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Retinal Cell Biology  |   February 2015
Evidence for Lymphatics in the Developing and Adult Human Choroid
Author Affiliations & Notes
  • Mark E. Koina
    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
  • Louise Baxter
    Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia
  • Samuel J. Adamson
    Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia
  • Frank Arfuso
    Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia
    School of Anatomy, Physiology and Human Biology, Faculty of Science, The University of Western Australia, Crawley, Western Australia, Australia
  • Ping Hu
    Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia
  • Michele C. Madigan
    School of Optometry, University of New South Wales, New South Wales, Australia
    Save Sight Institute, The University of Sydney, New South Wales, Australia
  • Tailoi Chan-Ling
    Discipline of Anatomy, School of Medical Sciences, Bosch Institute, The University of Sydney, Sydney, New South Wales, Australia
  • 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
Investigative Ophthalmology & Visual Science February 2015, Vol.56, 1310-1327. doi:10.1167/iovs.14-15705
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      Mark E. Koina, Louise Baxter, Samuel J. Adamson, Frank Arfuso, Ping Hu, Michele C. Madigan, Tailoi Chan-Ling; Evidence for Lymphatics in the Developing and Adult Human Choroid. Invest. Ophthalmol. Vis. Sci. 2015;56(2):1310-1327. doi: 10.1167/iovs.14-15705.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

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.

Introduction
The 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 antigen-presenting 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 lymphatic capillaries was found in the choroid of nonhuman primate eyes (n = 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-1+ 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-1+ 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-1+ 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.1518 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 +2 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 thin-walled 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.1618,21 
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-1+ 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 4°C. 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 performed on a Ventana Benchmark NexES machine (Ventana Medical Systems, Inc., Tucson, AZ, USA) using the streptavidin–biotin method on 4-μm 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. 
Table 1
 
Antibodies and Markers Used in the Current Study
Table 1
 
Antibodies and Markers Used in the Current Study
Antibody Description Manufacturer Species Subclass Reference
Podoplanin 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. R&D Systems (Minneapolis, MN, USA) Sheep IgG 22
D2-40 Clone D2-40 identifies the 38-kDa integral membrane glycoprotein podoplanin (description above). Dako (Australia Pty Ltd., North Sydney, New South Wales, Australia) Mouse IgG1 23
LYVE-1 Lymphatic-specific receptor for endothelial hyaluronan; expressed on lymphatic but not blood vascular endothelium. Abcam (Cambridge, MA, USA) Rabbit IgG 24
PROX-1 Prospero-homeobox 1 transcription factor, a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium, and lymphangioblasts. Reliatech (Wolfenbüttel, Germany) Rabbit IgG 25
VEGFR-3 Endothelium-specific receptor tyrosine kinase, expressed by immature blood vessels and not mature ones, constitutively expressed by the lymphatic endothelium. Reliatech Mouse IgG1 27
VEGF-C Participates in development of lymphatic vasculature by activation of VEGFR-3. R&D Systems Rabbit IgG 26
Endomucin Endothelial sialomucin expressed on lymphatic endothelium, veins, and venules, but not arteries. Gift from D. Vestweber Rat IgG2a 28
PECAM-1 (CD31) Platelet endothelial cell adhesion molecule-1. Integral membrane glycoprotein expressed on endothelial intercellular junctions. Santa-Cruz (Dallas, TX, USA) Rabbit IgG 37
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. Serotec (Raleigh, SC, USA) Mouse IgG2a 21
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. Novocastra (Newcastle-Upon-Tyne, UK) Mouse IgG2a 21
CD44 CD44 is a cell adhesion receptor widely expressed on hematopoietic and nonhematopoietic cells. Immunotech (Brea, CA, USA) Mouse IgG1 21
Collagen IV Basement membrane protein found on vessel walls. Abcam Rabbit IgG 4
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. Sigma-Aldrich Corp. (St. Louis, MO, USA) - - 35
Choroids were incubated overnight at 4°C 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 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 4°C 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 μm 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 = 15) were immersion fixed in 4% PFA for 1 hour at 4°C, 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 = 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 70°C. 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 μm from the ONH. Lymphatic vessels with a range of lumen diameters were typically characterized as initial (10–60 μm),42 precollector (35–150 μm),43 and collector lymphatics (>200 μm).42 The frequency was then expressed as a percentage of total vessel profiles counted per area of choroid at each level (Table 2). 
Table 2
 
