Current Eye Research

ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20

Expression of Lymphangiogenic Markers in Rejected Human Corneal Buttons after Penetrating Keratoplasty Yuri Seo, Mee Kum Kim, Joon H. Lee, Eun-Ju Chang, Eung Kweon Kim & Hyung Keun Lee To cite this article: Yuri Seo, Mee Kum Kim, Joon H. Lee, Eun-Ju Chang, Eung Kweon Kim & Hyung Keun Lee (2015) Expression of Lymphangiogenic Markers in Rejected Human Corneal Buttons after Penetrating Keratoplasty, Current Eye Research, 40:9, 902-912 To link to this article: http://dx.doi.org/10.3109/02713683.2014.969809

Published online: 20 Oct 2014.

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Date: 30 October 2015, At: 23:53

Current Eye Research, 2015; 40(9): 902–912 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.969809

ORIGINAL ARTICLE

Expression of Lymphangiogenic Markers in Rejected Human Corneal Buttons after Penetrating Keratoplasty Yuri Seo1*, Mee Kum Kim2*, Joon H. Lee3, Eun-Ju Chang4, Eung Kweon Kim5 and Hyung Keun Lee1,6

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1

Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea, 2Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea, 3 Myunggok Eye Research Institute, Kim’s Eye Hospital, College of Medicine, KonYang University, ChungNam, Korea, 4Department of Anatomy and Cell Biology, School of Medicine, Ulsan University, Seoul, Korea, 5 Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul, Korea and 6Severance Institute of Vascular Metabolism Research, Yonsei University College of Medicine, Seoul, Korea

ABSTRACT Purpose: To investigate the extent and distribution of lymphangiogenesis in the rejected corneal graft, we determined the expression of several lymphangiogenic markers in rejected human corneal buttons. Material and methods: Thirty-four corneal buttons were obtained from patients who underwent re-keratoplasty for graft rejection after penetrating keratoplasty. All corneas showed signs of rejection, such as, sudden muttonfat keratic precipitates (KPs) or lines before re-keratoplasty. The corneas were halved, and one half was used for immunostaining and the other half was used for RT-PCR. Expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3, LYVE-1 and podoplanin were measured as lymphangiogenic markers. Four non-operated normal corneas were used as controls. Results: Numerous podoplanin positive cells were found in the anterior and posterior stroma. However, LYVE-1 positive mature lymphatics were found only in herpetic keratitis (HK)-induced graft rejection, and not in pseudophakic bullous keratopathy (PBK). RT-PCR showed that levels of VEGF-A, VEGF-C, VEGFR-2, and VEGFR-3 mRNAs were elevated in rejected corneal buttons versus the non-operated control corneas. Based upon the pre-keratoplasty pathologic conditions, HK cases showed higher levels of VEGF-A and VEGFR-2 than PBK. The mRNA ratios (keratoplastic cornea/normal cornea) for VEGF-A and VEGFR-2 were 8.9 and 5.8, respectively. Conclusions: The results suggested that the VEGF-A and the VEGFR-2 may be a more important pathway for lymphangiogenesis in rejected corneal grafts than the VEGFR-3. In addition, organized lymphangiogenesis is more prominent in HK than PBK. Keywords: Lymphangiogenesis, penetrating keratoplasty, rejection, VEGF, VEGFR

INTRODUCTION

more than 70,000 corneal donor grafts used for transplantation every year. With improvements in surgical techniques, ocular pharmacology, and eye banking procedures, corneal allografts placed in

Corneal allografts are one of the most successful and frequently performed transplants in the world, with

Received 22 April 2014; revised 17 September 2014; accepted 22 September 2014; published online 20 October 2014 *These authors contributed equally to this work and manuscript as the first authors. Correspondence: Hyung Keun Lee, Department of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, 146-92 Dogok-dong, Kangnam-gu, Seoul 135-720, Korea. Tel: +82-2-2019-3444. Fax: +82-2-3463-1049. E-mail: [email protected]

