Download Evidence for Lymphatics in the Developing and

Retinal Cell Biology
Evidence for Lymphatics in the Developing and Adult
Human Choroid
Mark E. Koina,1,2 Louise Baxter,1 Samuel J. Adamson,1 Frank Arfuso,1,3 Ping Hu,1
Michele C. Madigan,4,5 and Tailoi Chan-Ling1
1
Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia
Department of Anatomical Pathology, ACT Pathology, The Canberra Hospital, Garran, Australian Capital Territory, Australia
3School of Anatomy, Physiology and Human Biology, Faculty of Science, The University of Western Australia, Crawley, Western
Australia, Australia
4
School of Optometry, University of New South Wales, New South Wales, Australia
5
Save Sight Institute, The University of Sydney, New South Wales, Australia
2
Correspondence: Tailoi Chan-Ling,
Discipline of Anatomy, School of
Medical Sciences, Bosch Institute,
Room S466, Anderson Stuart Building, F13, The University of Sydney,
New South Wales 2006 Australia;
tailoi@anatomy.usyd.edu.au.
MEK, LB, SJA and FA contributed
equally to the work presented here
and should therefore be regarded as
equivalent authors.
Submitted: September 19, 2014
Accepted: December 10, 2014
Citation: Koina ME, Baxter L, Adamson
SJ, et al. Evidence for lymphatics in the
developing and adult human choroid.
Invest Ophthalmol Vis Sci.
2015;56:1310–1327. DOI:10.1167/
iovs.14-15705
PURPOSE. Lymphatics subserve many important functions in the human body including
maintenance of fluid homeostasis, immune surveillance, and tumor metastasis. Our aim was
to provide structural and phenotypic evidence of lymphatic-like structures in the human
choroid, including details of its development.
METHODS. Using multiple-marker immunohistochemistry (IHC), choroids from human fetal
eyes (8–26 weeks gestation) and adults (17–74 years) were examined with lymphatic- and
vascular-specific markers: prospero homeobox-1 (PROX-1), lymphatic vascular endothelium
receptor-1 (LYVE-1), podoplanin, D2-40, endomucin, VEGF-C, vascular endothelial growth
factor receptor-3 (VEGFR-3 or Flt4), UEA lectin, platelet endothelial cell adhesion molecule-1
(PECAM-1), CD34, and CD39. Transmission electron microscopy (TEM) was used to establish
evidence for choroidal lymphatics, and to provide details of stratification and relative
frequency of lymphatics compared to choroidal blood vessels.
RESULTS. Immunohistochemistry and TEM indicated a central-to-peripheral topography of
lymphatic formation, with numerous blind-ended lymph sacs just external to the
choriocapillaris, as well as the presence of infrequent precollector and collector lymphatic
channels. Characteristic ultrastructural features of lymphatics in adult human choroid
included anchoring filaments, luminal flocculent protein but absence of erythrocytes,
fragmented and/or absent basal lamina, absence of intracellular Weibel-Palade bodies,
infrequent pericyte ensheathment, and lack of fenestrae.
CONCLUSIONS. The system of blind-ended initial lymphatic segments seen just external to the
fenestrated vessels of the choriocapillaris is ideally placed for recirculating extracellular fluid
and strategically placed for immune surveillance. The presence of a system of lymphatic-like
channels in the human choroid provides an anatomical basis for antigen presentation in the
posterior eye, with a possible route from the eye to the sentinel lymph nodes, similar to that
already described for anterior eye lymphatics.
Keywords: lymphangiogenesis, structural biology, anchoring filaments, VEGF-C, VEGFR-3,
transmission electron microscopy
he lymphatic system in most vertebrates plays a key role in
maintaining fluid homeostasis, macromolecular absorption
(including lipids), immune surveillance, and tumor metastasis. It is classically described as a network of permeable
vessels that collects excess interstitial fluid and proteins from
the tissue and recirculates these to the venous blood.1 These
vessels also transport various antigens and activated antigenpresenting cells to the lymph nodes. For these reasons,
lymphatic defects underlie many pathological processes. In
the eye, the uveal tract (comprising the iris, ciliary body, and
choroid) plays a critical role in maintaining fluid homeostasis
and the immune microenvironment of adjacent ocular tissues,
including the retina. Anterior eye lymphatics are involved in
the response to corneal injury,2 in the growth of conjunctival
tumors, 3 in the drainage of aqueous humor through
Schlemm’s canal,4 and in uveoscleral outflow of aqueous
humor likely via lymphatics in the ciliary body.5 A recent
study also reported peritumoral ciliary body lymphatics in
posterior uveal melanomas involving the ciliary body, with
and without extraocular extension.6 Schlereth et al.7 recently
concluded an absence of lymphatics in the human sclera
using double-label immunohistochemistry (IHC) on human
paraffin sections with markers CD31 to detect blood vessels
and lymphatic vessel endothelial receptor-1 (LYVE-1) and
podoplanin to detect lymphatic vessels. However, the
evidence for lymphatics in the mammalian choroid is
equivocal,8,9 and the proposal that there are human choroidal
lymphatics remains controversial.10 Earlier studies did not
find evidence for a classical lymphatic system in the human
choroid.11 More recently, limited ultrastructural evidence of
Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.
www.iovs.org j ISSN: 1552-5783
1310
T
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1311
Evidence of Lymphatics in the Human Choroid
lymphatic capillaries was found in the choroid of nonhuman
primate eyes (n ¼ 4), where distinct spaces that contain
amorphous material and no collagen, delineated by extremely thin and irregular cellular walls, were identified.12
Lymphatic capillaries have also been identified in the choroid
of birds.13
The application of lymphatic lineage markers LYVE-1 and
podoplanin,8 or LYVE-1 and vascular endothelial growth
factor receptor-3 (VEGFR-3 or Flt4),9 showed only LYVE-1þ
macrophages in the choroid. These studies concluded that
there is an absence of formed lymphatic channels in the aging
human choroid8 and in 8- to 12-week-old murine eyes,9 with
no evidence of classical lymphatic vessels in the normal adult
human choroid. However, Schroedl et al.8 reported netlike
structures with a pseudovessel appearance in the human
choroid, and suggested that while lymphatic vascular
precursor cells (represented as LYVE-1þ macrophages) in
the choroidal stroma do not form functional channels, they
may respond to inflammatory stimuli. Moreover, there is
functional evidence for a lymphatic system in the anterior
uvea5 and in the bulbar conjunctiva.14 Yucel5 provided
morphological and functional evidence for anterior uveal
lymphatics by injecting I125 radiolabeled human serum
albumin into sheep eyes, which drained into four head and
neck lymph nodes including the cervical, retropharyngeal,
submandibular, and preauricular nodes. Fluorescent nanospheres injected into the anterior chamber of sheep eyes
were also detected in LYVE-1þ channels of the ciliary body.5
While these data continue to be debated, it would appear
inconsistent for lymphatics to be present only in the anterior
eye, with no system for removal of excess interstitial fluid and
proteins from the posterior eye and no direct means for
antigen-presenting cells to exit the posterior eye and present
antigen at sentinel lymph nodes.
To date, the presence of anchoring filaments represents
the gold standard in ultrastructural evidence for lymphatic
capillaries.15–18 Numerous fine anchoring filaments (approximately 6 nm in diameter) can be seen extending from the
abluminal surface of lymphatic endothelial cells (LECs) and
are attached to the lymphatic endothelia at areas of increased
electron density. Fibrillin-containing anchoring filaments have
been shown to be between 4 and 10 nm in diameter.1 In our
study the anchoring filaments identified using TEM ultrastructural criteria were on average 6 nm in diameter, making our
anchoring filament structure consistent with the conclusion
that they contain fibrillin. They extend to the surrounding
collagen fibers for varying distances into adjoining connective
tissue, and anchor the lymphatic capillary.17,19,20 These
filaments attach the LECs to collagen fibers within the
surrounding stromal matrix and regulate the leaflike opening
into the lumen of the initial precollector segments of
lymphatics. In normal conditions, the lymphatic capillaries
are generally collapsed. Increased interstitial pressure from 7
to þ2 mm Hg distends the lymphatic vessels and increases
lymph flow.20 Anchoring filaments allow the lymphatic initial
segment to distend and expand the lymphatic lumen, and
open overlapping intracellular junctions to facilitate the
passage of fluid and macromolecules into the lymphatic
vessels.20
The lymphatic vasculature is an intricate network of thinwalled vessels, the capillaries of which are highly permeable
blind-ending sacs that allow the lymph easy access. However,
the end of the capillary is only one cell thick with loose
endothelial junctions, and these endothelial cells are arranged
in a slightly overlapping pattern (flap valves). Pressure from
the fluid surrounding the capillary forces these cells to
separate, allowing fluid to enter but not to leave the capillary,
so that increased interstitial pressure results in vessel dilation
rather than vessel collapse. The lack of a continuous
basement membrane or pericyte ensheathment facilitates
entry of fluid into these vessels and restoration of normal
interstitial volume, resulting in a slackening of the anchoring
filaments and eventual return of LECs to their overlapping
resting position.16–18,21
The availability of improved lymphatic-specific markers22 and
interest in the cellular and molecular mechanisms of lymphangiogenesis23 led us to re-examine the evidence for lymphatics in
the developing and adult human choroid. Using both multimarker immunolabeling for a comprehensive list of proteins
reported to be specific for lymphatic and vascular lineages and
established ultrastructural criteria to identify lymphatics,16,18 we
aimed to provide evidence for a system of lymphatic-like
structures and insights into the cellular processes of lymphatic
formation in the developing and adult human choroid. This
included spatial, temporal, and topographical details of lymphatic channel formation and the possible relationships with
LYVE-1þ macrophages and the venous circulation.