Relative Frequency of Lymphatic-Like Vessels Versus Blood Vessels in Adult Human Choroid, n = 6
Table 2
 
Relative Frequency of Lymphatic-Like Vessels Versus Blood Vessels in Adult Human Choroid, n = 6
Lymphatics N Blood Vessels N Total Vessels Counted % Lymphatics
Initial, 10–60 μm42 40 Capillaries 543 583 6.9, 40/583
Precollector, 35–150 μm43 1 Medium 88 89 1.1, 1/89
Collector, >200 μm42 1 Large 21 22 4.5, 1/22
Results
PROX-1+/Podoplanin+/D2-40+/VEGF-R3+/CD34+ Presumed Lymphatic Precursor Cells (LPCs) in Human Choroidal Stroma During Early Development
We identified single, isolated PROX-1+/podoplanin+/D2-40+/VEGF-R3+/CD34+ 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 CD34+ cells (presumed hematopoietic stem cells [HSCs; according to a previous study28]) (Figs. 1E–H). 
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-1+/podoplanin+/D-240+/VEGFR-3+ and constituted a subset of CD34+ HSC. (AD) Whole mounts of human fetal choroid (14 WG) immunolabeled for podoplanin, PROX-1, D-240. (EH) 20 WG choroidal whole mounts immunostained for CD34, PROX-1, and VEGFR-3 (E). A subset of CD34+ hematopoietic stem cells were also PROX-1+/VEGFR-3+ (F, H, respectively). The pink arrowed cells in (H) show CD34+ HSC, whereas the yellow arrows point to PROX-1+/VEGFR-3+/CD34+ LPCs. (IK) PROX-1 staining is nuclear in the retina, staining the somas within the germinal layer of the embryonic human retina at 20 WG. However, the choroid is devoid of PROX-1 nuclear staining, instead showing weak channel-like structures. LYVE-1+ macrophages were closely associated with forming lymphatics. The inner limiting membrane (ILM) of the retina is indicated in (K). (LN) 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-1+ cells were found in the stroma at 9, 16, and 19 WG (purple arrows). (L) The CD34+ 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 CD34+ capillaries were patent; however, the bulk of the LYVE-1+ 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 CD34+ choriocapillaris increased, and deeper, larger blood vessels were observed. The majority of LYVE-1+ macrophages remained either as single isolated cells (purple arrows) or aligned with CD34-negative lumens, presumably lymphatics. (OQ) LYVE-1+ macrophages closely associated with VEGFR-3+ vessels throughout the choroid at 15 WG, similar to observations in 16 WG choroidal sections (LN).
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-1+/podoplanin+/D-240+/VEGFR-3+ and constituted a subset of CD34+ HSC. (AD) Whole mounts of human fetal choroid (14 WG) immunolabeled for podoplanin, PROX-1, D-240. (EH) 20 WG choroidal whole mounts immunostained for CD34, PROX-1, and VEGFR-3 (E). A subset of CD34+ hematopoietic stem cells were also PROX-1+/VEGFR-3+ (F, H, respectively). The pink arrowed cells in (H) show CD34+ HSC, whereas the yellow arrows point to PROX-1+/VEGFR-3+/CD34+ LPCs. (IK) PROX-1 staining is nuclear in the retina, staining the somas within the germinal layer of the embryonic human retina at 20 WG. However, the choroid is devoid of PROX-1 nuclear staining, instead showing weak channel-like structures. LYVE-1+ macrophages were closely associated with forming lymphatics. The inner limiting membrane (ILM) of the retina is indicated in (K). (LN) 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-1+ cells were found in the stroma at 9, 16, and 19 WG (purple arrows). (L) The CD34+ 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 CD34+ capillaries were patent; however, the bulk of the LYVE-1+ 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 CD34+ choriocapillaris increased, and deeper, larger blood vessels were observed. The majority of LYVE-1+ macrophages remained either as single isolated cells (purple arrows) or aligned with CD34-negative lumens, presumably lymphatics. (OQ) LYVE-1+ macrophages closely associated with VEGFR-3+ vessels throughout the choroid at 15 WG, similar to observations in 16 WG choroidal sections (LN).
Large numbers of CD34+ HSCs were evident (identified with pink arrows in Fig. 1H), whereas the yellow arrows show a subset of CD34+ cells that represent PROX-1+/VEGFR-3+/CD34+ 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 CD34+ Blood Vessels Become PROX-1+/VEGFR-3+ Lymphatics
Despite reports stating that PROX-1 antibody should localize to the cell nucleus, we did not find any PROX-1+ nuclei in the human choroid even though PROX-1+ 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-1+/VEGFR-3+ 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-1+ macrophages were evident within the choroidal stroma. By 16 WG, LYVE-1+ macrophages were not associated with forming CD34+ blood vessels (purple arrows in Figs. 1L–N). Rather, the LYVE-1+ macrophages appeared to line the CD34 lumina, suggesting the involvement of LYVE-1+ macrophages in the formation of the earliest lymphatic channels (green arrows in Figs. 1L–N). This observation persisted at 19 WG. LYVE-1+ 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-1+ macrophages were associated with VEGFR3+ 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 D2-40+ macrophages were closely aligned along a UEA lectin+ 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-3+/CD34 initial lymphatic segments were evident dispersed among the forming CD34+ 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 VEGFR-3+/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 CD34+ HSCs/RBCs within the tear-shaped lymphatic sacs. Small numbers of CD34+ HSCs/RBCs were sometimes observed within the initial segments/lymphatic sacs of the forming lymphatics (Figs. 2D–I). Consistent with our observations of CD34+ 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-3+/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. 
Figure 2
 