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Lymphangiogenic Markers in Rejected Human Cornea 903 uncomplicated human eyes result in an overall first year survival rate as high as 90%.1,2 However, this promising success rate differs from corneal grafts placed in high-risk patients, for which success rates can decrease to 10–30%, even with maximal local and systemic immune suppression.1,3 In many high-risk in vivo corneal allograft models, corneal angiogenesis and lymphangiogenesis have been characterized by immune staining, which showed that the graft had a chance of contacting both the blood and lymphatic systems during allograft rejection.4,5 Whereas blood vessels provide a route of entry for immune effector cells (e.g. CD4+ alloreactive T cells), corneal lymphangiogenesis facilitates the exit of antigenic material and antigen-presenting cells (APCs) from the graft to the regional lymph node. This can induce alloimmunization and subsequent graft rejection.6–9 Also, lymphangiogenesis seems to be a sign of poor prognosis for the new allograft.4 The normal cornea is free of blood and lymphatic vessels, and its ability to actively inhibit ingrowth of vessels is called the ‘‘angiogenic privilege of the cornea,’’ in analogy with corneal ‘‘immune privilege’’.10 However, many pathologic conditions in the cornea interfere with this privilege and cause pathologic angio- and lymphangiogenesis. The ingrowths of pathologic blood and lymphatic vessels into the cornea not only reduce transparency which can eventually cause blindness, but also significantly increase the rate of graft rejections after subsequent corneal allografts.11 To promote graft survival, there has been great interest in reducing corneal angiogenesis. Because it has been known for hundreds of years that pathologic ingrowths of blood vessels into the cornea can occur, there have been many studies of the mechanisms and treatments for corneal angiogenesis. Recently, immune cell distribution and angiogenic cytokines for lymphangiogenesis were widely studied.11,12 However, the characteristics of the expression of several lymphangiogenic biomarkers as pathologic causes of graft rejections has not been studied. Based on this information, the purpose of this study was to investigate the differences in the expression of lymphangiogenic biomarkers depending on the pathologic causes that resulted in corneal graft rejections.

MATERIALS AND METHODS Human Cornea Preparation The investigation was performed at two independent locations, and designed as a prospective, nonrandomized, case control study. The institutional review boards of both Yonsei University College of Medicine (YUH, Seoul, South Korea) and Seoul National University Hospital (SNUH) approved the study protocol, and the protocol complied with the !

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tenets of the Declaration of Helsinki. After informed consent had been obtained for the research use of discarded buttons, a total of 34 corneal buttons in recipient beds were collected from 34 patients who underwent re-keratoplasty for graft rejection after penetrating keratoplasty. The patients were enrolled from two university hospitals (SNUH and YUH), and 12 males and 22 females were included. Inclusion criteria for the cases were as follows: previously performed keratoplasty needing re-keratoplasty for graft rejection events, stromal edema, and visual acuity less than 0.1 due to severe corneal edema and opacity. Patients were excluded for the following: (1) infection or trauma within the previous one month, (2) ocular or other surgery within the previous 6 months, (3) severe blepharitis with meibomian gland dysfunction, (4) blinking abnormalities (e.g. Parkinson’s disease, facial nerve palsy, etc.), (5) uncontrolled glaucoma, 6) severe pterygium or uncontrolled systemic disease, and (7) pregnancy or lactation. To differentiate the natural graft loss from sudden graft failure or rejection, the cases of slow and progressive loss of endothelium were excluded. Therefore, all the cases were experienced abrupt visual loss with graft edema and significant decreased endothelium. As a control group, four eyes with no history of ocular or systemic diseases and surgery were included. The mean age of control subjects was 54.2 ± 14.2 (range 38–75) years. The primary cause of transplantation was endothelial failure by herpetic keratopathy (HK) (n = 15), pseudophakic bullous keratopathy (PBK) (n = 17). All corneas showed signs of endothelial edema before rekeratoplasty or endothelial failure. Mean age of the patients was 55.4 years old (range, 24–79 years). The demographic data of the patients were summarized in Table 1. Each of the five corneal buttons from HK and PBK was used for FACS analysis. Others (10 buttons from HK and 12 buttons from PBK) were used for immunofluorescence staining and quantitative RTPCR. For RT-PCR and immunofluorescence staining, all tissues were obtained within 2 h after surgery, were rinsed three times with phosphate buffer saline (PBS) and divided into two pieces unless otherwise stated. One fragment of each sample was used for immunostaining, and the other fragment was used for RT-PCR. For qPCR analysis, the bisected corneas were collected and preserved at 80  C until RNA extraction.

