associated proliferating lymphatic vessels in malignant melanomas of the conjunctiva contain tumor cells. Active lymphatic vessels (A: LYVE-1 [brown]/Ki-67
[red] and B: podoplanin [brown]/Ki-67 [red]) with tumor cells (asterisk) within the vessel lumen (modied from Heindl et al. [27]).
 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
of lymphatic cancer spread [2127]. Therefore, latest therapeu-
tic strategies aim to block the prolymphangiogenic VEGF-C/-D/-R3
axis, and rst studies in mice could successfully reduce tumor-
associated lymphangiogenesis in non-ocular tumors [132135].
Another, more indirect approach aims to block the migration of
tumor cells to the lymph node in mice, e.g. by interfering with CCR7
[136,137].
However, as tumor cells are highly exible and can quickly
adjust to environmental changes and therapies, blocking just one
component can be compensated by other mechanisms. Therefore,
different blocking mechanisms at the same time might be required
for the best outcomes.
5. Concluding remarks and further directions
The pathological contribution of lymphangiogenesis to sev-
eral corneal diseases has recently been demonstrated. Specically,
corneal lymphatics have been shown to play an important role in
the development of dry eye disease, ocular allergy, herpetic kerati-
tis, and graft rejection in various experimental models [17,62,63].
Therefore, targeting the lymphatic system in these ocular sur-
face diseases is a very reasonable approach, and initial results are
promising [30]. Although there is so far no drug approved by the
FDA for the treatment of corneal neovascularization in patients,
pharmacological inhibitors are becoming increasingly available
and approvals are likely to be granted in the near future.
In the context of lymphangiogenesis research, the physio-
logically avascular cornea serves as an excellent in vivo model
to study mechanisms of lymphangiogenesis that also have rele-
vance outside the eye. Furthermore, most of our knowledge about
the role of lymphatic vessels in transplantation immunology is
deduced from grafting experiments at the cornea [56]. Based on
these ndings, several studies could provide evidence that lym-
phangiogenesis also occurs in solid organ grafting, such as renal
and cardiac transplantation [1820]: Kerjaschki and colleagues
demonstrated that human renal grafts contain numerous prolif-
erating CCL21+ lymphatic vessels, suggesting CCR7+ dendritic cell
trafcking to the draining lymph nodes via these neolymphat-
ics [18,19]. Additionally, Nyknen and coworkers investigated the
potential involvement of lymphatic vessels in cardiac grafting,
and demonstrated that chronic rejection-associated inammation
induces lymphangiogenesis in rat cardiac allografts. Furthermore,
these newly formed lymphatic vessels were functional in attract-
ing activated dendritic cells to the lymph nodes and in inducing
alloimmune responses [20]. Studies in further organ systems e.g.
in human allogenic liver and rat lung transplantation, could also
conrm the association of lymphangiogenesis with chronic allo-
graft rejection [138,139]. Based on these ndings, the impact of
anti(lymph)angiogenic treatment strategies on allograft survival is
currently assessed, and several preclinical studies in mice and rats
could already demonstrate benecial effects of this novel antilym-
phangiogenic concept also in solid organ grafting [20,140,141].
In the context of tumor-associated lymphangiogenesis, our
group has shown for different malignancies of the ocular surface,
including the conjunctival carcinoma and melanoma, as well as
the ciliary body melanoma with extraocular extension, that lym-
phangiogenesis occurs and correlates with an increased risk of local
recurrence, metastasis and tumor related death [2127]. Therefore,
targeting lymphangiogenesis in the context of ocular tumors may
be an additional treatment option for patients suffering from these
malignancies. Furthermore, ocular tumors may serve as models to
study tumor-associated lymphangiogenesis in general, and nd-
ings from anti(lymph)angiogenic strategies at the eye may also be
attributable to malignancies outside the eye. Nevertheless, these
potential therapeutical options are still in a very early phase and
127
have not reached clinical standard treatment yet, as optimized time
point, application scheme and form, exact indications or side effects
need to be established in further studies.
Last but not least, the cornea is a powerful tool to identify novel
endogenous regulators of lymphangiogenesis [42]. This approach
has already been used to identify TSP-1 as novel endogenous reg-
ulator, with others likely to follow.
Funding
This work was supported by the German Research Foundation:
DFG Cu 47/4-1 (CC), DFG Cu 47/6-1 (CC), DFG HE 6743/2-1 (LMH);
GEROK-Program, University of Cologne (DH, SLS and LMH); EU
COST BM1302 (DH, FB and CC); Helmut und Ruth Lingen-Stiftung,
Cologne (CC).
Conict of interest
No conicting relationship exists for any author.