MATERIALS
AND
METHODS
Human Tissue Collection
Fetal Eyes. Fourteen fetal human eyes (8–26 weeks
gestation [WG]) were collected in accordance with the
Declaration of Helsinki for the Use of Human Tissue, as
previously reported,24,25 with approval from the University of
Sydney Human Research Ethics Committee (HREC approval
numbers 2006/9060 and 2012/15186). Fetal age was determined from the date of last menstruation, or by using the
guidelines previously described by Potter and Craig for
measurement of antenatal growth.26 No sex determination
was undertaken.
Adult Eyes. Ten adult (17–54 years) postmortem eyes, with
no history of ocular disease or comorbidities, were obtained
from the Lions NSW Eye Bank, with consent and ethical
approval from the Human Research Ethics Committee of The
University of Sydney. The cause of death was trauma related in
the cases less than 30 years old. For comparative purposes,
additional aged eyes from a 74-year-old male with respiratory
failure, metastatic pancreatic cancer, and type 1 diabetes
mellitus and from a 50-year-old male with glioblastoma
multiforme were examined. Typically, postmortem delay
ranged from 12 to 22 hours. All eyes were prepared either as
whole mounts, paraffin or frozen sections, or resin sections for
transmission electron microscopy (TEM).
Preparation of Human Choroidal Whole Mounts
The choroids were dissected and prepared as whole mounts as
previously described.27 After carefully removing the retina,
four or five radial cuts were placed through the entire
thickness of the choroid and sclera to permit flattening of
the tissue. The human fetal choroid was dissected away from
any scleral attachments to ensure continuity of the choroid
through the optic nerve head (ONH), then immersion fixed in
4% paraformaldehyde (PFA) for 1 hour at 48C. Adult specimens
were immersion fixed overnight in 2% PFA. All observations
were confirmed on a minimum of three specimens for both
sections and whole mounts.
Preparation and Analysis of Human Choroidal
Transverse Paraffin-Embedded Sections
The tissue was fixed in 2% PFA, processed through graded
ethanols, and then paraffin embedded. Double-label IHC was
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1312
Evidence of Lymphatics in the Human Choroid
TABLE 1. Antibodies and Markers Used in the Current Study
Antibody
Podoplanin
D2-40
Description
Manufacturer
A mucin-type transmembrane glycoprotein with extensive
O-glycosylation specifically expressed by lymphatic
endothelial cells but not blood vascular endothelial cells.
Functions include regulation of lymphatic vascular
formation and platelet aggregation.
Clone D2-40 identifies the 38-kDa integral membrane
glycoprotein podoplanin (description above).
LYVE-1
Lymphatic-specific receptor for endothelial hyaluronan;
expressed on lymphatic but not blood vascular
endothelium.
PROX-1
Prospero-homeobox 1 transcription factor, a marker of
ectodermal placodes, endodermal compartments,
lymphatic endothelium, and lymphangioblasts.
VEGFR-3
Endothelium-specific receptor tyrosine kinase, expressed by
immature blood vessels and not mature ones,
constitutively expressed by the lymphatic endothelium.
VEGF-C
Participates in development of lymphatic vasculature by
activation of VEGFR-3.
Endomucin
Endothelial sialomucin expressed on lymphatic endothelium,
veins, and venules, but not arteries.
PECAM-1 (CD31) Platelet endothelial cell adhesion molecule-1. Integral
membrane glycoprotein expressed on endothelial
intercellular junctions.
CD34
CD34 is a single-chain transmembrane glycoprotein
selectively expressed on human lymphoid and myeloid
hematopoietic progenitor cells as well as on the filopodial
extensions and the luminal membrane of endothelial cells.
CD39
CD39 is an ecto-ADPase and a marker of VPCs and human
endothelial cells but is also expressed on mature B and
microglial cells.
CD44
CD44 is a cell adhesion receptor widely expressed on
hematopoietic and nonhematopoietic cells.
Collagen IV
Basement membrane protein found on vessel walls.
UEA lectin
UEA(1) lectin has been used to evaluate the antigen H that
corresponds to blood group O. UEA(1) strongly reacts with
endothelial cells from all lymphatics and blood vessels.
Species Subclass Reference
R&D Systems
(Minneapolis, MN,
USA)
Sheep
IgG
22
Dako (Australia Pty Ltd.,
North Sydney, New
South Wales, Australia)
Abcam (Cambridge, MA,
USA)
Mouse
IgG1
23
Rabbit
IgG
24
Reliatech (Wolfenbüttel,
Germany)
Rabbit
IgG
25
Reliatech
Mouse
IgG1
27
R&D Systems
Rabbit
IgG
26
Gift from D. Vestweber
Rat
IgG2a
28
Santa-Cruz (Dallas, TX,
USA)
Rabbit
IgG
37
Serotec (Raleigh, SC,
USA)
Mouse
IgG2a
21
Novocastra (NewcastleUpon-Tyne, UK)
Mouse
IgG2a
21
Immunotech (Brea, CA,
USA)
Abcam
Sigma-Aldrich Corp. (St.
Louis, MO, USA)
Mouse
IgG1
21
Rabbit
-
IgG
-
4
35
ADPase, adenosine 5c-diphosphatase; VPC, vascular precursor cells.
performed on a Ventana Benchmark NexES machine (Ventana
Medical Systems, Inc., Tucson, AZ, USA) using the streptavidin–biotin method on 4-lm paraffin sections collected on
Superfrost Plus slides (Menzel-Glaser, Menzel GmbH & Co. KG,
Braunschweig, Germany).
Multiple-Marker Immunofluorescence (Frozen
Sections and Whole Mounts)
Given the close temporal and spatial relationship between
formation of the vascular and lymphatic lineages,28 our analyses
involved combining the stem cell, vasculogenic, and angiogenic
markers detailed in Table 1 of our recent paper,28 as well as a
number of established lymphatic markers including podoplanin,29 D2-40,30 LYVE-1,31 prospero homeobox-1 (PROX-1),32
VEGF-C,33 and VEGFR-3,34 along with the pan-vascular/lymphatic marker UEA lectin35 and basement membrane protein marker
collagen IV.5 We also utilized human endomucin, which is
expressed on lymphatic endothelium, veins, and venules,36 and
platelet endothelial cell adhesion molecule-1 (PECAM-1), an
integral membrane glycoprotein constitutively expressed on
endothelial intercellular junctions.37 This approach made it
possible to provide unique insights regarding the relationship
between the formation of blood vessels and lymphatics. Vascular
endothelial growth factor C induces lymphangiogenesis through
VEGFR-3, which in the adult is expressed on lymphatic
endothelium34,38 but is also found on some proliferating
endothelial cells.39 Table 1 details the antibodies and lymphatic
and blood vascular markers utilized in this study. Human
choroidal whole mounts and transverse sections were incubated
overnight in UEA lectin35 FITC conjugated (L006, 1:100 diluted
in 0.05% Triton X-100 in PBS; Sigma-Aldrich Corp., St. Louis, MO,
USA) after blocking in 1% bovine serum albumin for 30 minutes.
Mild permeabilization with 0.1% Triton X-100 for 30 minutes
preceded the incubation.
Choroids were incubated overnight at 48C with appropriate
primary antibodies, washed with 0.1% nonionic surfactant
(Triton X-100; Sigma-Aldrich Corp.) in PBS, incubated for 2
hours at room temperature with the respective secondary
antibodies, and washed again. For double or triple labeling, this
procedure was repeated with different primary antibodies and
appropriate species- and class-specific secondary antibodies.
Negative controls included omission of the primary antibody
such that tissue was incubated in 1% bovine serum albumin in
PBS alone, and isotype control antibodies (Supplementary Fig.
S1). All primary and secondary antibodies were diluted with
1% bovine serum albumin in PBS, and washes were performed
with 0.1% Triton X-100 in PBS. Choroidal whole mounts were
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1313
Evidence of Lymphatics in the Human Choroid
mounted in antifade mounting medium (Vectashield Mounting
Medium H-1000; Vector Laboratories, Burlingame, CA, USA),
with the retinal pigmented epithelium (RPE) side up or down
depending on the layer of interest. For histological cross
sections, tissue was fixed in 4% PFA, washed several times in
PBS, and incubated at 48C in 30% (vol/vol) sucrose overnight,
then embedded in optimal cutting temperature mounting
medium (OCT) (Siemens Medical Solutions, Sydney, Australia)
using a dry ice and isopentane slush and cryosectioned on a
Leica CM3050S Research Cryostat (Leica Microsystems GmbH,
Wetzlar, Germany). Sections 12 lm thick were collected on
Superfrost Plus slides (Menzel GmbH & Co. KG), and primary
and secondary antibodies were applied and mounted as
described above.
Confocal Microscopy
Fluorochrome-immunostained choroidal whole mounts were
viewed using a Zeiss META LSM 510 confocal inverted
microscope (Carl Zeiss, Oberkochen, Germany) equipped
with appropriate excitation laser lines (405, 488, 561, and
633 nm). Captured images were processed with Adobe
Photoshop CS6 (Adobe Systems, Inc., San Jose, CA, USA).
The Tile Scan function in the Zeiss LSM software was used for
montaging adjacent maximum-intensity projections of z-stacks
taken from the optic disc to the periphery.
Transmission Electron Microscopy
Human fetal eyes aged between 8 and 26 WG (n ¼ 15) were
immersion fixed in 4% PFA for 1 hour at 48C, washed well in
0.1 M sodium phosphate buffer (Sorenson’s, pH 7.4), then
postfixed with 2% osmium tetroxide (Electron Microscopy
Sciences, Hatfield, PA, USA) in 0.1 M sodium phosphate buffer
for 2.5 hours. Young (17–33 years) adult human eyes (n ¼ 6)
were immersion fixed in 2% PFA for 2 hours, then postfixed in
2% osmium tetroxide in 0.1 M sodium phosphate buffer for 2.5
hours. En bloc staining with 2% uranyl acetate preceded
dehydration through a graded series of ethanols. Specimens
were infiltrated with low-viscosity epoxy resin (Spurr’s
replacement; TAAB Laboratory and Microscopy, Berkshire,
UK) using a mix of 50:50 ethanol to TAAB resin for 2 hours
followed by TAAB resin for 3 hours, embedded, and set
overnight at 708C. Ultrathin (100 nm) sections were cut from
each block of each eye, with a minimum of three levels per
block examined. Sections were mounted on 200-mesh copper/
palladium grids, stained with Reynold’s lead citrate, and
viewed on a transmission electron microscope (JEOL 1011;
JEOL Ltd, Tokyo, Japan). Images were captured using a digital
camera (MegaView G2; Olympus, Münster, Germany) and
software (iTEM; Olympus).