Clinical Examination and Classification of Vascularized Cornea Before keratoplasty, the recipient cornea was examined by slit-lamp microscopy, and photographs of the cornea were used to determine the vascularized

Sex

M

F

M

M

M M

M M

F

F

M

M F

F

F

F

F

F

F F

F

M

F F

Patients Number

1

2

3

4

5 6

7 8

9

10

11

12 13

14

15

16

17

18

19 20

21

22

23 24

57 39

66

24

48 39

45

71

72

63

61

72 65

55

54

27

58 37

26 48

62

54

55

81

Age

Bullous KP pseudophakic HK & endotheliitis

Traumatic opacity pseudophakic HK & endotheliitis

Bullous KP pseudophakic HK & endotheliitis

HK & endotheliitis

Bullous KP aphakic

HK & endotheliitis

Bullous KP pseudophakic

Traumatic opacity, aphakic

HK & endotheliitis HK & endotheliitis

ICE syndrome/pseudophakic bullous KP HK & endotheliitis

HK & endotheliitis

Bullous KP pseudophakic Bullous KP pseudophakic

Bullous KP pseudophakic HK & endotheliitis

Bullous KP pseudophakic

Bullous KP pseudophakic

Bullous KP pseudophakic

Bullous KP, aphakic

Preoperative diagnosis

TABLE 1 Demographic data of the subjects.

Rejection Rejection with ulcer (culture negative) Chronic rejection + traumatic hyphema Rejection

100 PKP, PE PCL 110 PPV, PKP

000 scleral fixation, 070 PKP 070 PKP

060 PKP

100 PKP 010 PE, 2006 PKP

080 PKP 040 , 080 PKP

Rejection with neurotrophic ulcer Rejection Rejection with neurotrophic ulcer Rejection

060 , 100 PKP (OS)

060 phakic IOL (OU) Recurrent HK

Rejection

050 PKP

Rejection, s/p suture related bacterial keratitis Rejection Rejection

Rejection with sterile ulcer Rejection

020 PKP

050 PKP c cL transplantation

Rejection

990 , 050 PKP 100 PKP

Recurrent HK, chronic uveitis PEX glaucoma Recurrent HK, chronic uveitis 2001 fungal keratitis, 2011, traumatic CP Recurrent HK, AKC

high myopia, anterior uveitis, 04 PE (OU) Aphakia, 850 ECCE

Recurrent HK Recurrent HK, chronic uveitis 96, traumatic corneal performation 97, RRD, PE, Glaucoma

Rejection s/p fungal keratitis Rejection Rejection

060 PKP

070 PKP

050 Ahmed 09 PE PCL DM, recurrent HK

980 , 000 , 020 , 070 PKP

Behcet uveitis, recurrent HK

DM, recurrent HK AKC, 110 RRD PPV PE

01, 06, 07, 08 PKP 97, 05, 07, 08, 10 PKP

02, 08 PKP

960 ACL removal Congenital glaucoma 050 PCL

090 PKP

05, 06, 09 PKP

Rejection s/p fungal keratitis Rejection s/p fungal keratitis Rejection s/p suture related bacterial keratitis Rejection s/p fungal keratitis Rejection Rejection

Major events post-KP

080 PKP (OD), 09 PKP(OS)

Previous KP

PEX glaucoma

Aniridia, 95 PE, 04 Ahmed

840 ECCE (OU)

Past history

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Diffuse edema (continued )

Diffuse edema with new vessel 3 to 7 o’clock

Diffuse edema, new vessels, 3 to 5 o’clock Diffuse edema with new vessels 3 to 7 o’clock Diffuse edema Diffuse edema, stormal opacity Diffuse edema

Diffuse edema, bullae

Diffuse edema, bullae

Diffuse edema, bullae

Diffuse edema Diffuse edema, bullae

Diffuse edema, bullae

Diffuse edema, new vessels, 3 to 7 o’clock Diffuse edema

Diffuse edema Diffuse edema, new vessels, 6 to 9 o’clock Diffuse edema Diffuse edema & opacity

