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Original Basic Science

VEGF-trap Aflibercept Significantly Improves Long-term Graft Survival in High-risk Corneal Transplantation

Dohlman, Thomas H.1; Omoto, Masahiro1; Hua, Jing1; Stevenson, William1; Lee, Sang-Mok1; Chauhan, Sunil K.1; Dana, Reza1

Author Information

1 Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, MA.

Received 7 April 2014. Revision requested 18 August 2014.

Accepted 22 September 2014.

This study was supported in part by the National Institutes of Health/National Eye Institute Grant EY012963 and Vegenics, Circadian Technologies Ltd. (Melbourne, Australia).

The authors declare no conflicts of interest.

T.H.D. and M.O. contributed equally to this work. T.H.D. contributed to the performance of the research, data analysis, and writing the article. M.O. contributed to the performance of the research and data analysis. J.H. contributed to the performance of the research. W.S. contributed to the performance of the research. S.M.L. contributed to the performance of the research. S.K.C. and R.D. contributed to the research design and writing the article.

Correspondence: Reza Dana, M.D., M.P.H., M.Sc., Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, 20 Staniford Street, Boston, MA 02114. ([email protected]).

doi: 10.1097/TP.0000000000000512
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Transplantation is an important therapeutic option for restoring tissue function, but graft failure because of immune rejection remains a major obstacle to the success of all transplantations.1,2 The immunologic fate of an allograft primarily depends on the induction and expression of the alloimmune response.3 Lymphatic vessels facilitate trafficking of antigen-presenting cells (APCs) from the graft site to regional draining lymphoid tissues where they present alloantigens to naive host T cells.3,4 In contrast, blood vessels facilitate the homing of primed effector T cells back to the graft site where they mediate allorejection.5–7 Although experimental attempts to suppress the afferent and efferent arms of the alloimmune response have both shown promise in preventing allorejection,8,9 the relative efficacy of targeting hemeangiogenesis and lymphangiogenesis in transplantation is unknown.

Because the cornea is an avascular and alymphatic tissue, corneal transplantation is an attractive model for assessing the relative contribution of graft site lymphangiogenesis and hemangiogenesis to alloimmunity. In standard “low-risk” corneal transplantation, where a donor cornea is grafted into an avascular and uninflamed host bed, grafts can enjoy survival rates greater than 90% at 2 years10,11 and greater than 70% at 10 years.12,13 In contrast, transplantation of an allograft into an inflamed host bed containing blood and lymphatic vessels (“high-risk” corneal transplantation), a situation akin to other solid organ transplantations, yields survival rates that can fall below 50%.14,15 It is now understood that the cornea’s normal “immune privilege” is lost in high-risk transplantation, and that preexisting blood and lymphatic vessels contribute to the high rate of rejection by promoting allosensitization and effector cell homing to the graft.3

Both hemeangiogenesis and lymphangiogenesis are principally coordinated by the VEGF family of receptors and ligands, with hemangiogenesis largely driven by VEGF-A binding to VEGFR-1 and VEGFR-2 and lymphangiogenesis by VEGF-C and VEGF-D binding to VEGFR-3.16 Studies targeting VEGF-A in both low-risk and high-risk corneal transplantation have demonstrated success in decreasing hemangiogenesis and improving transplant survival.8,17,18 In addition, several antilymphangiogenic strategies have also shown success in improving low-risk graft survival.9,19,20 Interestingly, findings from two studies have raised the question of whether lymphatic vessels may indeed be more important than blood vessels in mediating immune rejection.21,22 In both studies, the importance of the lymphatic vasculature in alloimmunity was highlighted by the observation that transplantation into an inflamed but alymphatic host bed significantly promoted allograft survival.

In the present study, we have directly compared the effects of antihemangiogenic and antilymphangiogenic agents on the induction and expression of alloimmunity and graft survival in high-risk corneal transplantation. Using a clinically relevant murine model of high-risk transplantation in which host-bed blood and lymphatic vessels are present at the time of transplantation, we evaluated the relative efficacy of treatment with VEGF-trap (Eylea, antihemangiogenic), anti-VEGF-C (VGX-100, antilymphangiogenic), and sVEGFR-3 (VGX-300, antilymphangiogenic) on allograft survival.