Ultrastructural Analysis of Lymphatic Distribution
and Density
Ultrastructural characteristics of human choroidal blood
vessels have been described in detail in our previous
studies.28,40 The ultrastructural criteria for lymphatic vessels
have also been established, including anchoring filaments,
absence of Weibel-Palade bodies (WPBs), lack of fenestrae,
fragmented and/or absent basal lamina, incomplete and
infrequent pericyte ensheathment, high endothelial cells, and
absence of red blood cells (RBCs).16,18,41 Using these
ultrastructural characteristics, an area of approximately 1.5
mm2 in six adult choroid specimens was examined, and the
frequency of all blood vessels and lymphatics for three levels of
ultrathin sections of choroid was counted; sections were taken
at 50, 100, and 150 lm from the ONH. Lymphatic vessels with
a range of lumen diameters were typically characterized as
initial (10–60 lm),42 precollector (35–150 lm),43 and collector
lymphatics (>200 lm).42 The frequency was then expressed as
a percentage of total vessel profiles counted per area of
choroid at each level (Table 2).
RESULTS
PROX-1þ/Podoplaninþ/D2-40þ/VEGF-R3þ/CD34þ
Presumed Lymphatic Precursor Cells (LPCs) in
Human Choroidal Stroma During Early
Development
We identified single, isolated PROX-1þ/podoplaninþ/D2-40þ/
VEGF-R3þ/CD34þ cells, which we presumed were LPCs due to
their antigenic phenotype and their single, isolated distribution
within the human choroidal stroma from an early age (Figs. 1A–
D, 1E–H). CD34/PROX-1/VEGFR3 triple-label IHC showed that
the LPCs constituted a subset of CD34þ cells (presumed
hematopoietic stem cells [HSCs; according to a previous
study28]) (Figs. 1E–H).
Large numbers of CD34þ HSCs were evident (identified
with pink arrows in Fig. 1H), whereas the yellow arrows show
a subset of CD34þ cells that represent PROX-1þ/VEGFR-3þ/
CD34þ LPCs. These observations suggest that LPCs may play a
role in human choroidal lymphatic formation. However,
evidence supporting a conclusion that presumed LPCs are
able to form lymphatic vasculatures is not available from
postmortem human fetal tissue, as time-lapse live cell imaging
is not possible.
PROX-1 Delineates Channel-Like Structures, and a
Subset of Formed CD34þ Blood Vessels Become
PROX-1þ/VEGFR-3þ Lymphatics
Despite reports stating that PROX-1 antibody should localize
to the cell nucleus, we did not find any PROX-1þ nuclei in
the human choroid even though PROX-1þ nuclei were
evident in the germinal layer (Fig. 1I, arrows) of the human
fetal retina at 20 WG (Figs. 1I–K). No nuclear staining was
evident in the choroid; instead, PROX-1 weakly delineated
structures that were tubular in nature (arrowheads in lower
right-hand corner, Fig. 1I). We are not the first to report this
observation with PROX-1 IHC. Truman et al.44 reported that
PROX-1 is predominantly located in the cytoplasm of
megakaryocytes, with a smaller amount in the nuclear
fraction, and is also expressed in some extra-lymphatic
tissues, suggesting that PROX-1 may move more freely
between the nucleus and cytoplasm than was initially
appreciated.
LYVE-1þ/VEGFR-3þ Macrophages Are Closely
Associated With Early Lymphatic Channel
Development in the Human Choroid
Consistent with earlier reports in human8 and rat choroid,9 the
lymphatic and macrophage marker, LYVE-1, identified a
significant population of macrophages in the developing
choroid (Figs. 1L–N). Using double-marker IHC for CD34 and
LYVE-1 on transverse sections of 9 to 19 WG choroids, many
isolated LYVE-1þ macrophages were evident within the
choroidal stroma. By 16 WG, LYVE-1þ macrophages were not
associated with forming CD34þ blood vessels (purple arrows in
Figs. 1L–N). Rather, the LYVE-1þ macrophages appeared to line
the CD34 lumina, suggesting the involvement of LYVE-1þ
macrophages in the formation of the earliest lymphatic
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
Evidence of Lymphatics in the Human Choroid
IOVS j February 2015 j Vol. 56 j No. 2 j 1314
FIGURE 1. Single isolated lymphatic precursor cells (LPCs) were evident within the stroma of the human choroid from an early age. Utilizing
different combinations of multimarker IHC, LPCs were determined to have an antigenic phenotype of PROX-1þ/podoplaninþ/D-240þ/VEGFR-3þ and
constituted a subset of CD34þ HSC. (A–D) Whole mounts of human fetal choroid (14 WG) immunolabeled for podoplanin, PROX-1, D-240. (E–H)
20 WG choroidal whole mounts immunostained for CD34, PROX-1, and VEGFR-3 (E). A subset of CD34þ hematopoietic stem cells were also PROX1þ/VEGFR-3þ (F, H, respectively). The pink arrowed cells in (H) show CD34þ HSC, whereas the yellow arrows point to PROX-1þ/VEGFR-3þ/CD34þ
LPCs. (I–K) PROX-1 staining is nuclear in the retina, staining the somas within the germinal layer of the embryonic human retina at 20 WG.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1315
Evidence of Lymphatics in the Human Choroid
However, the choroid is devoid of PROX-1 nuclear staining, instead showing weak channel-like structures. LYVE-1þ macrophages were closely
associated with forming lymphatics. The inner limiting membrane (ILM) of the retina is indicated in (K). (L–N) Sections of human fetal choroid
stained with LYVE-1 (brown) and CD34 ( pink). The retinal pigmented epithelium (RPE) is LYVE-1 and can be seen as a dark row of cells at the top
of each image. Many isolated LYVE-1þ cells were found in the stroma at 9, 16, and 19 WG ( purple arrows). (L) The CD34þ choriocapillaris is already
beginning to form at 9 WG, with both patent (blue arrows) and nonpatent vessels (black arrows). (M) At 16 WG many more CD34þ capillaries
were patent; however, the bulk of the LYVE-1þ macrophages appeared to line up with vessel lumens that are CD34 negative ( green arrows),
suggesting that they were associating with the forming lymphatic channels rather than the forming blood vessels. (N) By 19 WG, the density of the
CD34þ choriocapillaris increased, and deeper, larger blood vessels were observed. The majority of LYVE-1þ macrophages remained either as single
isolated cells ( purple arrows) or aligned with CD34-negative lumens, presumably lymphatics. (O–Q) LYVE-1þ macrophages closely associated with
VEGFR-3þ vessels throughout the choroid at 15 WG, similar to observations in 16 WG choroidal sections (L–N).
channels (green arrows in Figs. 1L–N). This observation
persisted at 19 WG. LYVE-1þ macrophages around vessel
lumina were predominantly located just external to the
choriocapillaris, and coincided with the predominant location
of the lymphatic sacs and lymphatic initial collector channels
observed with TEM and characterized using ultrastructural
criteria (discussed further below). These conclusions were
supported in whole-mount choroids, where LYVE-1þ macrophages were associated with VEGFR3þ channels at 15 WG
(Figs. 1O–Q). Our observations are consistent with those of
Schroedl et al.,8 who reported netlike structures with a
pseudovessel appearance in the adult human choroid. Our
D2-40þ macrophages were closely aligned along a UEA lectinþ
channel-like structure, which was collagen IV negative,
supportive of a lymphatic phenotype.
Numerous Initial Lymphatic Segments Evident in
Strata Just External to the Choriocapillaris
During human choroidal development, VEGFR-3þ/CD34
initial lymphatic segments were evident dispersed among
the forming CD34þ blood vessels in the midperipheral
choroidal region, as shown in a 19 WG eye (Figs. 2A–C).
We have previously shown that CD34 is expressed in the
human choroid from 8 WG28 (as seen in Figs. 9G–L in an
earlier study28). In addition, numerous round-shaped VEGFR3þ/CD34 lymphatic sacs just external to the choriocapillaris
were evident, shown here at 19 WG in a region adjacent to
the ONH; note the co-incidence of CD34þ HSCs/RBCs within
the tear-shaped lymphatic sacs. Small numbers of CD34þ
HSCs/RBCs were sometimes observed within the initial
segments/lymphatic sacs of the forming lymphatics (Figs.
2D–I). Consistent with our observations of CD34þ HSCs/RBCs
within the initial segments of lymphatics, previous studies45,46 have provided evidence that the venous system and
lymphatic sacs are directly connected and ‘‘communicate via
a small aperture,’’ 47(p92) and that the lymphatic sacs
sometimes contain RBCs.46 Representative views of the
VEGFR-3þ/CD34weak ‘‘tear-shaped’’ lymphatic sacs are shown
in Figures 2G through 2I. These tear-shaped or round-shaped
lymphatic sacs were blind-ended, consistent with the
interpretation that they constitute initial lymphatic segments,
and were continuous with precollector lymphatics in the
developing human choroid. While this phenomenon was
described in the terminal vein and our system represents
terminal lymphatics, both systems describe phenomena very
early in embryonic development and could explain the
similarities observed.