Diffuse edema

Diffuse endo opacity, inf horizontal NV Diffuse edema

Diffuse edema

Cornea examination

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F

F F

M F

F

F

M

F

F

25

26 27

28 29

30

31

32

33

34

67

71

65

51

52

79 56

52 56

41

Age

HK & endotheliitis‘

HK & endotheliitis

Bullous KP pseudophakic

HK & endotheliitis

Bullous KP aphakic

Bullous KP pseudophakic HK & endotheliitis

Bullous KP pseudophakic Bullous KP pseudophakic

HK & endotheliitis

Preoperative diagnosis

Recurrent HK, chronic uveitis POAG, recurrent uveitis, 980 PE (OU)

Recurrent HK, chronic uveitis POAG, 060 Ahmed valve

Recurrent HK, chronic uveitis high myopia, 990 PE high mypopia, AKC, 970 PE PCIOL (OU) 910 AC IOL removal AS, chronic uveitis, 960 PE ACIOL aniridia, 950 PE PCIOL

Past history

Rejection Rejection with neurotrophic ulcer Rejection Rejection Rejection with neurotrophic ulcer Rejection with neurotrophic ulcer Rejection, s/p fungal keratitis Rejection Rejection

070 , 110 PKP 960 PE (OU), 07 PKP

02, 05, 07 PKP 100 PKP with cLimbal transplantation 02, 08 PKP

04 PKP

06, 10, PKP

000 PKP 020 , 060 , 080 PKP

Rejection

Major events post-KP

060 PKP

Previous KP

Diffuse edema Diffuse edema with new vessel 9 to 2 o’clock Diffuse edema with focal new vessel Diffuse edema with new vessel 3 to 8 o’clock Diffuse edema with new vessel 10 to 12 o’clock Diffuse edema with stromal opacity Diffuse edema

Diffuse edema with stromal opacity Diffuse edema with stomal opacity Diffuse edema Diffuse edema

Cornea examination

KP: keratoplasty, ECCE: external capsular cataract extraction, PKP: penetrating keratoplasty, PEX: pseudoexfoliation, PCL: posterior chamber IOL, ACL: anterior chamber IOL, AKC: atopic keratoconjunctivitis, RRD: rhegmatogenous retinal detachment, HK: herpetic keratitis, PE: phacoemulsification, cLimbal: conjunctivolimbal transplantation, CP: corneal performation, POAG: primary open angle glaucoma.

Sex

Patients Number

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Lymphangiogenic Markers in Rejected Human Cornea 905

906 Y. Seo et al. corneal area. If corneal vascularization was found, image analysis software was used to calculate the vascularized area.

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Immunofluorescence Staining for VEGF-A, VEGFR2, Podoplanin, and LYVE-1 Immunofluorescence staining of the human corneal button was performed as previously described. Briefly, the cornea was fixed in 10% neutral formalin for 24 h, and then embedded in paraffin. To examine the whole layers of the cornea, 5–7 mm sections from epithelium to endothelium were also made for hematoxylin and eosin (H&E) and immunofluorescence staining. Then, to obtain a similar image with mouse whole mount corneas, the paraffin embedded cornea was serially cut into 70 mm thickness from the epithelium to the endothelium. Mouse monoclonal anti-human antibodies against VEGF-A (1:100 Abcam, Cambridge, MA, USA), VEGFR2 (1:100, Abcam), LYVE-1 (1:200, Dako, Copenhagen, Denmark), and anti-human podoplanin antibody (1:200, Abcam) were used as primary antibodies for incubating overnight. Then, sections were incubated with TRITC- or FITC-conjugated secondary antibodies for three hours. After rinsing with PBS, samples were observed under a fluorescence microscope (a Nikon Eclipse TE200 instrument equipped with a Nikon digital camera, model DXM 1200; Nikon, Tokyo, Japan) and laser confocal microscopy (LSM 710 equipped with AxioCam ERc5S, Carl Zeiss Microscopy GmbH, Jena, Germany) using filters appropriate for fluorescence visualization.