RESULTS

VEGF Neutralization Reduces Clinical Neovascularization

High-risk corneal transplant recipients were randomly divided into four experimental groups: anti-VEGF-C, sVEGFR-3, VEGF-trap, or no treatment. Given the importance of neovascularization (NV) in the prognosis of corneal transplant survival,14 we first evaluated clinical NV in each group 14 days after transplantation. Grafts were evaluated by slit-lamp biomicroscopy and clinical NV scored according to an established 0 to 8+ scale (Figure 1).23 Treatment with sVEGFR-3 (NV score, 5; P < 0.005) and VEGF-trap (NV score, 3; P < 0.005) yielded a significant decrease in clinical NV when compared to the untreated control group (NV score, 7.5), whereas treatment with anti-VEGF-C led to a decrease in clinical NV that approached statistical significance (NV score, 6; P = 0.06). Treatment with VEGF-trap was significantly more effective in reducing clinical NV when compared to both anti-VEGF-C (P < 0.005) and sVEGFR-3 (P < 0.005).

F1-10
FIGURE 1:
Clinical neovascularization after high-risk corneal transplantation. Fourteen days after high-risk allogeneic corneal transplantation, mice were evaluated using slit lamp biomicroscopy for the appearance of clinical NV according to a standard 0 to 8+ scale. Mice were treated with anti-VEGF-C, sVEGFR-3, VEGF-trap or remained untreated. Each group consists of n=10 mice, **P < 0.005, *P < 0.05, error bars represent standard error of the mean (SEM). Data from one experiment of two are shown. NV, neovascularization.

VEGF-Trap Reduces Hemangiogenesis in High-Risk Corneal Transplantation

To further evaluate the presence of blood vessels in transplant recipients, corneal grafts were stained for CD31 (platelet endothelial cell adhesion molecule, a marker for vascular endothelial cells) and analyzed using epifluorescent microscopy (Figure 2A), after which the area of graft-infiltrating blood vessels was calculated using ImageJ software (Figure 2B). Treatment with VEGF-trap was most effective at reducing graft hemangiogenesis and led to a significantly lower area of vessel invasion (2%) than anti-VEGF-C (10%, P < 0.05), sVEGFR-3 (9.5%, P < 0.005), and no treatment (11.7%, P < 0.05) (Figure 2B).

F2-10
FIGURE 2:
Evaluation of graft hemeangiogenesis and lymphangiogenesis. Corneal grafts were immunostained for CD31 and LYVE-1, and the area of vessel invasion of the graft was analyzed with ImageJ software. A, representative micrographs (4× magnification) of CD31 staining demonstrate the presence of blood vessels. B, the area (%) of blood vessel invasion in anti-VEGF-C, sVEGFR-3, VEGF-trap, or untreated mice is shown. C, representative micrographs (4× magnification) of LYVE-1 staining demonstrate the presence of lymphatic vessels. D, the area (%) of lymphatic vessel invasion of anti-VEGF-C, sVEGFR-3, VEGF-trap, or untreated mice is shown. Each group consists of n=5 mice, **P < 0.005, *P < 0.05, error bars represent standard error of the mean (SEM). Data from one experiment of two are shown.

Anti-VEGF-C and sVEGFR-3 Reduce Lymphangiogenesis in High-Risk Corneal Transplantation

To assess graft lymphangiogenesis, grafts were immunohistochemically stained for lymphatic vessel endothelial hyaluronan receptor-1, a marker for lymphatic endothelial cells) (Figure 2C), and the area of lymphatic vessels was calculated (Figure 2D). Treatment with anti-VEGF-C (1.9%, P < 0.05) and sVEGFR-3 (1.2%, P < 0.05) was most effective at reducing graft lymphangiogenesis when compared to the untreated control group (3.2%) (Figure 2D), and sVEGFR-3 was significantly more effective than VEGF-trap (2.4%, P < 0.05).