VEGFR-3þ Lymphatics Constitute a Network
Distinct From the CD34þ Vasculature
We saw VEGFR-3þ and CD34þ structures nearest to the formed
choriocapillaris in a region adjacent to the ONH, shown here at
19 WG (Figs. 2D–F). Confocal imaging showed that VEGFR-3þ
lymphatics appeared in a deeper plane of focus to the CD34þ
choriocapillaris, and the caliber of the lymphatic capillaries
was broader than that of vessels in the choriocapillaris (not
shown), while the lymphatic sacs are VEGFR3þ/CD34weak
(Figs. 2D–F). Figures 2J through 2L show wispy CD34þ/VEGFR3þ structures in the outer choroidal stroma. Figures 2M
through 2O show a choroid from a 50-year-old patient who
died from complications arising from glioblastoma multiforme.
Using differential expression of endomucin and CD39 on
arteries, veins, and LECs, we show lymphatic-like channels in
the adult choroid.
Lymphatic-Like Collector Channels External to
Choriocapillaris
Taking advantage of the fact that collagen IV is found only on
blood vessel walls, we combined vascular markers with D2-40
and UEA lectin to visualize lymphatic-like structures on adult
human choroidal whole mounts and sections. Figures 3A
through 3C show a UEA lectin- and D2-40-stained cryosection
from a 74-year-old adult human choroid. This specimen is
from a patient who died of respiratory failure, metastatic
pancreatic cancer, and type 1 diabetes mellitus (T1D), with
both T1D and cancer assumed to cause some level of
inflammation, which would lead to activation and increase
in these lymphatic channels. UEA lectinþ/D2-40 blood
vessels are evident just adjacent to the RPE (RPE discernible
via autofluorescence in both green and red channels). D240þ/UEA lectin lymphatic structures are visible just below
the UEA lectinþ choriocapillaris in Figure 3C. Utilizing triplemarker whole-mount IHC (UEA lectin, D2-40, and collagen
IV), we were able to discern a D2-40þ/UEA lectin/collagen
IV structure with a netlike morphology that outlined a tubelike structure just external to a wide, UEA lectinþ/collagen IVþ
blood vessel (see Figs. 3D–K). Further, utilizing UEA lectinþ/
D2-40/LYVE-1 frozen sections, blood vessels are visible just
adjacent to the RPE (RPE discernible by autofluorescence
across all channels) and also in the mid and large vessel layers
of the choroid. In contrast, D2-40þ/LYVE-1þ structures are
visible only in a strata just external to the choriocapillaris,
shown in Figure 3O as a magenta band below the
choroiocapillaris and internal to mid- and large-sized choroidal blood vessels (see Figs. 3L–O). This stratification of
lymphatic-like structures is consistent with their location as
determined by TEM in this study.
Choroidal Blood Vessel Development Precedes
Lymphatic Development, Both Systems Displaying
an Optic Nerve Head-to-Periphery Topography of
Formation
At 19 WG, VEGFR-3þ lymphatic sacs and vessels were seen at
the ONH, spreading centrifugally toward the periphery in
the human choroid (Fig. 4A). The extent of VEGFR-3þ
immunolabeling at 13 WG in the region of the ONH,
midperiphery, and peripheral retina at the leading edge of
formation of lymphatics, respectively, is shown (Figs. 4C,
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
Evidence of Lymphatics in the Human Choroid
IOVS j February 2015 j Vol. 56 j No. 2 j 1316
FIGURE 2. Precollector lymphatic-like channels connect to dead-ended round or tear-shaped lymphatic sacs in whole mounts of the developing
human choroid. (A–C) VEGFR-3þ/CD34 buds were seen interspersed among the CD34þ choroidal vasculature. Note the frequent association of the
VEGFR-3þ lymphatic initial collector channels with intraluminal CD34þ HSCs. (D–F) Two representative round lymphatic sacs just external to the
choriocapillaris. (G–I) A representative tear-shaped VEGFR-3þ lymphatic sac located just external to the choriocapillaris in a 19 WG human fetal
choroid, adjacent to the ONH. Note the numerous CD34þ HSCs within the lymphatic sacs. (J–L) Wispy CD34þ/VEGFR-3þ structures in the outer
choroidal stroma. (M–O) Choroid from a 50-year-old with history of glioblastoma.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1317
Evidence of Lymphatics in the Human Choroid
FIGURE 3. D2-40þ/UEA lectin/collagen IV lymphatic-like channels external to the choriocapillaris in 74-year-old human adult choroid. (A–C) A 12lm-thick cryosection taken at 340 magnification from a 74-year-old adult human choroid double stained for UEA lectin and D2-40. (A) UEA lectinþ/
D2-40 blood vessels are shown just adjacent to the RPE (RPE discernible via autofluorescence in both green and red channels). D2-40þ/UEA lectin
structures are visible just below the UEA lectinþ blood vessels in (B). (D–G) Orthographic projections; (H–K) triple-marker immunohistochemistry
(UEA lectin, D2-40, and collagen IV) of a 74-year-old human choroidal whole mount. The yellow boxes denote the level at which the maximumintensity projections displayed in (H–K) were created. (E) The level at which a tube-like D2-40þ/UEA lectin/collagen IV structure is present just
adjacent to a wide, UEA lectinþ/collagen IVþ blood vessel. (L–O) UEA lectin, D2-40, and LYVE-1 staining on a 12-lm-thick cryosection in the same
74-year-old human specimen at 340 magnification. UEA lectinþ/D2-40/LYVE-1 blood vessels are visible just adjacent to the RPE (RPE discernible by
autofluorescence across all channels) and also in the mid and large vessel layers of the choroid. In contrast, D2-40þ/LYVE-1þ structures are visible
only in (O) as a magenta streak below the vessels in the choroiocapillaris and internal to the mid and large vascular layers of the choroidal blood
vessels. This stratification of lymphatic-like structures is supportive of their location as determined by TEM in this study.
4D). By montaging a large area imaged at low magnification
(Fig. 4A), we discerned that the earliest formation of
lymphatic structures appeared centered on the ONH and
spread to the midperipheral choroid at 19 WG. Figure 4A
shows an entire segment of a whole-mount human choroid
from ONH to the periphery immunostained for VEGFR-3 at
19 WG. Insets show the regions of interest at higher
magnification, with the outer limit of lymphatic sacs in the
midperipheral choroid. The four insets (a–d) seen at higher
magnification show the outer limit of lymphatic sacs, as the
sacs are visible only on the left-hand half of all insets. The
transitional zone between formed sacs to the left and
absence to the right is also shown (Fig. 4A, inset d).
Lymphatic sacs are evident from the ONH to the midperiphery, with mid- to large-sized lymphatics less evident
peripherally. A higher density of the VEGFR3þ lymphatic sacs
can be seen in the midperiphery, tapering off at the leading
edge. The topography of the developing lymphatic vessel
structures showed that the VEGFR-3þ structures emanate
centrifugally from the ONH of the human choroid at 13 WG.
Formation of the blood vessels showed a disc-to-periphery
topography that had reached the periphery of the choroid as
TABLE 2. Relative Frequency of Lymphatic-Like Vessels Versus Blood Vessels in Adult Human Choroid, n ¼ 6
Lymphatics
N
Blood Vessels
N
Total Vessels Counted
% Lymphatics
Initial, 10–60 lm42
Precollector, 35–150 lm43
Collector, >200 lm42
40
1
1
Capillaries
Medium
Large
543
88
21
583
89
22
6.9, 40/583
1.1, 1/89
4.5, 1/22
Ultrathin sections were cut from each block of each eye (n ¼ 6), with a minimum of three levels per eye examined by TEM. The relative
frequency of lymphatics was determined by counting all blood vessels and lymphatics (Total Vessels) in a choroidal area of 1.5 mm2 (n ¼ 3). The
numbers of lymphatics counted per total number of vessels of that size31 in 1.5 mm2 (n ¼ 3) was used to estimate the relative frequency of
lymphatics.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
Evidence of Lymphatics in the Human Choroid
IOVS j February 2015 j Vol. 56 j No. 2 j 1318
FIGURE 4. Whole mount showing the optic nerve head (ONH)-to-periphery topography of formation of VEGFR-3þ and endomucinþ lymphatics during
human choroidal development: (A) An entire sector of a 19 WG human choroid from ONH to the periphery immunostained for VEGFR-3. Multiple fields
of view were montaged for topographical analysis at high magnification. Insets a through d show the transitional zone with formed sacs to the left and
none to the right. Thus, joining these insets shows that the outer limits of the lymphatic sacs are in midperipheral choroid at 19 WG. Two representative
formed lymphatic sacs are arrowed in inset a. (B–D) Representative fields of view from the central, midperiphery, and periphery of a 13 WG choroid.
The earliest formation of lymphatic-like structures was seen around the ONH, and extended to the midchoroid at 13 WG. (E–H) Thin-walled
endomucinþ structures with a tree-like morphology and very wide, irregular calibers were seen in adult choroid (54 years); these are atypical of choroidal
blood vessels, supporting an earlier report that endomucin stains lymphatics.28 Flocculent material, probably protein, is seen within the larger-caliber
lymphatic collector channel in (E) (arrowed). (F) Endomucinþ lymphatic channels showed additional ‘‘flaps’’ not seen in blood vessels. These smaller
initial lymphatic segments fed into the collector lymphatic at the site arrowed in (F). (G) An empty lymphatic precollector. (H) Examination of the
endomucinþ endothelial junctions lining the lymphatic channels showed a flap-like morphology (arrowed) as described elsewhere (see Fig. 4H48). (I)
High-magnification view of junctions between neighboring vascular endothelial cells in a human choroidal vein stained with PECAM-1. Note the marked
difference in the shape of VECs as outlined with PECAM-1 versus the apparent flap/petals shown in (H) for a presumed lymphatic.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1319
Evidence of Lymphatics in the Human Choroid
FIGURE 5. (A–D) Confocal images of vessels in midperipheral choroid (40 years) showing a presumed lymphatic valve (CD34þ, VEGFR-3þ, VEGFCþ) in a lymphatic precollector channel (arrow in [D] points to the precollector channel). (E–H) Confocal images of vessels near the optic nerve
head (40 years) showing lymphatic endothelial cells (LECs) (CD34þ, D2-40þ, LYVE-1þ, arrows in [H]) with lower density and much wider gaps along
the vessel lumen than typically seen on blood vessels, (I–L) Human 30-year-old choroid triple immunolabeled for CD34/D2-40 and VEGF-C.