Precollector lymphatic-like channels connect to dead-ended round or tear-shaped lymphatic sacs in whole mounts of the developing human choroid. (AC) VEGFR-3+/CD34 buds were seen interspersed among the CD34+ choroidal vasculature. Note the frequent association of the VEGFR-3+ lymphatic initial collector channels with intraluminal CD34+ HSCs. (DF) Two representative round lymphatic sacs just external to the choriocapillaris. (GI) A representative tear-shaped VEGFR-3+ lymphatic sac located just external to the choriocapillaris in a 19 WG human fetal choroid, adjacent to the ONH. Note the numerous CD34+ HSCs within the lymphatic sacs. (JL) Wispy CD34+/VEGFR-3+ structures in the outer choroidal stroma. (MO) Choroid from a 50-year-old with history of glioblastoma.
Figure 2
 
Precollector lymphatic-like channels connect to dead-ended round or tear-shaped lymphatic sacs in whole mounts of the developing human choroid. (AC) VEGFR-3+/CD34 buds were seen interspersed among the CD34+ choroidal vasculature. Note the frequent association of the VEGFR-3+ lymphatic initial collector channels with intraluminal CD34+ HSCs. (DF) Two representative round lymphatic sacs just external to the choriocapillaris. (GI) A representative tear-shaped VEGFR-3+ lymphatic sac located just external to the choriocapillaris in a 19 WG human fetal choroid, adjacent to the ONH. Note the numerous CD34+ HSCs within the lymphatic sacs. (JL) Wispy CD34+/VEGFR-3+ structures in the outer choroidal stroma. (MO) Choroid from a 50-year-old with history of glioblastoma.
VEGFR-3+ Lymphatics Constitute a Network Distinct From the CD34+ Vasculature
We saw VEGFR-3+ and CD34+ 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-3+ lymphatics appeared in a deeper plane of focus to the CD34+ choriocapillaris, and the caliber of the lymphatic capillaries was broader than that of vessels in the choriocapillaris (not shown), while the lymphatic sacs are VEGFR3+/CD34weak (Figs. 2D–F). Figures 2J through 2L show wispy CD34+/VEGFR-3+ 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 lectin+/D2-40 blood vessels are evident just adjacent to the RPE (RPE discernible via autofluorescence in both green and red channels). D2-40+/UEA lectin lymphatic structures are visible just below the UEA lectin+ choriocapillaris in Figure 3C. Utilizing triple-marker whole-mount IHC (UEA lectin, D2-40, and collagen IV), we were able to discern a D2-40+/UEA lectin/collagen IV structure with a netlike morphology that outlined a tube-like structure just external to a wide, UEA lectin+/collagen IV+ blood vessel (see Figs. 3D–K). Further, utilizing UEA lectin+/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, D2-40+/LYVE-1+ 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. 
Figure 3
 