References
[1] Alitalo K, Tammela T, Petrova TV. Lymphangiogenesis in development and
human disease. Nature 2005;438:94653.
[2] Tammela T, Alitalo K. Lymphangiogenesis: molecular mechanisms and future
promise. Cell 2010;140:46076.
[3] Cursiefen C, Schlotzer-Schrehardt U, Kuchle M, Sorokin L, Breiteneder-Geleff
S, Alitalo K, et al. Lymphatic vessels in vascularized human corneas: immuno-
histochemical investigation using LYVE-1 and podoplanin. Invest Ophthalmol
Vis Sci 2002;43:212735.
[4] Cursiefen C. Immune privilege and angiogenic privilege of the cornea. Chem
Immunol Allergy 2007;92:507.
[5] Cursiefen C, Chen L, Dana MR, Streilein JW. Corneal lymphangiogenesis: evi-
dence, mechanisms, and implications for corneal transplant immunology.
Cornea 2003;22:27381.
[6] Steven P, Bock F, Huttmann G, Cursiefen C. Intravital two-photon
microscopy of immune cell dynamics in corneal lymphatic vessels. PLoS ONE
2011;6:e26253.
[7] Albuquerque RJ, Hayashi T, Cho WG, Kleinman ME, Dridi S, Takeda A,
et al. Alternatively spliced vascular endothelial growth factor receptor-2
is an essential endogenous inhibitor of lymphatic vessel growth. Nat Med
2009;15:102330.
[8] Chang LK, Garcia-Cardena G, Farnebo F, Fannon M, Chen EJ, Buttereld C, et al.
Dose-dependent response of FGF-2 for lymphangiogenesis. Proc Natl Acad Sci
U S A 2004;101:1165863.
[9] Cursiefen C, Maruyama K, Bock F, Saban D, Sadrai Z, Lawler J, et al. Throm-
bospondin 1 inhibits inammatory lymphangiogenesis by CD36 ligation on
monocytes. J Exp Med 2011;208:108392.
[10] Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C, et al. VEGF-A
stimulates lymphangiogenesis and hemangiogenesis in inammatory neo-
vascularization via macrophage recruitment. J Clin Invest 2004;113:104050.
[11] Bos FL, Caunt M, Peterson-Maduro J, Planas-Paz L, Kowalski J, Karpanen T,
et al. CCBE1 is essential for mammalian lymphatic vascular development and
enhances the lymphangiogenic effect of vascular endothelial growth factor-C
in vivo. Circ Res 2011;109:48691.
[12] Niederkorn JY. The immune privilege of corneal allografts. Transplantation
1999;67:15038.
[13] Streilein JW, Yamada J, Dana MR, Ksander BR. Anterior chamber-associated
immune deviation, ocular immune privilege, and orthotopic corneal allo-
grafts. Transplant Proc 1999;31:14725.
[14] Niederkorn JY. High-risk corneal allografts and why they lose their immune
privilege. Curr Opin Allergy Clin Immunol 2010;10:4937.
[15] Dana MR, Streilein JW. Loss and restoration of immune privilege in eyes with
corneal neovascularization. Invest Ophthalmol Vis Sci 1996;37:248594.
[16] Bachmann B, Taylor RS, Cursiefen C. Corneal neovascularization as a risk factor
for graft failure and rejection after keratoplasty: an evidence-based meta-
analysis. Ophthalmology 2010;117, 1300-5.e7.
[17] Dietrich T, Bock F, Yuen D, Hos D, Bachmann BO, Zahn G, et al. Cutting edge:
lymphatic vessels, not blood vessels, primarily mediate immune rejections
after transplantation. J Immunol 2010;184:5359.
[18] Kerjaschki D, Regele HM, Moosberger I, Nagy-Bojarski K, Watschinger B,
Soleiman A, et al. Lymphatic neoangiogenesis in human kidney transplants
is associated with immunologically active lymphocytic inltrates. J Am Soc
Nephrol 2004;15:60312.
[19] Kerjaschki D, Huttary N, Raab I, Regele H, Bojarski-Nagy K, Bartel G, et al. Lym-
phatic endothelial progenitor cells contribute to de novo lymphangiogenesis
in human renal transplants. Nat Med 2006;12:2304.
[20] Nykanen AI, Sandelin H, Krebs R, Keranen MA, Tuuminen R, Karpanen T,
et al. Targeting lymphatic vessel activation and CCL21 production by vascular
128 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
endothelial growth factor receptor-3 inhibition has novel immunomod-
ulatory and antiarteriosclerotic effects in cardiac allografts. Circulation
2010;121:141322.
[21] Heindl LM, Hofmann TN, Knorr HL, Rummelt C, Schrodl F, Schlotzer-
Schrehardt U, et al. Intraocular lymphangiogenesis in malignant melanomas
of the ciliary body with extraocular extension. Invest Ophthalmol Vis Sci
2009;50:198895.