Macrophages (M) were frequently seen at the junction between lymphatics (L) (arrowheads) that had no red blood cells (RBCs) and blood vessels
(tailed arrows) with intraluminal RBCs and lymphocytes.
described previously at 19 WG,28 whereas the lymphatic
vessels were found only from the ONH to the midperiphery.
This topography of formation suggests that choroidal blood
vessel development precedes lymphatic development.
Endomucinþ Lymphatic-Like Channels With
Unique Branching Patterns and Irregular Caliber
in Adult Choroid and Lymphatic Valves
The adult endomucinþ lymphatic channels (Figs. 4E–H) were
thin-walled structures with a branching morphology atypical of
choroidal veins, with an extremely wide, irregular caliber (128
lm at its widest point, Fig. 4G). These smaller initial lymphaticlike segments fed into the larger presumptive collector
lymphatics (Fig. 4F). Flocculent material, likely protein, was
evident within the larger-caliber, presumptive lymphatic collector channels (Fig. 4E, arrow). Endomucinþ presumptive
lymphatic channels showed additional ‘‘flaps’’ not seen in blood
vessels. With high magnification, endomucinþ-stained endothelial junctions lining these presumptive lymphatic channels
showed a flap-like morphology as previously described48 (Fig.
4H, arrows), compared to the regular hexagonal junctions
observed between vascular endothelial cells (Fig. 4I). Further,
these presumptive lymphatic-like collector channels did not
show the bifurcations typically observed on blood vessel branch
points. A lymphatic valve (CD34þ, VEGFR-3þ, VEGF-Cþ) is
shown in a presumptive lymphatic precollector channel in the
midperiphery of a human choroid (40 years) (Figs. 5A–D; arrow
in Fig. 5D). Lymphatic valves in other organs and species have
been previously reported with the molecular identity of PROX1þþþ, Foxc2þþþ, VEGFR3þþ, podoplaninþ, GATA2þ, integrin-a2þ,
laminin-a5þ, integrin-a9þ, Cx37þ, Cx43þ, Cx47þ, LYVE-1.49,50
High Lymphatic Endothelial Cells
In adult human choroid (40 years), high LECs (CD34þ, D2-40þ,
LYVE-1þ) with a lower density and much wider gaps between
neighboring cells lining the lymphatic lumen were evident (Figs.
5E–H). Also Figures 5I through 5L show a representative
example of a 30-year-old adult choroid, with a lack of cellular
content in the large-caliber lymphatic collector channel
compared to the adjacent vein, which is full of RBCs and
leukocytes. While our data are not definitive, the LYVE-1þ
macrophages (labeled M in Fig. 5L) suggest a possible site of
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
Evidence of Lymphatics in the Human Choroid
IOVS j February 2015 j Vol. 56 j No. 2 j 1320
FIGURE 6. Ultrastructural evidence of lymphatic channels in young adult human choroid (29 and 33 years). (A) Representative lymphatic capillary
external to the choriocapillaris, showing no evidence of fenestrae, Weibel-Palade bodies (WPB), or intraluminal red blood cells (RBCs). Inset shows
cytoplasmic content in detail (polyribosomes and portion of the nucleus). Pericyte ensheathment is not present for any of the smaller ("capillary’’)sized lymph vessels. High endothelial cell nuclei protrude into the lumen. (B) A lymphatic collecting channel located among arterioles and venules.
These vessels do not appear to display fenestrae or WPB, and have a fragmented or absent basal lamina. Pericyte ensheathment is present but
incomplete in this midsized lymph vessel. (C) A representative lymphatic showing endothelial cell and no features of typical arteriole or vein.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1321
Evidence of Lymphatics in the Human Choroid
Pericyte ensheathment is most complete in (C). Higher-magnification examination confirmed three cell components in this area around the vessel:
the outer pericyte processes and inner luminal endothelial cell. (D) This micrograph shows intraluminal flocculent material within a putative
lymphatic vessel. (E) In this high-power micrograph, Weibel-Palade bodies are not visible within the cytoplasm of a lymphatic endothelial cell. (F)
In this micrograph, a vascular capillary is shown with endothelial fenestrae (arrowheads) and a complete basal lamina (arrows). An intravascular
leukocyte can also be observed. Inset shows WPB. (G) In this electron micrograph, the lumen of a choroidal arteriole is lined with endothelial cells
and displays a complete basal lamina. Smooth muscle cells packed with thin filaments with focal densities can also be seen (SMA). (H) The lumen of
a choroidal venule can be seen here with an adjacent pericyte. BrM, Bruch’s membrane; E, endothelial cell; Vc, vascular capillary; Pi, pigmented cell;
WPB, Weibel-Palade body; M, mitochondria; RER, rough endothelial reticulum; SMA, smooth muscle actin; VEC, vascular endothelial cell; C,
collagen.
interaction between the two systems requiring further investigation in future studies.
Ultrastructural Evidence for Lymphatics in Young
Adult Human Choroid
Transmission electron microscopy of adult human choroid
showed features typical of lymphatic vessels (see Methods).
Lymphatic vessels with a range of lumen diameters, typically
characterized as initial (10–60 lm),42 precollector (35–150
lm),43 and collector lymphatics (>200 lm),42 were evident.
Consistent with their function of collecting fluid, the
lymphatic vessels examined often displayed evidence of
luminal flocculent material (most likely proteins) but not
luminal cells (Figs. 6A–E). Figure 6B shows a lymphatic
collecting channel located in between the choriocapillaris
and Sattler’s layer, with features typical of lymphatic vessels
including an absence of WPBs, thin or absent basal lamina, and
absence of any cellular content within its lumen. Similar to
what was seen in our earlier study in human choriocapillaris,40
pericyte ensheathment was not apparent for the smaller
("capillary’’)-sized lymph vessels; however, it was present but
incomplete for some midsized lymphatic vessels (Fig. 6B).
Larger-caliber lymphatic vessels displayed high endothelial
cells with large gaps between neighboring cells, and an
apparent absence of ultrastructural features that typically
characterize arterioles or veins (Figs. 6C, 6D). Figure 6D (high
magnification) shows the lumens of these lymphatic channels
filled with a flocculent material (most likely proteins) and an
absence of basal lamina along the entire capillary, as well as an
absence of cytoplasmic WPBs in the endothelium. The absence
of RBCs, together with the presence of flocculent material,
provides further support for these channels as lymphatics;
most blood vessels normally present with intraluminal RBCs
and some leukocytes. In contrast, ultrastructural characteristics
of the adult human choriocapillaris included luminal RBCs, a
substantial basal lamina (Figs. 7C–F), a presence of WPBs (Fig.
7D), and fenestrae (Fig. 7C).
A representative lymphatic initial segment (lymphatic
capillary) external to the choriocapillaris, with no fenestrae
FIGURE 7. Capillaries in the choroid of a 33-year-old at low magnification. (A, B) Pericytes (marked) are visible partially ensheathing the capillaries.
Well-formed basal lamina is present entirely surrounding the capillaries and is thinner on the Bruch’s membrane side. (C) Fenestrae (F) are present
on all of the vascular capillaries. (D) WPB is demonstrated in the VEC but was absent in LEC (previous image). High-power micrographs of regions
in (A, B) are shown in (E, F), illustrating the vessel basal lamina.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1322
Evidence of Lymphatics in the Human Choroid
FIGURE 8. (A, B) Electron micrographs showing anchoring filaments attached to the lymphatic endothelial cells. Poorly formed junctions were
observed between endothelial cells (B). The diagram in (C) illustrates how poorly formed endothelial junctions can be separated by increased
interstitial pressure, with a subsequent influx of extracellular fluids from the surrounding tissue into the lymphatic vessel. Diagram where the
drawbridge analogy is described is reprinted with permission from Skobe M, Detmar M. Structure, function and molecular control of the skin
lymphatic system. J Investig Dermatol Symp Proc. 2000;5:14–19. Copyright 2000.
and no intracellular WPBs, is shown in Figure 8A. The human
choriocapillaris is characterized by the presence of fenestrae
(Fig. 7C); and the absence, or relative paucity, in these vessels
is consistent with a lymphatic capillary (Fig. 6A). Both the
choriocapillaris (Figs. 7A, 7B, 7F) and lymphatic initial
segments showed a relative paucity of pericyte ensheathment
(Figs. 6B, 6C) compared to the retinal capillaries (but not an
absolute absence of pericyte ensheathment).40
Anchoring Filaments, Definitive Evidence of
Lymphatic Initial Segment Structure, Are Present
in Human Choroid
Anchoring filaments, a hallmark feature of lymphatic capillaries, were observed attached from the extracellular matrix to
the LECs in the human choroid using TEM (Figs. 8A, 8C; inset
in Fig. 8B). Anchoring filaments are resolvable on originals but
have been marked in red to facilitate their visualization (Fig.
8C). These images at full resolution are also included as
Supplementary Figure S2.
Ultrastructural Evidence That Blood Vessels and
Lymphatic Initial Segments Are Located at Distinct
Substrata Within the Adult Human Choroid
Although the endothelial cells that line the initial segments of
lymphatics displayed some features in common with blood
vessel endothelium, they also showed many distinct structural
characteristics reflecting their specific functions as noted
above (compare Figs. 6A–E to Figs. 6F–H). A comparison of
multiple-immunomarker choroidal whole-mount confocal zstacks (Fig. 2) and TEM montages of choroidal cross sections,
where the entire choroidal cross section is imaged (Fig. 9),
showed that lymphatic initial segments were most frequently
located in a stratum just external to the choriocapillaris. The
relative depths of the choroidal blood vessels, compared to the
lymphatic structures identified in the current study using
ultrastructural criteria (see above), are shown in Figure 9. The
similarities in the stratification observed between venous
plexuses and lymphatic channels in the human choroid lead
us to suggest possible communication under normal circumstances between the lymphatic and vascular system, but
further tracing and imaging studies are required to confirm
this suggestion.