FACS Analysis Each of the five human corneal buttons from HK and PBK was prepared for FACS analysis. Corneal buttons were dissected into more than 10 pieces, and incubated in Hanks’ balanced salt solution containing 0.2% collagenase (Gibco, Carlsbad, CA) and 0.01% hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 1 h with rotation (70 rpm) at 37  C. Then, the cells and debris were strained through a 80 mm filter and the filtrates collected. The samples were centrifuged and resuspended in FACS buffer (1% BSA, 10 mM NaN3 in PBS) and aliquots were prepared for further staining. After blocking with anti-FcR mAb (CD16/CD32), cells were labeled with anti-human CD11b, CD11c, and F4/ 80 (all antibodies from eBioscience, San Diego, CA). For the isotype control, cells were labeled with the respective fluorochrome-conjugated human IgG antibodies (BD Pharmaceuticals, Franklin Lakes, NJ). Cells were subsequently washed and analyzed using a flow cytometer (FACS Caliber, BD Biosciences, San Jose, CA).

RNA Preparation The method of human PKP corneal button preparation was already described and published before.13 Briefly, the cryopreserved bisected corneas were rinsed with sterile RNase free water, cut into small pieces, minced, and subjected to homogenization using a handheld homogenizer for 1 min, followed by 1 min of rest (this was repeated several times to confirm the completely minced cornea without large debris). Then, total RNA was isolated from the samples using TRIzol Reagent (GIBCO-BRL, Gaithersburg, MD). RNA was prepared following the manufacturer’s protocol.

RT-PCR RNA was isolated with the RNeasy Micro Kit (Qiagen, Valencia, CA) and reverse transcribed using the Superscript III Kit (Invitrogen, Carlsbad, CA). RT-PCR was performed using TaqMan Universal PCR Mastermix and preformulated primers for VEGF-A (assay ID Hs00900055_m1), VEGF-C (Hs01099203_m1), VEGFR-2 (Hs00911700_m1), VEGFR-3 (Hs01047677_m1), and GAPDH (assay ID Hs02758991_g1) (Applied Biosystems, Foster City, CA). The VEGF-D primer was designed as follows: forward, 50 -GTATGGACTCTCGCTCAGCAT-30 ; 0 reverse, 5 -AGGCTCTCTTCATTGCAA CAG-30 . The results were analyzed using StepOne software (Applied Biosystems, Carlsbad, CA) by the comparative threshold cycle method and normalized by GAPDH as an internal control. The average value from the normal sample was considered as 100, and the mean value from KC was calculated as percent increase or decrease from the normal, and then the graphs were plotted.

Statistics All data were expressed as mean ± standard deviation (SD). Differences between groups were examined by multivariate analyses using the Newman–Keuls test or ANOVA followed by the Bonferroni procedure for comparison of means using SPSS 18.0 software (Chicago, IL). Values of p50.05 were considered statistically significant.

RESULTS Determination of Lymphatic Vessels in Rejected Recipient Corneal Buttons Based upon immunofluorescence staining, not all cases showed LVs (lymphatic vessels) in the rejected graft. In PBK cases, LVs were found in only 4 out of 17 Current Eye Research

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Lymphangiogenic Markers in Rejected Human Cornea 907

FIGURE 1 Determination of lymphatic vessels in rejected recipient corneal buttons. (A) and (B) Percentage of PECAM stained blood vessels (A) and LYVE-1 stained lymphatic vessels (B) found cases from total HK (15 cases), PBK (17 cases) related rejected corneal button. (C) Representative immunofluroscence staining for LYVE-1 (200) of herpetic keratopathy (HK) or pseudophakic bullous keratopathy (PBK) induced rejected corneal button.

corneas. However, all HK cases were found to have LVs (Figure 1A and B). Interestingly, all the cases containing LVs were positive for PECAM (Platelet Endothelial Cell Adhesion Molecule), a BV (Blood vessels) marker. There were no cases of LV staining buttons without BVs. Based upon fluorescence microscope images of thick preparations (70 mm) from the corneal button, LVs were mainly found in the peripheral area of subbasal and anterior stromal layer of the graft in HK (Figure 1C). The direction of LVs was towards the central cornea with branching or budding from the main stem. The microscope views of the LVs from PBK involved a few, short, interrupted staining patterns. In both HK and PBK, LVs were only found in the superficial stromal area and were not found in the posterior stroma.

mRNA Expression of VEGF-A, VEGF-C, VEGF-D, VEGF Receptor-2, and VEGF Receptor-3 Because the VEGF and VEGF receptor systems have been known to be responsible for lymphangiogenesis, !