D2-40+/UEA lectin/collagen IV lymphatic-like channels external to the choriocapillaris in 74-year-old human adult choroid. (AC) A 12-μm-thick cryosection taken at ×40 magnification from a 74-year-old adult human choroid double stained for UEA lectin and D2-40. (A) UEA lectin+/D2-40 blood vessels are shown just adjacent to the RPE (RPE discernible via autofluorescence in both green and red channels). D2-40+/UEA lectin structures are visible just below the UEA lectin+ blood vessels in (B). (DG) Orthographic projections; (HK) 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 maximum-intensity projections displayed in (HK) were created. (E) The level at which a tube-like D2-40+/UEA lectin/collagen IV structure is present just adjacent to a wide, UEA lectin+/collagen IV+ blood vessel. (LO) UEA lectin, D2-40, and LYVE-1 staining on a 12-μm-thick cryosection in the same 74-year-old human specimen at ×40 magnification. UEA lectin+/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, D2-40+/LYVE-1+ 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.
Figure 3
 
D2-40+/UEA lectin/collagen IV lymphatic-like channels external to the choriocapillaris in 74-year-old human adult choroid. (AC) A 12-μm-thick cryosection taken at ×40 magnification from a 74-year-old adult human choroid double stained for UEA lectin and D2-40. (A) UEA lectin+/D2-40 blood vessels are shown just adjacent to the RPE (RPE discernible via autofluorescence in both green and red channels). D2-40+/UEA lectin structures are visible just below the UEA lectin+ blood vessels in (B). (DG) Orthographic projections; (HK) 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 maximum-intensity projections displayed in (HK) were created. (E) The level at which a tube-like D2-40+/UEA lectin/collagen IV structure is present just adjacent to a wide, UEA lectin+/collagen IV+ blood vessel. (LO) UEA lectin, D2-40, and LYVE-1 staining on a 12-μm-thick cryosection in the same 74-year-old human specimen at ×40 magnification. UEA lectin+/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, D2-40+/LYVE-1+ 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.
Choroidal Blood Vessel Development Precedes Lymphatic Development, Both Systems Displaying an Optic Nerve Head-to-Periphery Topography of Formation
At 19 WG, VEGFR-3+ lymphatic sacs and vessels were seen at the ONH, spreading centrifugally toward the periphery in the human choroid (Fig. 4A). The extent of VEGFR-3+ 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, 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 VEGFR3+ 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-3+ 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 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. 
Figure 4
 
Whole mount showing the optic nerve head (ONH)-to-periphery topography of formation of VEGFR-3+ and endomucin+ 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. (BD) 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. (EH) Thin-walled endomucin+ 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) Endomucin+ 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 endomucin+ 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.
Figure 4
 
Whole mount showing the optic nerve head (ONH)-to-periphery topography of formation of VEGFR-3+ and endomucin+ 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. (BD) 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. (EH) Thin-walled endomucin+ 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) Endomucin+ 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 endomucin+ 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.
Endomucin+ Lymphatic-Like Channels With Unique Branching Patterns and Irregular Caliber in Adult Choroid and Lymphatic Valves
The adult endomucin+ lymphatic channels (Figs. 4E–H) were thin-walled structures with a branching morphology atypical of choroidal veins, with an extremely wide, irregular caliber (128 μm at its widest point, Fig. 4G). These smaller initial lymphatic-like 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). Endomucin+ presumptive lymphatic channels showed additional “flaps” not seen in blood vessels. With high magnification, endomucin+-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 (CD34+, VEGFR-3+, VEGF-C+) 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 PROX-1+++, Foxc2+++, VEGFR3++, podoplanin+, GATA2+, integrin-α2+, laminin-α5+, integrin-α9+, Cx37+, Cx43+, Cx47+, LYVE-1.49,50 
Figure 5
 
(AD) Confocal images of vessels in midperipheral choroid (40 years) showing a presumed lymphatic valve (CD34+, VEGFR-3+, VEGF-C+) in a lymphatic precollector channel (arrow in [D] points to the precollector channel). (EH) Confocal images of vessels near the optic nerve head (40 years) showing lymphatic endothelial cells (LECs) (CD34+, D2-40+, LYVE-1+, arrows in [H]) with lower density and much wider gaps along the vessel lumen than typically seen on blood vessels, (IL) 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.
Figure 5
 