Low Frequency of Lymphatic Channels in Young
Adult Human Eye
Using ultrastructural criteria described in Methods to differentiate blood vessels from lymphatic channels, the relative
frequencies of initial, precollector, and large lymphatic
channels were compared to those of vascular capillaries,
midsized arterioles or venules, and large arteries/veins in a
representative region of young adult human choroids (n ¼ 6
eyes, n ¼ 3 levels of tissue) (Table 2). The relative frequency of
lymphatic vessels in healthy young adult eyes was approximately 7%, 1%, and 5%, respectively (Table 2).
DISCUSSION
Our aim was to examine for structural and phenotypic
evidence of a lymphatic system in the human choroid and to
provide the first details of its development using more recently
available lymphatic specific markers on choroidal whole
mounts and sections. Transmission electron microscopy
provided ultrastructural confirmation of a system of lymphatic-like structures within the human choroid and an estimate of
the relative frequency of these structures compared to blood
vessels in adult human choroid. The classical concept of
lymphatics refers to structures that represent blind-ending
capillaries building a netlike framework throughout the tissue,
converging to larger lymphatic vessels and collector vessels,
eventually entering lymph nodes.21 While we were able to
demonstrate the presence of blind-ended lymphatic capillary
sacs just external to the choriocapillaris, we were only able to
demonstrate limited larger lymphatic channels using recognized lymphatic markers. However, we were able to discriminate lymphatic channels in the outer human choroid using
TEM. Similarly to Schroedl et al.,8 who reported LYVE-1þ
‘‘netlike structures with a pseudovessel-like appearance,’’(p5226)
we also recognized very fine, netlike CD34þ/VEGFR-3þ
structures in developing choroid, along with tube-like D240þ/collagen IV/UEA lectin structures in aged choroid.
Further, Schroedl et al.8 noted that ‘‘by virtue of their tubular
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1323
Evidence of Lymphatics in the Human Choroid
FIGURE 9. Montaged TEM of the entire choroidal thickness in an adult human eye aged 25 years showing ultrastructural features that differentiate
blood vessels from lymphatics. (A) This image shows layers of blood vessels of various sizes located at different depths within the choroid, as well as
the locations of lymphatic channels of various sizes. Note the capillary-sized lymphatic located just external to the choriocapillaris. The lymphatic
lumen is filled with a flocculent material (most likely proteins); no basal lamina is apparent for the capillary; no Weibel-Palade bodies (WPBs) are
seen in the endothelium; and no fenestrae are apparent. Insets B, C show high-power images of anchoring filaments attached to the vessel, identified
as such, as a lymphatic capillary. (Anchoring filaments were resolvable on originals but have been marked in red to facilitate visualization). In inset
C, the junction between endothelial cells can be observed, and is consistent with a poorly formed junctional structure reported in lymphatic
vessels.32 This vessel also shows flocculent material within the lumen and a fragmented basal lamina, indicative that this is not a blood capillary.
processes, some structures appeared lymphatic-like at a first
glance, they neither converged nor formed larger compounds
in the periphery.’’(p5226) Our netlike structures and formed
lymphatic channels, when observed in nonpathological eyes,
predominated in the choroid in regions adjacent to the ONH,
but like Schroedl et al.8, we could not track continuous
lymphatic channels using immunohistochemical markers.
Further, our findings are consistent with those of Cursiefen
and Schroedl (unpublished observations, 2006), referred to in
Schroedl et al.,8 who reported that it was possible to identify
lymphatic vessel-like structures with a clear vessel lumen in
fetal human eyes. Our previously unpublished observations
(Chan-Ling T, et al. IOVS 2013;54;ARVO E-Abstract D0348) lead
us to suggest that in disease conditions involving inflammation
and cancer, the larger lymphatic collector channels are more
numerous and have a larger lumen diameter. These could be
the wispy, netlike structures we observed in deeper choroid in
development (Figs. 2J–L), that have become engorged and may
respond to inflammatory stimuli as shown in tissue from a
cancer (Figs. 2M–O) and a T1D patient (Figs. 3A–O). However,
this requires significant further study in order to be substantiated.
The recent report by Park et al.,4 in Schlemm’s canal, shows
a novel class of endothelial cell that displays both blood
endothelial cell and LEC phenotypes, supporting our observations in the human choroid. Furthermore, Schlemm’s canal
endothelial cells expressed key lymphatic-signature markers,
such as PROX-1 and VEGFR-3, along with the venous marker
endomucin but, most notably, lacked expression of podoplanin
and LYVE-1. Our observations of lymphatic channels in the
human choroid are consistent with these observations in that
the channels display both lymphatic and blood endothelial cell
phenotype.
Taken together, our observations provide the first evidence
for a system of lymphatic-like structures, as well as an
extensive array of LYVE-1þ/CD39þ/D2-40þ macrophages closely
associated with these lymphatic-like channels, in the developing and adult human choroid. We found that PROX-1þ/
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1324
Evidence of Lymphatics in the Human Choroid
podoplaninþ/D2-40þ LPCs are evident in the developing
choroid from 9 to 20 WG. By montaging a large area of a 19
WG specimen imaged at high magnification, we were able to
discern that the earliest formation of lymphatic structures
begins at the ONH and spreads to the midperipheral choroid,
consistent with observations in the current study (using a
panel of lymphatic antibody markers) that lymphatic formation
has a disc-to-periphery topography of formation.
Furthermore, light microscope and ultrastructural evidence
consistent with lymphatics included the presence of the
hallmark anchoring filaments and lymphatic sacs, luminal
flocculent material, tall LECs, absence of WPBs, patchy or
absent basal lamina, and an absence of luminal RBCs. Formed
endomucinþ lymphatics were characterized by irregularcaliber, very broad lumens ranging from 56 to 80 lm, with
irregular branching patterns. Wide-caliber lymphatic collector
channels with luminal flocculent proteins and lined with a low
density of high LECs were evident in adult choroid. Based on
ultrastructural criteria, we also showed that the initial collector
lymphatics are predominantly located just external to the
choriocapillaris and among the arteries and veins of Sattler’s
layer. The system of blind-ended initial lymphatic segments
seen just external to the fenestrated vessels of the choriocapillaris is ideally placed for recirculating extracellular fluid and
strategically placed for immune surveillance. The choriocapillaris has fenestrated capillaries that allow for high fluid/cellular
transfer within the tissue, and the presence of a lymphatic
system in the vicinity of the choriocapillaris would further
facilitate the recirculation of extracellular fluid and immune
cells into the blood system.
Lymphatics in the choroid occurred at a much lower
frequency compared to blood vessels in young adult eyes (7%
initial lymphatics, 1% precollector, 5% collector channels),
with initial lymphatic segments predominantly located just
external to the choriocapillaris. Having recently completed a
study on the formation of human choroidal blood vessels,28,40
we undertook this study in the context of lymphatic formation
relative to vascular formation. The similarity of some of the
lymphatic structures to those of the blood vessels, combined
with the relative infrequency of lymphatic structures, could
also explain the failure of earlier investigators8,9 to definitively
identify lymphatics in the human choroid. Our finding of a
system of channels with ultrastructural features consistent
with initial lymphatic channels (just external to the choriocapillaris), precollector, and collector lymphatics (in Haller’s
layer) places them in an ideal position to subserve their
functions of fluid homeostasis, macromolecular absorption,
and immune surveillance in the human choroid.
Relationship Between the Current Study and
Recent Recommendations for
Immunohistochemical and Ultrastructural
Detection/Assessment of Ocular Lymphatics
A recently published consensus statement51 provides several
recommendations and criteria for the detection and reporting
of lymphatics in ocular tissue. It is pertinent to discuss the
criteria for assessing the presence of lymphatics in the current
study in the context of these recent recommendations.
We applied the gold standard criteria for ultrastructural
identification of lymphatics utilizing standard TEM with the
following combined features, consistent with a lymphatic
versus a vascular phenotype: anchoring filaments16–18; luminal
flocculent protein but absence of erythrocytes; fragmented
and/or absent basal lamina; absence of intracellular WPBs;
infrequent pericyte ensheathment; and lack of fenestrae.12
Schroedl et al.51 proposed that TEM showing a lack of RBCs or
luminal cell-free homogenous material is insufficient to
discriminate lymphatic vessels from blood vessels, and
suggested the additional application of immune-electron
microscopy to identify lymphatics. However, these authors
do not mention other well-established lymphatic ultrastructural features, in particular anchoring filaments, long recognized
as a hallmark ultrastructural feature of lymphatic vessels.16,17
Further, they conclude that no single immunomarker can
definitively identify a lymphatic; implicit in this is the
requirement for the application of multiple-immunomarker
TEM.
This may be possible for animal studies in which
postmortem delay and fixation can be controlled; however,
the application of this approach for human postmortem
material, including material for diagnostic pathology, is limited.
There may be delays before tissue is fixed, and routine fixation
in neutral buffered formalin can affect the sensitivity and
specificity of certain antibodies.52–54 The importance of
continuing to investigate for lymphatics in human ocular
postmortem tissue requires further consideration and open
debate, including the recognition of all established TEM-based
criteria. Failure to do so may limit ongoing investigations into
the role of lymphatics within human ocular biology, especially
in relation to the role of lymphatics in antigen presentation and
cancer metastasis in the posterior eye.
The second and third recommendations indicate that a
panel of immunomarkers is required to identify lymphatics in
areas of ocular tissue where the presence of lympathics is
currently equivocal; however, there appears to be limited
consensus on exactly what markers are appropriate. Currently,
antibodies to podoplanin (clone D2-40) represent the best
available marker for formalin-fixed material, as this antibody
was produced using a formalin-modified antigen for podoplanin.55 We remain concerned about the use of LYVE-1 as a
principal marker for lymphatic identification, instead of PDPN/
D2-40, as it counters our extensive observations in the human
posterior eye: LYVE-1 has failed to visualize any lymphatic
structures including channels and tear-shaped initial segments
and labels only a subpopulation of macrophages in human
developmental and adult tissue.