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lymphangiogenic markers were determined by RTPCR. Compared to normal corneas, rejected corneal buttons, whether PBK or HK, showed significantly elevated mRNA levels of VEGF-A, VEGFR-2, and VEGFR-3 (Figure 2A–E). VEGF-A and VEGFR-2 mRNA levels in HK were found to be 8.9-fold and 5.8-fold higher, respectively, than normal controls. However, VEGF-C and VEGF-D mRNAs in PBK were not significantly elevated compared to controls. We then compared VEGF family markers with the cause of rejection. Besides VEGF-D, all the VEGF mRNAs were significantly higher in HK than in PBK. VEGF-A and VEGF-C from the rejected buttons in HK were elevated compared with those in PBK (3.5-fold and 2.3-fold, respectively, **p50.01, Student’s t-test, Figure 2A and B). Additionally, VEGFR-3 mRNA was also increased to 1.3-fold. (*p = 0.039, Student’s t-test, Figure 2E). By immunofluorescence staining of VEGF-A and VEGFR2, normal and PBK cases showed faintly stained VEGF-A and VEGFR2 in epithelium. Few stromal cells were stained by both VEGF-A and VEGFR2. However, in HK cases, entire epithelial and stromal layers strongly stained with VEGF-A.

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FIGURE 2 The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3. (A)–(E) Three to five recipient corneal buttons were minced and digested with reagent solution. Specific mRNAs of each VEGF and their receptor expression in HK and PBK were compared (*p50.05, **p50.01, Student’s t-test). (F) VEGF-A and VEGFR2 expression in rejected corneal button was determined by immunofluroescence microscopy (scale bar = 50mm). White arrows indicate VEGFR2 expressed cells in stromal layer.

Moreover, many cells in stromal layer were expressed VEGFR2 (white arrow, Figure 2F).

Podoplanin Positive Cells Identified by Confocal Microscopy Podoplanin is a cytoplasmic marker for lymphatic endothelial cells.14 Normal corneal endothelial cells and keratocytes remained unstained with podoplanin.15 To determine their possible involvement in lymphangiogeneic processes in rejected corneal buttons, podoplanin positive cells were stained and observed by confocal microscopy. Not only the organized LVs, but also many cells in the anterior stroma, were podoplanin positive. Some podoplanin stained cells were found in tubular structures which might be composed of LVs. (White arrow, Figure 3A). Cursiefen et al. reported that LYVE-1 and podoplanin

antigen were found on endothelial cells lining vessels with ultrastructural features of lymph vessels.16 However, many other cells were localized in the anterior stroma without constructing tubular structures. Based upon the pathological condition, podoplanin positive cells were found in 14% of cells in PBK, and 29% of cells in HK, whereas only 3% podoplanin positive cells were found in normal corneas (Figure 3B).

Comparison of CD11b+, CD11c+, and F4/80+ Cell Populations in Rejected Corneal Buttons Since mononuclear cells are involved in lymphangiogenesis as well as angiogenesis in the cornea,17 the frequencies of CD11b+, CD11c+, and F4/80+ cells were determined in rejected corneal buttons by flow Current Eye Research

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Lymphangiogenic Markers in Rejected Human Cornea 909

FIGURE 3 Immunofluroescence staining of podoplanin+ cells in herpetic keratitis-induced rejected corneal bed. (A) Podoplanin positive cells are observed in the peripheral region from two herpetic keratitis-induced rejected corneal buttons by confocal microscopy (scale bar = 20mm). Lower row is a Z-stacked reconstructed image of each buttons. Some tubular or network-like structures are observed (white arrows). (B) Podoplanin positive cell population is higher in the HK-induced rejected button (**p50.01, One-way ANOVA). Cells were counted from five independent high power fields (HPF, 400) for each condition.