[22] Heindl LM, Hofmann-Rummelt C, Adler W, Bosch JJ, Holbach LM, Naumann
GO, et al. Tumor-associated lymphangiogenesis in the development of con-
junctival melanoma. Invest Ophthalmol Vis Sci 2011;52:707483.
[23] Heindl LM, Hofmann-Rummelt C, Adler W, Holbach LM, Naumann GO, Kruse
FE, et al. Tumor-associated lymphangiogenesis in the development of con-
junctival squamous cell carcinoma. Ophthalmology 2010;117:64958.
[24] Zimmermann P, Dietrich T, Bock F, Horn FK, Hofmann-Rummelt C, Kruse
FE, et al. Tumour-associated lymphangiogenesis in conjunctival malignant
melanoma. Br J Ophthalmol 2009;93:152934.
[25] Heindl LM, Hofmann TN, Adler W, Knorr HL, Holbach LM, Naumann GO,
et al. Intraocular tumor-associated lymphangiogenesis a novel prognostic fac-
tor for ciliary body melanomas with extraocular extension? Ophthalmology
2010;117:33442.
[26] Heindl LM, Hofmann TN, Schrodl F, Holbach LM, Kruse FE, Cursiefen C.
Intraocular lymphatics in ciliary body melanomas with extraocular exten-
sion: functional for lymphatic spread? Arch Ophthalmol 2010;128:10018.
[27] Heindl LM, Hofmann-Rummelt C, Adler W, Bosch JJ, Holbach LM,
Naumann GO, et al. Prognostic signicance of tumor-associated lymphan-
giogenesis in malignant melanomas of the conjunctiva. Ophthalmology
2011;118:235160.
[28] Hos D, Bachmann B, Bock F, Onderka J, Cursiefen C. Age-related changes in
murine limbal lymphatic vessels and corneal lymphangiogenesis. Exp Eye Res
2008;87:42732.
[29] Cursiefen C, Maruyama K, Jackson DG, Streilein JW, Kruse FE. Time course
of angiogenesis and lymphangiogenesis after brief corneal inammation.
Cornea 2006;25:4437.
[30] Bock F, Maruyama K, Regenfuss B, Hos D, Steven P, Heindl LM, et al. Novel
anti(lymph)angiogenic treatment strategies for corneal and ocular surface
diseases. Prog Retin Eye Res 2013;34:89124.
[31] Schroedl F, Brehmer A, Neuhuber WL, Kruse FE, May CA, Cursiefen C. The
normal human choroid is endowed with a signicant number of lymphatic
vessel endothelial hyaluronate receptor 1 (LYVE-1)-positive macrophages.
Invest Ophthalmol Vis Sci 2008;49:52229.
[32] Schlereth SL, Neuser B, Caramoy A, Grajewski RS, Koch KR, Schrodl F, et al.
Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-
positive macrophages around blood vessels in the normal human sclera.
Invest Ophthalmol Vis Sci 2014;55:86572.
[33] Schlereth SL, Neuser B, Herwig MC, Muller AM, Koch KR, Reitsamer HA, et al.
Absence of lymphatic vessels in the developing human sclera. Exp Eye Res
2014;125C:2039.
[34] Wessel JM, Hofmann-Rummelt C, Kruse FE, Cursiefen C, Heindl LM. Invasion
of lymphatic vessels into the eye after open globe injuries. Invest Ophthalmol
Vis Sci 2012;53:371725.
[35] Cursiefen C, Rummelt C, Junemann A, Vorwerk C, Neuhuber W, Kruse FE, et al.
Absence of blood and lymphatic vessels in the developing human cornea.
Cornea 2006;25:7226.
[36] Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, et al. Corneal
avascularity is due to soluble VEGF receptor-1. Nature 2006;443:9937.
[37] Singh N, Tiem M, Watkins R, Cho YK, Wang Y, Olsen T, et al. Soluble vascular
endothelial growth factor receptor-3 is essential for corneal alymphaticity.
Blood 2013;121:42429.
[38] Cursiefen C, Chen L, Saint-Geniez M, Hamrah P, Jin Y, Rashid S, et al. Nonvascu-
lar VEGF receptor 3 expression by corneal epithelium maintains avascularity
and vision. Proc Natl Acad Sci U S A 2006;103:1140510.
[39] Armstrong LC, Bornstein P. Thrombospondins 1 and 2 function as inhibitors
of angiogenesis. Matrix Biol: J Int Soc Matrix Biol 2003;22:6371.