Most importantly, there is no argument within the literature
as to the existence of lymphatics in the healthy cornea and
conjunctiva, and more recently the expression of PROX-1 and a
lymphatic-like molecular signature within Schlemm’s canal.4
The emphasis of the consensus recommendations51 reflects
predominantly anterior eye experience and observations,
which may differ from the posterior human eye. Most
importantly, the corneal models often used for studying
lymphatics represent a pathological context since the cornea
is avascular and does not have lymphatics under physiological
conditions. The formation of lymphatics and angiogenesis in
the cornea in these paradigms reflects a response to immune,
toxic, or foreign body challenge. This differs markedly from the
human choroid in the current study and from observed PROX-1
expression in Schlemm’s canal, where the evidence for
lymphatic-like structures reflects physiological conditions.
Mechanism of Lymphangiogenesis in the Human
Choroid
Earlier studies in mice have shown that lymphatic vessel
differentiation and budding are initially under the control of
PROX-1 and Sox-18 genes,32 with subsequent migration,
growth, and survival being controlled predominantly by
VEGF-C. 22,56 We also took advantage of the fact that
endomucin stains only capillaries, venules, and lymphatic
vessels, and is only weakly expressed on arterial endothelial
cells,57 in order to determine whether lymphatic formation can
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1325
Evidence of Lymphatics in the Human Choroid
occur via PROX-1 specification from existing veins or via
coalescence from LPCs in the human choroid. Consistent with
this mechanism of lymphatic formation, vasculogenesis and
angiogenesis appear to precede lymphangiogenesis, although
both showed an ONH-to-periphery topography of formation in
the human choroid. We were unable to confirm the role of
PROX-1; nuclear PROX-1 staining was not seen in the human
choroid with the exception of single-isolated PROX-1þ/
podoplaninþ/D-240þ/VEGFR-3þ/CD34þ presumed LPCs scattered throughout the choroidal stroma. Presumed LPCs within
the stroma of the human choroid early in fetal embryological
development are supportive of the alternative theory of
lymphatic formation, which is more ‘‘vasculogenesis-like,’’
where the LPCs form islands and coalesce into vessels
independent of the blood vasculature, though later forming
venous connections.58,59 This interpretation is supported by
the work of Buttler et al.,58,60 who identified mesenchymal
cells in the developing mammalian embryo that express
lymphatic markers. Using human eyes, we are unable to
provide conclusive evidence of the mechanism by which
lymphatic-like structures form in the human choroid; however,
our findings suggest that both processes may occur.61,62
Species Difference—the Need for Human Studies
While experimental studies in other species can offer valuable
insights as to the molecular determinants of lymphatic
formation, it is not possible to fully elucidate the complexity
of human development or pathology from studying animal
models and tissues alone. Our earlier work clearly shows major
species differences in the mechanisms of retinal vascular
formation in human and other mammalian species.63 Further,
while studies in mice offer opportunities for experimental
manipulation, researchers need to be cognizant of the
increasing understanding of the limitations of animal models
in driving translational outcomes for humans as discussed
previously.64
Functional Implications and Relevance of
Lymphatics to Posterior Eye Disease
The system of blind-ended initial lymphatic-like segments
identified in the current study, just external to the fenestrated
choriocapillaris, is ideally placed to recirculate extracellular
fluids from these vessels and strategically placed for immune
surveillance. The presence of a system of lymphatic-like
channels in the posterior eye provides an anatomical basis
for antigen presentation in the posterior eye, with a possible
(as yet undefined) route to leave the eye and travel to sentinel
lymph nodes.
Some studies have explored the role of the lymphatic
system in the pathogenesis of human diseases involving
inflammation and modulation of inflammatory responses,65,66
and the presence of a choroidal lymphatic system may be
relevant for posterior eye diseases including uveitis, choroidal
neovascularization, diabetic retinopathy, retinal vein occlusion,
and macular edema.67
Lymphatics in the human choroid may also play an
important role in the homeostatic control of eye growth and
in the etiology of refractive error. For example, our findings
may have direct relevance to myopia (short-sightedness)
(reviewed in Ref. 10), where dramatic changes in choroidal
thickness can move the retina forward and back, bringing the
photoreceptors into the plane of focus. Earlier investigators
have speculated that there could be lymphatics in the choroid
that underlie this phenomenon,68–70 as no other structure can
explain dramatic changes in ocular length in such a short
period of time.
Future Directions
The lymphatic system is a network composed of initial
absorptive capillary-sized vessels and larger collecting channels
specialized for lymph transport, which function to return
lymph to the bloodstream. Our observations provide evidence
of a system of lymphatic-like structures within the human
choroid consistent with this function in developing and adult
human eyes. These findings provide the foundation for further
studies exploring the roles of lymphatics in posterior eye
diseases and investigations of novel therapeutic targets such as
known lymphangiogenic factors VEGF-C and VEGF-D. Future
studies are also required to determine the pathways of
lymphatic drainage from the human choroid and how this
relates to the pathogenesis of glaucoma and other human
posterior eye diseases.
Acknowledgments
The authors thank Dietmar Vestweber and Kari Alitalo for the gifts
of endomucin and VEGFR-3 antibodies, respectively; Louise Cole at
the Bosch Institute Advanced Microscopy Facility at the University
of Sydney for support with confocal imaging; Jane Dahlstrom and
Elaine G. Bean for undertaking LYVE-1/CD34 transverse staining;
and Irwin Ting and Katherine Ling for outstanding technical
assistance. The authors also thank Raj Devashayam and Meidong
Zhu, senior scientists at the Lions NSW Eye Bank, for their
assistance in accessing adult human postmortem eyes for this
study.
Supported by grants from the National Health and Medical
Research Council of Australia (Nos. 1005730 and 571100; TC-L),
the Baxter Charitable Foundation, the Alma Hazel Eddy Trust, and
the Rebecca L. Cooper Medical Research Foundation (Sydney,
Australia). SJA and PH are Brian M. Kirby Foundation Gift of Sight
Initiative Scholarship holders (Sydney, Australia); MCM is funded
by the National Foundation for Medical Research and Innovation.
Disclosure: M.E. Koina, None; L. Baxter, None; S.J. Adamson,
None; F. Arfuso, None; P. Hu, None; M.C. Madigan, None; T.
Chan-Ling, None
References
1. Alitalo K, Tammela T, Petrova TV. Lymphangiogenesis in
development and human disease. Nature. 2005;438:946–953.
2. Cursiefen C, Schlotzer-Schrehardt U, Kuchle M, et al.
Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin.
Invest Ophthalmol Vis Sci. 2002;43:2127–2135.
3. Heindl LM, Hofmann-Rummelt C, Adler W, et al. Tumorassociated lymphangiogenesis in the development of conjunctival melanoma. Invest Ophthalmol Vis Sci. 2011;52:7074–
7083.
4. Park DY, Lee J, Park I, et al. Lymphatic regulator PROX1
determines Schlemm’s canal integrity and identity. J Clin
Invest. 2014;124:3960–3974.
5. Yucel YH, Johnston MG, Ly T, et al. Identification of lymphatics
in the ciliary body of the human eye: a novel ‘‘uveolymphatic’’
outflow pathway. Exp Eye Res. 2009;89:810–819.
6. Khan AM, Kagan DB, Gupta N, Navajas EV, Jin YP, Yucel YH.
Ciliary body lymphangiogenesis in uveal melanoma with and
without extraocular extension. Ophthalmology. 2013;120:
306–310.
7. Schlereth SL, Neuser B, Herwig MC, et al. Absence of
lymphatic vessels in the developing human sclera. Exp Eye
Res. 2014;125:203–209.
8. Schroedl F, Brehmer A, Neuhuber WL, Kruse FE, May CA,
Cursiefen C. The normal human choroid is endowed with a
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1326
Evidence of Lymphatics in the Human Choroid
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
significant number of lymphatic vessel endothelial hyaluronate
receptor 1 (LYVE-1)-positive macrophages. Invest Ophthalmol
Vis Sci. 2008;49:5222–5229.
Xu H, Chen M, Reid D, Forrester J. LYVE-1-positive macrophages are present in normal murine eyes. Invest Ophthalmol
Vis Sci. 2007;48:2161–2171.
Nickla DL, Wallman J. The multifunctional choroid. Prog Retin
Eye Res. 2010;29:144–168.
Bill A. Blood circulation and fluid dynamics in the eye. Physiol
Rev. 1975;55:383–417.
Krebs W, Krebs IP. Ultrastructural evidence for lymphatic
capillaries in the primate choroid. Arch Ophthalmol. 1988;
106:1615–1616.
De Stefano ME, Mugnaini E. Fine structure of the choroidal
coat of the avian eye. Lymphatic vessels. Invest Ophthalmol
Vis Sci. 1997;38:1241–1260.
Sugar HS, Riazi A, Schaffner R. The bulbar conjunctival
lymphatics and their clinical significance. Trans Am Acad
Ophthalmol Otolaryngol. 1957;61:212–223.
Weber E, Rossi A, Solito R, Sacchi G, Agliano M, Gerli R. Focal
adhesion molecules expression and fibrillin deposition by
lymphatic and blood vessel endothelial cells in culture.
Microvasc Res. 2002;64:47–55.
Leak LV, Burke JF. Ultrastructural studies on the lymphatic
anchoring filaments. J Cell Biol. 1968;36:129–149.
Leak LV, Burke JF. Fine structure of the lymphatic capillary and
the adjoining connective tissue area. Am J Anat. 1966;118:
785–809.
Collin HB. Ocular lymphatics. Am J Optom Arch Am Acad
Optom. 1966;43:96–106.
Collin HB. The ultrastructure of conjunctival lymphatic
anchoring filaments. Exp Eye Res. 1969;8:102–105.