(AD) Confocal images of vessels in midperipheral choroid (40 years) showing a presumed lymphatic valve (CD34+, VEGFR-3+, VEGF-C+) in a lymphatic precollector channel (arrow in [D] points to the precollector channel). (EH) Confocal images of vessels near the optic nerve head (40 years) showing lymphatic endothelial cells (LECs) (CD34+, D2-40+, LYVE-1+, arrows in [H]) with lower density and much wider gaps along the vessel lumen than typically seen on blood vessels, (IL) 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.
High Lymphatic Endothelial Cells
In adult human choroid (40 years), high LECs (CD34+, D2-40+, LYVE-1+) 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-1+ macrophages (labeled M in Fig. 5L) suggest a possible site of 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 μm),42 precollector (35–150 μm),43 and collector lymphatics (>200 μm),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). 
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. 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.
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. 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.
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.
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.
A representative lymphatic initial segment (lymphatic capillary) external to the choriocapillaris, with no fenestrae 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 
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.
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.
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.
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.
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 z-stacks (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 = 6 eyes, n = 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-1+ “netlike structures with a pseudovessel-like appearance,”(p5226) we also recognized very fine, netlike CD34+/VEGFR-3+ structures in developing choroid, along with tube-like D2-40+/collagen IV/UEA lectin structures in aged choroid. Further, Schroedl et al.8 noted that “by virtue of their tubular 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-1+/CD39+/D2-40+ macrophages closely associated with these lymphatic-like channels, in the developing and adult human choroid. We found that PROX-1+/podoplanin+/D2-40+ 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 endomucin+ lymphatics were characterized by irregular-caliber, very broad lumens ranging from 56 to 80 μm, 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 filaments1618; 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.5254 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 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-1+/podoplanin+/D-240+/VEGFR-3+/CD34+ 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,6870 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 
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Footnotes
 MEK, LB, SJA and FA contributed equally to the work presented here and should therefore be regarded as equivalent authors.
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-1+/podoplanin+/D-240+/VEGFR-3+ and constituted a subset of CD34+ HSC. (AD) Whole mounts of human fetal choroid (14 WG) immunolabeled for podoplanin, PROX-1, D-240. (EH) 20 WG choroidal whole mounts immunostained for CD34, PROX-1, and VEGFR-3 (E). A subset of CD34+ hematopoietic stem cells were also PROX-1+/VEGFR-3+ (F, H, respectively). The pink arrowed cells in (H) show CD34+ HSC, whereas the yellow arrows point to PROX-1+/VEGFR-3+/CD34+ LPCs. (IK) PROX-1 staining is nuclear in the retina, staining the somas within the germinal layer of the embryonic human retina at 20 WG. However, the choroid is devoid of PROX-1 nuclear staining, instead showing weak channel-like structures. LYVE-1+ macrophages were closely associated with forming lymphatics. The inner limiting membrane (ILM) of the retina is indicated in (K). (LN) 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-1+ cells were found in the stroma at 9, 16, and 19 WG (purple arrows). (L) The CD34+ 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 CD34+ capillaries were patent; however, the bulk of the LYVE-1+ 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 CD34+ choriocapillaris increased, and deeper, larger blood vessels were observed. The majority of LYVE-1+ macrophages remained either as single isolated cells (purple arrows) or aligned with CD34-negative lumens, presumably lymphatics. (OQ) LYVE-1+ macrophages closely associated with VEGFR-3+ vessels throughout the choroid at 15 WG, similar to observations in 16 WG choroidal sections (LN).
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-1+/podoplanin+/D-240+/VEGFR-3+ and constituted a subset of CD34+ HSC. (AD) Whole mounts of human fetal choroid (14 WG) immunolabeled for podoplanin, PROX-1, D-240. (EH) 20 WG choroidal whole mounts immunostained for CD34, PROX-1, and VEGFR-3 (E). A subset of CD34+ hematopoietic stem cells were also PROX-1+/VEGFR-3+ (F, H, respectively). The pink arrowed cells in (H) show CD34+ HSC, whereas the yellow arrows point to PROX-1+/VEGFR-3+/CD34+ LPCs. (IK) PROX-1 staining is nuclear in the retina, staining the somas within the germinal layer of the embryonic human retina at 20 WG. However, the choroid is devoid of PROX-1 nuclear staining, instead showing weak channel-like structures. LYVE-1+ macrophages were closely associated with forming lymphatics. The inner limiting membrane (ILM) of the retina is indicated in (K). (LN) 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-1+ cells were found in the stroma at 9, 16, and 19 WG (purple arrows). (L) The CD34+ 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 CD34+ capillaries were patent; however, the bulk of the LYVE-1+ 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 CD34+ choriocapillaris increased, and deeper, larger blood vessels were observed. The majority of LYVE-1+ macrophages remained either as single isolated cells (purple arrows) or aligned with CD34-negative lumens, presumably lymphatics. (OQ) LYVE-1+ macrophages closely associated with VEGFR-3+ vessels throughout the choroid at 15 WG, similar to observations in 16 WG choroidal sections (LN).
Figure 2
 