[40] Cursiefen C, Masli S, Ng TF, Dana MR, Bornstein P, Lawler J, et al. Roles of
thrombospondin-1 and -2 in regulating corneal and iris angiogenesis. Invest
Ophthalmol Vis Sci 2004;45:111724.
[41] Makino Y, Cao R, Svensson K, Bertilsson G, Asman M, Tanaka H, et al. Inhibitory
PAS domain protein is a negative regulator of hypoxia-inducible gene expres-
sion. Nature 2001;414:5504.
[42] Regenfuss B, Onderka J, Bock F, Hos D, Maruyama K, Cursiefen C. Genetic
heterogeneity of lymphangiogenesis in different mouse strains. Am J Pathol
2010;177:50110.
[43] Bock F, Onderka J, Hos D, Horn F, Martus P, Cursiefen C. Improved semiauto-
matic method for morphometry of angiogenesis and lymphangiogenesis in
corneal atmounts. Exp Eye Res 2008;87:46270.
[44] Blacher S, Detry B, Bruyere F, Foidart JM, Noel A. Additional parameters for the
morphometry of angiogenesis and lymphangiogenesis in corneal at mounts.
Exp Eye Res 2009;89:2746.
[45] DAmato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of
angiogenesis. Proc Natl Acad Sci U S A 1994;91:40825.
[46] Schulz MM, Reisen F, Zgraggen S, Fischer S, Yuen D, Kang GJ, et al. Phenotype-
based high-content chemical library screening identies statins as inhibitors
of in vivo lymphangiogenesis. Proc Natl Acad Sci U S A 2012;109:E266574.
[47] Bock F, Onderka J, Dietrich T, Bachmann B, Kruse FE, Paschke M, et al. Beva-
cizumab as a potent inhibitor of inammatory corneal angiogenesis and
lymphangiogenesis. Invest Ophthalmol Vis Sci 2007;48:254552.
[48] Hos D, Bock F, Dietrich T, Onderka J, Kruse FE, Thierauch KH, et al. Inam-
matory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase
inhibitor ZK 261991, resulting in improved graft survival after corneal trans-
plantation. Invest Ophthalmol Vis Sci 2008;49:183642.
[49] Gross J, Azizkhan RG, Biswas C, Bruns RR, Hsieh DS, Folkman J. Inhibition
of tumor growth, vascularization, and collagenolysis in the rabbit cornea by
medroxyprogesterone. Proc Natl Acad Sci U S A 1981;78:117680.
[50] Kenyon BM, Voest EE, Chen CC, Flynn E, Folkman J, DAmato RJ. A
model of angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci
1996;37:162532.
[51] Chauhan SK, Jin Y, Goyal S, Lee HS, Fuchsluger TA, Lee HK, et al. A novel pro-
lymphangiogenic function for Th17/IL-17. Blood 2011;118:46304.
[52] Lee H, Schlereth SL, Park EY, Emami-Naeini P, Chauhan SK, Dana R. A novel
pro-angiogenic function for interferon-gamma-secreting natural killer cells.
Invest Ophthalmol Vis Sci 2014;55:288592.
[53] Sano Y, Ksander BR, Streilein JW. Fate of orthotopic corneal allografts in eyes
that cannot support anterior chamber-associated immune deviation induc-
tion. Invest Ophthalmol Vis Sci 1995;36:217685.
[54] Hos D, Saban DR, Bock F, Regenfuss B, Onderka J, Masli S, et al. Suppres-
sion of inammatory corneal lymphangiogenesis by application of topical
corticosteroids. Arch Ophthalmol 2011;129:44552.
[55] Ecoifer T, Yuen D, Chen L. Differential distribution of blood and lym-
phatic vessels in the murine cornea. Invest Ophthalmol Vis Sci 2010;51:
243640.
[56] Hos D, Cursiefen C. Lymphatic vessels in the development of tissue and organ
rejection. Adv Anat Embryol Cell Biol 2014;214:11941.
[57] Hos D, Regenfuss B, Bock F, Onderka J, Cursiefen C. Blockade of insulin receptor
substrate-1 inhibits corneal lymphangiogenesis. Invest Ophthalmol Vis Sci
2011;52:577885.
[58] Cursiefen C, Bock F, Horn FK, Kruse FE, Seitz B, Borderie V, et al. GS-
101 antisense oligonucleotide eye drops inhibit corneal neovascularization:
interim results of a randomized phase II trial. Ophthalmology 2009;116:
16307.
[59] Cursiefen C, Viaud E, Bock F, Geudelin B, Ferry A, Kadlecova P, et al. Aganirsen
antisense oligonucleotide eye drops inhibit keratitis-induced corneal neo-
vascularization and reduce need for transplantation: the I-CAN study.
Ophthalmology 2014.