Skobe M, Detmar M. Structure, function, and molecular
control of the skin lymphatic system. J Investig Dermatol
Symp Proc. 2000;5:14–19.
Sleeman JP, Krishnan J, Kirkin V, Baumann P. Markers for the
lymphatic endothelium: in search of the holy grail? Microsc
Res Tech. 2001;55:61–69.
Karpanen T, Alitalo K. Molecular biology and pathology of
lymphangiogenesis. Annu Rev Pathol. 2008;3:367–397.
Alitalo K. The lymphatic vasculature in disease. Nat Med.
2011;17:1371–1380.
Hughes S, Yang H, Chan-Ling T. Vascularization of the human
fetal retina: roles of vasculogenesis and angiogenesis. Invest
Ophthalmol Vis Sci. 2000;41:1217–1228.
Chan-Ling T, McLeod DS, Hughes S, et al. Astrocyte-endothelial
cell relationships during human retinal vascular development.
Invest Ophthalmol Vis Sci. 2004;45:2020–2032.
Potter EL, Craig JM. Rate of Antenatal Growth. 3rd ed.
Chicago: Year Book Medical Publishers; 1975:19.
Chan-Ling T. Glial, vascular, and neuronal cytogenesis in
whole-mounted cat retina. Microsc Res Tech. 1997;36:1–16.
Chan-Ling T, Dahlstrom JE, Koina ME, et al. Evidence of
hematopoietic differentiation, vasculogenesis and angiogenesis in the formation of human choroidal blood vessels. Exp Eye
Res. 2011;92:361–376.
Breiteneder-Geleff S, Soleiman A, Kowalski H, et al. Angiosarcomas express mixed endothelial phenotypes of blood and
lymphatic capillaries: podoplanin as a specific marker for
lymphatic endothelium. Am J Pathol. 1999;154:385–394.
Britto AV, Schenka AA, Moraes-Schenka NG, et al. Immunostaining with D2-40 improves evaluation of lymphovascular
invasion, but may not predict sentinel lymph node status in
early breast cancer. BMC Cancer. 2009;9:109.
31. Banerji S, Ni J, Wang SX, et al. LYVE-1, a new homologue of the
CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol. 1999;144:789–801.
32. Wigle JT, Harvey N, Detmar M, et al. An essential role for Prox1
in the induction of the lymphatic endothelial cell phenotype.
EMBO J. 2002;21:1505–1513.
33. Karkkainen MJ, Haiko P, Sainio K, et al. Vascular endothelial
growth factor C is required for sprouting of the first lymphatic
vessels from embryonic veins. Nat Immunol. 2004;5:74–80.
34. Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the
fms-like tyrosine kinase 4 gene becomes restricted to
lymphatic endothelium during development. Proc Natl Acad
Sci U S A. 1995;92:3566–3570.
35. Ordonez NG, Brooks T, Thompson S, Batsakis JG. Use of Ulex
europaeus agglutinin-I in the identification of lymphatic and
blood-vessel invasion in previously stained microscopic slides.
Am J Surg Pathol. 1987;11:543–550.
36. Samulowitz U, Kuhn A, Brachtendorf G, et al. Human
endomucin: distribution pattern, expression on high endothelial venules, and decoration with the MECA-79 epitope. Am
J Pathol. 2002;160:1669–1681.
37. Albelda SM, Muller WA, Buck CA, Newman PJ. Molecular and
cellular properties of PECAM-1 (endoCAM/CD31): a novel
vascular cell-cell adhesion molecule. J Cell Biol. 1991;114:
1059–1068.
38. Lymboussaki A, Achen MG, Stacker SA, Alitalo K. Growth
factors regulating lymphatic vessels. Curr Top Microbiol
Immunol. 2000;251:75–82.
39. Partanen TA, Alitalo K, Miettinen M. Lack of lymphatic
vascular specificity of vascular endothelial growth factor
receptor 3 in 185 vascular tumors. Cancer. 1999;86:2406–
2412.
40. Chan-Ling T, Koina ME, McColm JR, et al. Role of CD44þ stem
cells in mural cell formation in the human choroid: evidence
of vascular instability due to limited pericyte ensheathment.
Invest Ophthalmol Vis Sci. 2011;52:399–410.
41. Collin HB. The fine structure of growing corneal lymphatic
vessels. J Pathol. 1971;104:99–113.
42. Scavelli C, Weber E, Agliano M, et al. Lymphatics at the
crossroads of angiogenesis and lymphangiogenesis. J Anat.
2004;204:433–449.
43. Sacchi G, Weber E, Agliano M, Raffaelli N, Comparini L. The
structure of superficial lymphatics in the human thigh:
precollectors. Anat Rec. 1997;247:53–62.
44. Truman LA, Bentley KL, Smith EC, et al. ProxTom lymphatic
vessel reporter mice reveal Prox1 expression in the adrenal
medulla, megakaryocytes, and platelets. Am J Pathol. 2012;
180:1715–1725.
45. Uhrin P, Zaujec J, Breuss JM, et al. Novel function for blood
platelets and podoplanin in developmental separation of blood
and lymphatic circulation. Blood. 2010;115:3997–4005.
46. van der Putte SC. The early development of the lymphatic
system in mouse embryos. Acta Morphol Neerl Scand. 1975;
13:245–286.
47. Francois M, Short K, Secker GA, et al. Segmental territories
along the cardinal veins generate lymph sacs via a ballooning
mechanism during embryonic lymphangiogenesis in mice.
Dev Biol. 2012;364:89–98.
48. Baluk P, Fuxe J, Hashizume H, et al. Functionally specialized
junctions between endothelial cells of lymphatic vessels. J Exp
Med. 2007;204:2349–2362.
49. Vittet D. Lymphatic collecting vessel maturation and valve
morphogenesis. Microvasc Res. 2014;96:31–37.
50. Yang Y, Oliver G. Development of the mammalian lymphatic
vasculature. J Clin Invest. 2014;124:888–897.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017
IOVS j February 2015 j Vol. 56 j No. 2 j 1327
Evidence of Lymphatics in the Human Choroid
51. Schroedl F, Kaser-Eichberger A, Schlereth SL, et al. Consensus
statement on the immunohistochemical detection of ocular
lymphatic vessels. Invest Ophthalmol Vis Sci. 2014;55:6440–
6442.
52. Penfold PL, Provis JM, Liew SC. Human retinal microglia
express phenotypic characteristics in common with dendritic
antigen-presenting cells. J Neuroimmunol. 1993;45:183–191.
53. Wu KH, Penfold PL, Billson FA. Effects of post-mortem delay
and storage duration on the expression of GFAP in normal
human adult retinae. Clin Experiment Ophthalmol. 2002;30:
200–207.
54. Hilbig H, Bidmon HJ, Oppermann OT, Remmerbach T.
Influence of post-mortem delay and storage temperature on
the immunohistochemical detection of antigens in the CNS of
mice. Exp Toxicol Pathol. 2004;56:159–171.
55. Kahn HJ, Marks A. A new monoclonal antibody, D2-40, for
detection of lymphatic invasion in primary tumors. Lab
Invest. 2002;82:1255–1257.
56. Maby-El Hajjami H, Petrova TV. Developmental and pathological lymphangiogenesis: from models to human disease.
Histochem Cell Biol. 2008;130:1063–1078.
57. Kuhn A, Brachtendorf G, Kurth F, et al. Expression of
endomucin, a novel endothelial sialomucin, in normal and
diseased human skin. J Invest Dermatol. 2002;119:1388–
1393.
58. Buttler K, Kreysing A, von Kaisenberg CS, et al. Mesenchymal
cells with leukocyte and lymphendothelial characteristics in
murine embryos. Dev Dyn. 2006;235:1554–1562.
59. Huntington GS, McClure CFW. The anatomy and development
of the jugular lymph sacs in the domestic cat (Felis domestica).
Am J Anat. 1910;10:177–312.
60. Buttler K, Ezaki T, Wilting J. Proliferating mesodermal cells in
murine embryos exhibiting macrophage and lymphendothelial
characteristics. BMC Dev Biol. 2008;8:43.
61. Jeltsch M, Tammela T, Alitalo K, Wilting J. Genesis and
pathogenesis of lymphatic vessels. Cell Tissue Res. 2003;314:
69–84.
62. van der Jagt ER. Memoirs: the origin and development of the
anterior lymph-sacs in the sea-turtle (Thalassochelys caretta).
Q J Microsc Sci. 1932;s2-s75:151–163.
63. Chan-Ling T, McLeod D, Hughes S, et al. Astrocyte-endothelial
cell relationships during human retinal vascular development.
Invest Ophthalmol Vis Sci. 2004;45:2020–2032.
64. Of men, not mice. Nat Med. 2013;19:379.
65. Jurisic G, Detmar M. Lymphatic endothelium in health and
disease. Cell Tissue Res. 2009;335:97–108.
66. Kerjaschki D. Lymphatic neoangiogenesis in renal transplants:
a driving force of chronic rejection? J Nephrol. 2006;19:403–
406.
67. Nakao S, Hafezi-Moghadam A, Ishibashi T. Lymphatics and
lymphangiogenesis in the eye. J Ophthalmol. 2012;2012:
783163.
68. Junghans BM, Crewther SG, Crewther DP, Pirie B. Lymphatic
sinusoids exist in chick but not in rabbit choroid. Aust N Z J
Ophthalmol. 1997;25(suppl 1):S103–S105.
69. Junghans BM, Crewther SG, Liang H, Crewther DP. A role for
choroidal lymphatics during recovery from form deprivation
myopia? Optom Vis Sci. 1999;76:796–803.
70. Westbrook AM, Crewther SG, Liang H, et al. Formoguanamineinduced inhibition of deprivation myopia in chick is accompanied by choroidal thinning while retinal function is retained.
Vision Res. 1995;35:2075–2088.
Downloaded From: http://arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933680/ on 06/18/2017