Precollector lymphatic-like channels connect to dead-ended round or tear-shaped lymphatic sacs in whole mounts of the developing human choroid. (AC) VEGFR-3+/CD34 buds were seen interspersed among the CD34+ choroidal vasculature. Note the frequent association of the VEGFR-3+ lymphatic initial collector channels with intraluminal CD34+ HSCs. (DF) Two representative round lymphatic sacs just external to the choriocapillaris. (GI) A representative tear-shaped VEGFR-3+ lymphatic sac located just external to the choriocapillaris in a 19 WG human fetal choroid, adjacent to the ONH. Note the numerous CD34+ HSCs within the lymphatic sacs. (JL) Wispy CD34+/VEGFR-3+ structures in the outer choroidal stroma. (MO) Choroid from a 50-year-old with history of glioblastoma.
Figure 2
 
Precollector lymphatic-like channels connect to dead-ended round or tear-shaped lymphatic sacs in whole mounts of the developing human choroid. (AC) VEGFR-3+/CD34 buds were seen interspersed among the CD34+ choroidal vasculature. Note the frequent association of the VEGFR-3+ lymphatic initial collector channels with intraluminal CD34+ HSCs. (DF) Two representative round lymphatic sacs just external to the choriocapillaris. (GI) A representative tear-shaped VEGFR-3+ lymphatic sac located just external to the choriocapillaris in a 19 WG human fetal choroid, adjacent to the ONH. Note the numerous CD34+ HSCs within the lymphatic sacs. (JL) Wispy CD34+/VEGFR-3+ structures in the outer choroidal stroma. (MO) Choroid from a 50-year-old with history of glioblastoma.
Figure 3
 
D2-40+/UEA lectin/collagen IV lymphatic-like channels external to the choriocapillaris in 74-year-old human adult choroid. (AC) A 12-μm-thick cryosection taken at ×40 magnification from a 74-year-old adult human choroid double stained for UEA lectin and D2-40. (A) UEA lectin+/D2-40 blood vessels are shown just adjacent to the RPE (RPE discernible via autofluorescence in both green and red channels). D2-40+/UEA lectin structures are visible just below the UEA lectin+ blood vessels in (B). (DG) Orthographic projections; (HK) 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 maximum-intensity projections displayed in (HK) were created. (E) The level at which a tube-like D2-40+/UEA lectin/collagen IV structure is present just adjacent to a wide, UEA lectin+/collagen IV+ blood vessel. (LO) UEA lectin, D2-40, and LYVE-1 staining on a 12-μm-thick cryosection in the same 74-year-old human specimen at ×40 magnification. UEA lectin+/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, D2-40+/LYVE-1+ 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.
Figure 3
 
D2-40+/UEA lectin/collagen IV lymphatic-like channels external to the choriocapillaris in 74-year-old human adult choroid. (AC) A 12-μm-thick cryosection taken at ×40 magnification from a 74-year-old adult human choroid double stained for UEA lectin and D2-40. (A) UEA lectin+/D2-40 blood vessels are shown just adjacent to the RPE (RPE discernible via autofluorescence in both green and red channels). D2-40+/UEA lectin structures are visible just below the UEA lectin+ blood vessels in (B). (DG) Orthographic projections; (HK) 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 maximum-intensity projections displayed in (HK) were created. (E) The level at which a tube-like D2-40+/UEA lectin/collagen IV structure is present just adjacent to a wide, UEA lectin+/collagen IV+ blood vessel. (LO) UEA lectin, D2-40, and LYVE-1 staining on a 12-μm-thick cryosection in the same 74-year-old human specimen at ×40 magnification. UEA lectin+/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, D2-40+/LYVE-1+ 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.
Figure 4
 