cytometry (Figure 4). For CD11c+ cells, there was no significant difference in rejected corneal buttons between PBK and HK (Figure 4C). However, CD11b+ and F4/80+ mononuclear cells were significantly increased in corneal buttons in HK. The median frequency of CD11b+ cells in PBK was 7.1% (range 1.1–10.8%) and 13.3% (range, 6.5–22.9%) in HK (*p = 0.021, One-way ANOVA, Figure 4A). For median F4/80 frequencies, 4.1% (range, 2.9–7.2%) were found in PBK, and 5.9 % (range, 3.5–9.7%) were found in HK (**p = 0.003, One-way ANOVA, Figure 4B).

DISCUSSION Lymphangiogenesis by VEGF-A and VEGFR-2 Lymphangiogenesis may be the initial step for allosensitization and graft rejection in keratoplasty. Dietrich et al. reported that presence of pathologic blood and lymphatic vessels in corneal recipient beds prior to transplantation significantly increased the rate of subsequent graft rejections.12 However, because of low detectability and the paucity of available markers, lymphangiogenesis studies have not been conclusive, so little is known of the role of LV growth in corneal allograft rejection in patients. Moreover, most studies have used an in vivo model, and these in vivo mouse and rabbit corneal transplantation rejection models are quite different from !

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humans. Recently, Zheng et al., reported lymphangiogenesis in rejected human corneal buttons.4 In vascularized corneal buttons, only 26% (6 of 23) had lymphatic vessels, which seemed to be evidence of poor prognosis.4 In addition, Ling et al. reported a similar rate of lymphatic vessels in rejected corneal buttons (26%, 5 of 19 cases).18 Both studies reported that all lymphatic vessels were concurrently found with blood vessels. However, these studies did not include lymphangiogenic cytokine expression data or pre-PKP pathologic conditions. The results of our present study demonstrated differences in lymphangiogenesis between pre-operative conditions, which may be affected by activation of different VEGF systems. Most rejected corneal button caused by HK showed few blood vessels using a slit-lamp. However, expression of VEGF-A and VEGFR-2 were significantly higher than VEGF-C and VEGFR-3. It is well known that the VEGFR-3/VEGF-C pathway is important for lymphangiogenesis.12,16,19,20 Cursiefen et al. described that VEGFR-3 and VEGF-C were co-localized on the endothelial lining of lymphatic vessels in human cornea.16 In addition, the blockade of VEGFR-3 reduced the corneal graft rejection in mice.19,20 However, VEGFR-2 and VEGF-A are also important in lymphangiogenesis during inflammatory conditions, such as after injections and corneal sutures.21,22 Albuquerque et al. reported that soluble VEGFR-2 inhibited lymphangiogenesis induced by corneal suture injury.23 Also, Uehara et al. reported that the injection of the splice-shifting morpholinos targeting

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FIGURE 4 Comparison of CD11b+, CD11c+, and F4/80+ cell populations in rejected corneal buttons caused by HK versus PBK. The rejected corneal buttons were minced and treated with collagenase and hyaluronidase enzyme cocktail. The cells were separated from the digested debris, then labeled with specific FACS antibodies, and the number of cells determined by FACS analysis (*p50.05, **p50.01, One-way ANOVA).

VEGFR-2 suppressed graft rejection in mouse corneal transplantation.24 Additionally, Wuest and Carr also reported that HSV-1-activated lymphangiogenesis was strictly dependent upon VEGF-A/VEGFR-2 signaling, but not on VEGFR-3 ligands.25 Also, the source of most of the VEGF-A was from the corneal epithelium, which caused vasculogenesis in HSV-infected conditions. These results support our findings, which showed higher level of VEGF-A/VEGFR-2 in HK caused by corneal allograft rejection, compared to VEGFR-3/VEGF-C. Taken together, the above results could explain why HK cases showed higher immune rejection rates than other pre-keratoplasty pathologic conditions.