[60] Bock F, Matthaei M, Reinhard T, Bohringer D, Christoph J, Ganslandt T,
et al. High-dose subconjunctival cyclosporine A implants do not affect
corneal neovascularization after high-risk keratoplasty. Ophthalmology
2014;121:167782.
[61] Cursiefen C, Kuchle M, Naumann GO. Angiogenesis in corneal diseases:
histopathologic evaluation of 254 human corneal buttons with neovascular-
ization. Cornea 1998;17:6113.
[62] Goyal S, Chauhan SK, El Annan J, Nallasamy N, Zhang Q, Dana R. Evidence of
corneal lymphangiogenesis in dry eye disease: a potential link to adaptive
immunity? Arch Ophthalmol 2010;128:81924.
[63] Wuest TR, Carr DJ. VEGF-A expression by HSV-1-infected cells drives corneal
lymphangiogenesis. J Exp Med 2010;207:10115.
[64] Stern ME, Schaumburg CS, Pugfelder SC. Dry eye as a mucosal autoimmune
disease. Int Rev Immunol 2013;32:1941.
[65] Barabino S, Chen Y, Chauhan S, Dana R. Ocular surface immunity: homeo-
static mechanisms and their disruption in dry eye disease. Prog Retin Eye Res
2012;31:27185.
[66] Stevenson W, Chauhan SK, Dana R. Dry eye disease: an immune-mediated
ocular surface disorder. Arch Ophthalmol 2012;130:90100.
[67] De Paiva CS, Chotikavanich S, Pangelinan SB, Pitcher 3rd JD, Fang B, Zheng
X, et al. IL-17 disrupts corneal barrier following desiccating stress. Mucosal
Immunol 2009;2:24353.
[68] Niederkorn JY, Stern ME, Pugfelder SC, De Paiva CS, Corrales RM, Gao J, et al.
Desiccating stress induces T cell-mediated Sjogrens Syndrome-like lacrimal
keratoconjunctivitis. J Immunol 2006;176:39507.
[69] Schaumburg CS, Siemasko KF, De Paiva CS, Wheeler LA, Niederkorn JY,
Pugfelder SC, et al. Ocular surface APCs are necessary for autoreactive
T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis. J
Immunol 2011;187:365362.
[70] Goyal S, Chauhan SK, Dana R. Blockade of prolymphangiogenic vascu-
lar endothelial growth factor C in dry eye disease. Arch Ophthalmol
2012;130:849.
[71] Zgraggen S, Ochsenbein AM, Detmar M. An important role of blood and lym-
phatic vessels in inammation and allergy. J Allergy 2013;2013:672381.
[72] Saban DR. The chemokine receptor CCR7 expressed by dendritic cells:
a key player in corneal and ocular surface inammation. Ocular Surf
2014;12:8799.
[73] Schlereth S, Lee HS, Khandelwal P, Saban DR. Blocking CCR7 at the ocular
surface impairs the pathogenic contribution of dendritic cells in allergic con-
junctivitis. Am J Pathol 2012;180:235160.
[74] Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H, Tomita M, et al.
Inammation-induced lymphangiogenesis in the cornea arises from CD11b-
positive macrophages. J Clin Invest 2005;115:236372.
[75] Dana MR. Angiogenesis and lymphangiogenesis-implications for corneal
immunity. Semin Ophthalmol 2006;21:1922.
[76] Patel SP, Dana R. Corneal lymphangiogenesis: implications in immunity.
Semin Ophthalmol 2009;24:1358.
[77] Yamagami S, Dana MR. The critical role of lymph nodes in corneal alloimmu-
nization and graft rejection. Invest Ophthalmol Vis Sci 2001;42:12938.
 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
[78] Yamagami S, Dana MR, Tsuru T. Draining lymph nodes play an essential role
in alloimmunity generated in response to high-risk corneal transplantation.
Cornea 2002;21:4059.
[79] Bock F, Onderka J, Dietrich T, Bachmann B, Pytowski B, Cursiefen C. Blockade
of VEGFR3-signalling specically inhibits lymphangiogenesis in inamma-
tory corneal neovascularisation. Albrecht von Graefes Archiv fur klinische
und experimentelle Ophthalmologie 2008;246:1159.
[80] Dietrich T, Onderka J, Bock F, Kruse FE, Vossmeyer D, Stragies R, et al. Inhibition
of inammatory lymphangiogenesis by integrin alpha5 blockade. Am J Pathol
2007;171:36172.
[81] Bock F, Konig Y, Kruse F, Baier M, Cursiefen C. Bevacizumab (Avastin) eye drops
inhibit corneal neovascularization. Albrecht von Graefes Archiv fur klinische
und experimentelle Ophthalmologie 2008;246:2814.