Whole mount showing the optic nerve head (ONH)-to-periphery topography of formation of VEGFR-3+ and endomucin+ 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. (BD) 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. (EH) Thin-walled endomucin+ 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) Endomucin+ 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 endomucin+ 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.
Figure 4
 
Whole mount showing the optic nerve head (ONH)-to-periphery topography of formation of VEGFR-3+ and endomucin+ 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. (BD) 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. (EH) Thin-walled endomucin+ 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) Endomucin+ 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 endomucin+ 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.
Figure 5
 
(AD) Confocal images of vessels in midperipheral choroid (40 years) showing a presumed lymphatic valve (CD34+, VEGFR-3+, VEGF-C+) in a lymphatic precollector channel (arrow in [D] points to the precollector channel). (EH) Confocal images of vessels near the optic nerve head (40 years) showing lymphatic endothelial cells (LECs) (CD34+, D2-40+, LYVE-1+, arrows in [H]) with lower density and much wider gaps along the vessel lumen than typically seen on blood vessels, (IL) 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.
Figure 5
 
(AD) Confocal images of vessels in midperipheral choroid (40 years) showing a presumed lymphatic valve (CD34+, VEGFR-3+, VEGF-C+) in a lymphatic precollector channel (arrow in [D] points to the precollector channel). (EH) Confocal images of vessels near the optic nerve head (40 years) showing lymphatic endothelial cells (LECs) (CD34+, D2-40+, LYVE-1+, arrows in [H]) with lower density and much wider gaps along the vessel lumen than typically seen on blood vessels, (IL) 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.
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. 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.
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. 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.
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.
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.
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.
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.
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.
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.
Table 1
 
Antibodies and Markers Used in the Current Study
Table 1
 
Antibodies and Markers Used in the Current Study
Antibody Description Manufacturer Species Subclass Reference
Podoplanin 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. R&D Systems (Minneapolis, MN, USA) Sheep IgG 22
D2-40 Clone D2-40 identifies the 38-kDa integral membrane glycoprotein podoplanin (description above). Dako (Australia Pty Ltd., North Sydney, New South Wales, Australia) Mouse IgG1 23
LYVE-1 Lymphatic-specific receptor for endothelial hyaluronan; expressed on lymphatic but not blood vascular endothelium. Abcam (Cambridge, MA, USA) Rabbit IgG 24
PROX-1 Prospero-homeobox 1 transcription factor, a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium, and lymphangioblasts. Reliatech (Wolfenbüttel, Germany) Rabbit IgG 25
VEGFR-3 Endothelium-specific receptor tyrosine kinase, expressed by immature blood vessels and not mature ones, constitutively expressed by the lymphatic endothelium. Reliatech Mouse IgG1 27
VEGF-C Participates in development of lymphatic vasculature by activation of VEGFR-3. R&D Systems Rabbit IgG 26
Endomucin Endothelial sialomucin expressed on lymphatic endothelium, veins, and venules, but not arteries. Gift from D. Vestweber Rat IgG2a 28
PECAM-1 (CD31) Platelet endothelial cell adhesion molecule-1. Integral membrane glycoprotein expressed on endothelial intercellular junctions. Santa-Cruz (Dallas, TX, USA) Rabbit IgG 37
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. Serotec (Raleigh, SC, USA) Mouse IgG2a 21
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. Novocastra (Newcastle-Upon-Tyne, UK) Mouse IgG2a 21
CD44 CD44 is a cell adhesion receptor widely expressed on hematopoietic and nonhematopoietic cells. Immunotech (Brea, CA, USA) Mouse IgG1 21
Collagen IV Basement membrane protein found on vessel walls. Abcam Rabbit IgG 4
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. Sigma-Aldrich Corp. (St. Louis, MO, USA) - - 35
Table 2
 
Relative Frequency of Lymphatic-Like Vessels Versus Blood Vessels in Adult Human Choroid, n = 6
Table 2
 
Relative Frequency of Lymphatic-Like Vessels Versus Blood Vessels in Adult Human Choroid, n = 6
Lymphatics N Blood Vessels N Total Vessels Counted % Lymphatics
Initial, 10–60 μm42 40 Capillaries 543 583 6.9, 40/583
Precollector, 35–150 μm43 1 Medium 88 89 1.1, 1/89
Collector, >200 μm42 1 Large 21 22 4.5, 1/22
Supplementary Figures
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