upon these results, different therapeutic regimens should be used to treat different pathologic conditions. For example, for cases of PBK-induced graft failure, which showed minimal lymphatic vessels as well as BVs, endothelial cell protective therapies would be more effective than anti-VEGF or antiinflammatory treatment for preventing and treating graft rejection. However, for HK cases, which have well-developed lymphatic vessels in the donor corneal button, anti-VEGF-A treatment (e.g. avastin injection) may be effective to prevent, treat, or reverse the graft rejection process during postoperative period as anti- VEGFR-3 and VEGFR-2 treatment were effective in graft rejection of mouse model.20,27

Differentially Expressed VEGFs as a Causative Factor in Corneal Allograft Rejection

Expression of VEGFR-2 and VEGF-A in Stromal Keratocytes

Although various conditions can cause graft failure and vision loss, the molecular mechanisms of the pathologic processes in each condition could differ. In a human study, PBK or aphakic bullous keratopathy (ABK) resulted in reduced VEGF mRNA levels, elevated insulin-like growth factor (IGF)-1 levels, and elevated bone morphogenic protein (BMP)-4 levels.26 In addition, diabetic corneas showed increased levels of VEGFs,26 and keratoconic corneas had decreased nerve growth factor receptors.13 Based

We found that for stromal cells in rejected corneal buttons caused by HK, expression of VEGFR-2 and VEGF-A were greater than in control buttons or in PBK cases. Although there is considerable evidence of expression of VEGF-A in normal and pathologic corneal epithelium, consistent with anti-VEGF-A treatment resulting in decreased angiogenic activities, the expression of VEGF-A/VEGFR-2 in human corneal keratocytes has not been thoroughly studied. Because our immunostaining and RT-PCR data Current Eye Research

Lymphangiogenic Markers in Rejected Human Cornea 911 showed that few keratocytes expressed VEGF-A/ VEGFR-2 in normal human corneas, VEGF-A/ VEGFR-2 expression must have been induced in keratocytes or infiltration of blood cells by pathologic conditions such as HK. This finding may therefore explain the deep stromal angio- and lymphangiogenesis present in herpetic keratitis. Furthermore, it is well known that keratocytes are not a type of fibroblast, such as those in the skin. The keratocytes act like macrophages28 or dendritic cells29,30 in various pathologic conditions. Therefore, it is possible that the VEGF-A/VEGFR-2 positive stromal cells actively participate in lymphangiogenesis as well as angiogenesis in the allograft rejection process.

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Limitations of the Study Although this study used human corneal buttons, there are some issues which should be addressed in future studies. The present work used a relatively small number of rejected corneal buttons. In the future, a large-scale and well-designed study should be performed to clearly determine the role of the VEGF systems in corneal allograft rejection. Additionally, although it was inevitable because each individual had different disease histories, the difference of anti-rejection or anti-viral medication history of each patient may affect the results of lymphatic growth. Another issue in the present work is its descriptive analysis of VEGF expression in rejected corneas. Future studies should be addressed to explaining how this change in VEGF expression causes graft rejection. Moreover, although we found that VEGF-A/VEGFR-2 is elevated in HK-induced rejection, and although the involvement of VEGF-C/D and VEGFR-3 are also well-known in the lymphangiogenic system, how these two systems cooperatively or independently work together in lymphangiogenesis needs to be better characterized. Also, the function of infiltrating CD11b+, F4/80+ and podoplanin+ cells for the lymphangiogeneisis should be addressed. Lastly, according to the in vivo mouse study, the corneal lymphangiogenesis after corneal allograft is age-dependent.31 The older aged cornea was less lymphangiogenic. Therefore, the effect of onset age on lymphangiogenesis should be considered and studied in the future. Conclusively, the novelty of this study is to show lymphangiogenic molecular patterns and immunological populations in rejected human corneal button, which demonstrates the development and progression of lymphatic vessels were different, depending on the corneal pathology. These results suggest that customized treatment protocols could be developed based upon the different causative conditions of the rejection to prevent or treat graft rejection in future studies. !

2015 Informa Healthcare USA, Inc.

DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by Advanced Science Research Program (grant no.: NRF-2012R1A2A2A 02009081) through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology and partially by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant no.: HI13C0055).

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