[82] Ferrari G, Dastjerdi MH, Okanobo A, Cheng SF, Amparo F, Nallasamy N, et al.
Topical ranibizumab as a treatment of corneal neovascularization. Cornea
2013;32:9927.
[83] Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Curr Opin
Ophthalmol 2001;12:2429.
[84] Koenig Y, Bock F, Horn F, Kruse F, Straub K, Cursiefen C. Short- and long-
term safety prole and efcacy of topical bevacizumab (Avastin) eye drops
against corneal neovascularization. Albrecht von Graefes Archiv fur klinische
und experimentelle Ophthalmologie 2009;247:137582.
[85] Detry B, Blacher S, Erpicum C, Paupert J, Maertens L, Maillard C, et al. Sunitinib
inhibits inammatory corneal lymphangiogenesis. Invest Ophthalmol Vis Sci
2013;54:308293.
[86] Bucher F, Parthasarathy A, Bergua A, Onderka J, Regenfuss B, Cursiefen C, et al.
Topical ranibizumab inhibits inammatory corneal hem- and lymphangio-
genesis. Acta Ophthalmol (Copenh) 2012;92:1438.
[87] Cursiefen C, Wenkel H, Martus P, Langenbucher A, Nguyen NX, Seitz B, et al.
Impact of short-term versus long-term topical steroids on corneal neovascu-
larization after non-high-risk keratoplasty. Albrecht von Graefes Archiv fur
klinische und experimentelle Ophthalmologie 2001;239:51421.
[88] Dastjerdi MH, Sadrai Z, Saban DR, Zhang Q, Dana R. Corneal penetration
of topical and subconjunctival bevacizumab. Invest Ophthalmol Vis Sci
2011;52:871823.
[89] Dastjerdi MH, Al-Arfaj KM, Nallasamy N, Hamrah P, Jurkunas UV, Pineda 2nd
R, et al. Topical bevacizumab in the treatment of corneal neovascularization:
results of a prospective, open-label, noncomparative study. Arch Ophthalmol
2009;127:3819.
[90] Cheng SF, Dastjerdi MH, Ferrari G, Okanobo A, Bower KS, Ryan DS, et al.
Short-term topical bevacizumab in the treatment of stable corneal neovascu-
larization. Am J Ophthalmol 2012;154, 940-8.e1.
[91] Andrieu-Soler C, Berdugo M, Doat M, Courtois Y, BenEzra D, Behar-Cohen F.
Downregulation of IRS-1 expression causes inhibition of corneal angiogene-
sis. Invest Ophthalmol Vis Sci 2005;46:40728.
[92] Cloutier F, Lawrence M, Goody R, Lamoureux S, Al-Mahmood S, Colin S, et al.
Antiangiogenic activity of aganirsen in nonhuman primate and rodent models
of retinal neovascular disease after topical administration. Invest Ophthalmol
Vis Sci 2012;53:1195203.
[93] Bachmann BO, Luetjen-Drecoll E, Bock F, Wiegand SJ, Hos D, Dana R, et al. Tran-
sient postoperative vascular endothelial growth factor (VEGF)-neutralisation
improves graft survival in corneas with partly regressed inammatory neo-
vascularisation. Br J Ophthalmol 2009;93:107580.
[94] Koenig Y, Bock F, Kruse FE, Stock K, Cursiefen C. Angioregressive
pretreatment of mature corneal blood vessels before keratoplasty: ne-
needle vessel coagulation combined with anti-VEGFs. Cornea 2012;31:
88792.
[95] Brooks BJ, Ambati BK, Marcus DM, Ratanasit A. Photodynamic therapy
for corneal neovascularisation and lipid degeneration. Br J Ophthalmol
2004;88:840.
[96] Morisada T, Oike Y, Yamada Y, Urano T, Akao M, Kubota Y, et al.
Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation. Blood
2005;105:464956.
[97] Song SH, Kim KL, Lee KA, Suh W. Tie1 regulates the Tie2 agonistic role of
angiopoietin-2 in human lymphatic endothelial cells. Biochem Biophys Res
Commun 2012;419:2816.
[98] Holzer MP, Solomon KD, Vroman DT, Sandoval HP, Margaron P, Kasper TJ,
et al. Photodynamic therapy with verteporn in a rabbit model of corneal
neovascularization. Invest Ophthalmol Vis Sci 2003;44:29548.
[99] Yoon KC, Ahn KY, Lee SE, Kim KK, Im SK, Oh HJ, et al. Experimental inhibition of
corneal neovascularization by photodynamic therapy with verteporn. Curr
Eye Res 2006;31:21524.
[100] Tammela T, Saaristo A, Holopainen T, Yla-Herttuala S, Andersson LC, Viro-
lainen S, et al. Photodynamic ablation of lymphatic vessels and intralymphatic
cancer cells prevents metastasis. Sci Transl Med 2011;3, 69ra11.
[101] Bucher F, Bi Y, Gehlsen U, Hos D, Cursiefen C, Bock F. Regression of mature
lymphatic vessels in the cornea by photodynamic therapy. Br J Ophthalmol
2014;98:3915.
[102] Gausas RE, Gonnering RS, Lemke BN, Dortzbach RK, Sherman DD. Iden-
tication of human orbital lymphatics. Ophthal Plast Reconstr Surg
1999;15:2529.
[103] Dithmar S, Diaz CE, Grossniklaus HE. Intraocular melanoma spread to regional
lymph nodes: report of two cases. Retina 2000;20:769.
[104] Padera TP, Kadambi A, di Tomaso E, Carreira CM, Brown EB, Boucher Y, et al.
Lymphatic metastasis in the absence of functional intratumor lymphatics.
Science 2002;296:18836.
129
[105] Achen MG, Jeltsch M, Kukk E, Makinen T, Vitali A, Wilks AF, et al. Vascu-
lar endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases
VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc Natl Acad Sci U S A
1998;95:54853.
[106] Cohen VM, Tsimpida M, Hungerford JL, Jan H, Cerio R, Moir G. Prospective
study of sentinel lymph node biopsy for conjunctival melanoma. Br J Oph-
thalmol 2013;97:15259.
[107] Pfeiffer ML, Savar A, Esmaeli B. Sentinel lymph node biopsy for eyelid and
conjunctival tumors: what have we learned in the past decade? Ophthal Plast
Reconstr Surg 2013;29:5762.
[108] Fidler IJ. Cancer metastasis. Br Med Bull 1991;47:15777.
[109] Schlereth SL, Refaian N, Iden S, Cursiefen C, Heindl LM. Impact of the prolym-
phangiogenic crosstalk in the tumor microenvironment on lymphatic cancer
metastasis. BioMed Res Int 2014;2014:639058.
[110] Ungefroren H, Sebens S, Seidl D, Lehnert H, Hass R. Interaction of tumor cells
with the microenvironment. Cell Commun Signal 2011;9:18.
[111] Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of
human monocytes in response to vascular endothelial growth factor (VEGF)
is mediated via the VEGF receptor t-1. Blood 1996;87:333643.
[112] Lin EY, Nguyen AV, Russell RG, Pollard JW. Colony-stimulating factor 1
promotes progression of mammary tumors to malignancy. J Exp Med
2001;193:72740.
[113] Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization:
tumor-associated macrophages as a paradigm for polarized M2 mononuclear
phagocytes. Trends Immunol 2002;23:54955.
[114] Duong T, Koopman P, Francois M. Tumor lymphangiogenesis as a potential
therapeutic target. J Oncol 2012;2012:204946.
[115] Schoppmann SF, Birner P, Stockl J, Kalt R, Ullrich R, Caucig C, et al. Tumor-
associated macrophages express lymphatic endothelial growth factors and
are related to peritumoral lymphangiogenesis. Am J Pathol 2002;161:94756.
[116] Bronkhorst IH, Ly LV, Jordanova ES, Vrolijk J, Versluis M, Luyten GP, et al.
Detection of M2-macrophages in uveal melanoma and relation with survival.
Invest Ophthalmol Vis Sci 2011;52:64350.
[117] Ly LV, Baghat A, Versluis M, Jordanova ES, Luyten GP, van Rooijen N, et al.
In aged mice, outgrowth of intraocular melanoma depends on proangiogenic
M2-type macrophages. J Immunol 2010;185:34818.
[118] Makitie T, Summanen P, Tarkkanen A, Kivela T. Tumor-inltrating
macrophages (CD68(+) cells) and prognosis in malignant uveal melanoma.
Invest Ophthalmol Vis Sci 2001;42:141421.
[119] Bronkhorst IH, Vu TH, Jordanova ES, Luyten GP, Burg SH, Jager MJ. Differ-
ent subsets of tumor-inltrating lymphocytes correlate with macrophage
inux and monosomy 3 in uveal melanoma. Invest Ophthalmol Vis Sci
2012;53:53708.
[120] Quezada SA, Peggs KS, Simpson TR, Allison JP. Shifting the equilibrium in
cancer immunoediting: from tumor tolerance to eradication. Immunol Rev
2011;241:10418.
[121] Cao R, Bjorndahl MA, Religa P, Clasper S, Garvin S, Galter D, et al. PDGF-BB
induces intratumoral lymphangiogenesis and promotes lymphatic metasta-
sis. Cancer Cell 2004;6:33345.
[122] Chang L, Kaipainen A, Folkman J. Lymphangiogenesis new mechanisms. Ann
NY Acad Sci 2002;979:1119.
[123] Gale NW, Thurston G, Hackett SF, Renard R, Wang Q, McClain J, et al.
Angiopoietin-2 is required for postnatal angiogenesis and lymphatic pat-
terning, and only the latter role is rescued by angiopoietin-1. Dev Cell
2002;3:41123.
[124] Kubo H, Cao R, Brakenhielm E, Makinen T, Cao Y, Alitalo K. Blockade of vascu-
lar endothelial growth factor receptor-3 signaling inhibits broblast growth
factor-2-induced lymphangiogenesis in mouse cornea. Proc Natl Acad Sci U S
A 2002;99:886873.
[125] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell
2011;144:64674.
[126] Dadras SS, Paul T, Bertoncini J, Brown LF, Muzikansky A, Jackson DG,
et al. Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous
melanoma metastasis and survival. Am J Pathol 2003;162:195160.
[127] Cao Y, Opinion:. emerging mechanisms of tumour lymphangiogenesis and
lymphatic metastasis. Nat Rev Cancer 2005;5:73543.
[128] Streit M, Detmar M. Angiogenesis, lymphangiogenesis, and melanoma metas-
tasis. Oncogene 2003;22:31729.
[129] Darash-Yahana M, Pikarsky E, Abramovitch R, Zeira E, Pal B, Karplus R, et al.
Role of high expression levels of CXCR4 in tumor growth, vascularization, and
metastasis. FASEB J: Off Publ Fed Am Soc Exp Biol 2004;18:12402.
[130] Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al. Involvement
of chemokine receptors in breast cancer metastasis. Nature 2001;410:506.
[131] Arya M, Patel HR, McGurk C, Tatoud R, Klocker H, Masters J, et al. The
importance of the CXCL12CXCR4 chemokine ligandreceptor interaction in
prostate cancer metastasis. J Exp Ther Oncol 2004;4:291303.
[132] Achen MG, Stacker SA. Molecular control of lymphatic metastasis. Ann NY
Acad Sci 2008;1131:22534.
[133] He Y, Kozaki K, Karpanen T, Koshikawa K, Yla-Herttuala S, Takahashi T, et al.
Suppression of tumor lymphangiogenesis and lymph node metastasis by
blocking vascular endothelial growth factor receptor 3 signaling. J Natl Cancer
Inst 2002;94:81925.
[134] Zhang D, Li B, Shi J, Zhao L, Zhang X, Wang C, et al. Suppression of tumor growth
and metastasis by simultaneously blocking vascular endothelial growth fac-
tor (VEGF)-A and VEGF-C with a receptorimmunoglobulin fusion protein.
Cancer Res 2010;70:2495503.
130 D. Hos et al. / Seminars in Cell & Developmental Biology 38 (2015) 117130
[135] Thiele W, Sleeman JP. Tumor-induced lymphangiogenesis: a target for cancer
therapy? J Biotechnol 2006;124:22441.
[136] Alfonso-Perez M, Lopez-Giral S, Quintana NE, Loscertales J, Martin-Jimenez P,
Munoz C. Anti-CCR7 monoclonal antibodies as a novel tool for the treatment
of chronic lymphocyte leukemia. J Leukoc Biol 2006;79:115765.
[137] Somovilla-Crespo B, Alfonso-Perez M, Cuesta-Mateos C, Carballo-de Dios C,
Beltran AE, Terron F, et al. Anti-CCR7 therapy exerts a potent anti-tumor activ-
ity in a xenograft model of human mantle cell lymphoma. J Hematol Oncol
2013;6:89.
[138] Dashkevich A, Heilmann C, Kayser G, Germann M, Beyersdorf F, Passlick B,
et al. Lymph angiogenesis after lung transplantation and relation to acute
organ rejection in humans. Ann Thoracic Surg 2010;90:40611.
[139] Ishii E, Shimizu A, Kuwahara N, Arai T, Kataoka M, Wakamatsu K, et al.
Lymphangiogenesis associated with acute cellular rejection in rat liver trans-
plantation. Transplant Proc 2010;42:42825.
[140] Palin NK, Savikko J, Koskinen PK. Sirolimus inhibits lymphangiogenesis
in rat renal allografts, a novel mechanism to prevent chronic kidney
allograft injury. Transpl Int: Off J Eur Soc Organ Transplant 2013;26:
195205.
[141] Yin N, Zhang N, Xu J, Shi Q, Ding Y, Bromberg JS. Targeting lymphangiogenesis
after islet transplantation prolongs islet allograft survival. Transplantation
2011;92:2530.