Anti-VEGF-C and sVEGFR-3 Minimize Th1 Cell Induction in the Draining Lymph Nodes

We assessed the induction of alloimmunity by quantifying the frequency of CD4 + IFN-γ + Th1 cells in ipsilateral draining lymphoid tissue by flow cytometry (Figure 3A and B). High-risk corneal transplantation without treatment led to a 3.5-fold increase in the frequency of Th1 cells as compared to naive mice, whereas treatment with anti-VEGF-C (P < 0.05), sVEGFR-3 (P < 0.05), and VEGF-trap (P = 0.06) all reduced the frequency of Th1 cells to a level similar to that seen in naive mice.

F3-10
FIGURE 3:
Quantification of Th1 cells in the regional draining LN of high-risk allografted mice. LNs of mice from each group were harvested and analyzed for CD4 + IFN-γ + Th1 cell frequencies. A, representative flow cytometry dot-plots showing the frequencies of Th1 cells in the draining LNs of anti-VEGF-C, sVEGFR-3, VEGF-trap, or untreated transplant recipients. B, the analysis of Th1 cell frequencies shows reduced Th1 cell frequencies in all VEGF treated mice compared to untreated mice. Each group consists of n=5 mice, **P < 0.005, *P < 0.05, error bars represent standard error of the mean (SEM). Data from one experiment of two are shown. LN, lymph nodes.

VEGF-Trap Is Most Effective at Reducing Immune Cell Infiltration of the Corneal Graft

The efferent arm of the alloimmune response entails the homing of immune cells to transplanted tissue through blood vessels. To evaluate the effect of anti-VEGF-C, sVEGFR-3, VEGF-trap, or no treatment on immune cell infiltration of the corneal graft in each of our experimental groups, we used flow cytometry to quantify the frequency of CD45+ leukocytes, CD3+ T cells, and CD11b + macrophages in the corneas of transplant recipients and naive mice. As expected, the frequency of CD45+ cells was highest in the untreated control group (20-fold increase compared to naive) (Figure 4A). Compared to the untreated group, treatment with anti-VEGF-C (12-fold, P < 0.05), sVEGFR-3 (13-fold, P < 0.05), and VEGF-trap (4-fold, P < 0.005) all reduced the frequency of graft-infiltrating CD45+ cells. However, VEGF-trap was significantly more effective than both anti-VEGF-C (P < 0.005) and sVEGFR-3 (P < 0.005) (Figure 4B). To further characterize infiltrating immune cells, the frequencies of graft-infiltrating CD45 + CD11b + monocytes/macrophages and CD45 + CD3+ T cells were quantified (Figure 4C). Corneal infiltration by CD45 + CD11b + cells was highest in the untreated group (sixfold increase compared to naive), and treatment with anti-VEGF-C (3.8-fold, P < 0.05), sVEGFR-3 (2.7-fold, P < 0.05), and VEGF-trap (2.2-fold, P < 0.05) were all successful in reducing the frequency of CD45 + CD11b + cells. CD45 + CD3+ T cell infiltration was highest in the corneas of the untreated control group (55-fold increase compared to naive). We found that VEGF-trap was most effective in reducing the frequency of infiltrating T cells because treatment led to a significant decrease in T cell frequency (7-fold increase) as compared to the anti-VEGF-C (35-fold increase, P < 0.005), sVEGFR-3 (42-fold increase, P < 0.005), and untreated control groups (P < 0.005) (Figure 4E).

F4-10
FIGURE 4:
Quantification of graft-infiltrating immune cells. Transplant recipients were euthanized and corneal grafts plus host corneal beds were collected and analyzed for the presence of CD45+, CD11b + and CD3+ cells by flow cytometry. A, representative flow cytometry dot-plots (n=5 pooled mice) and B, analysis of CD45+ cell frequencies of anti-VEGF-C, sVEGFR-3, VEGF-trap, or untreated transplant recipients presented as fold change relative to naive mice. C, representative dot-plots of CD11b + and CD3+ cell frequencies, gated on CD45+ cells (n=5 pooled mice, percentages are the percent positive of all cells). D, analysis of CD11b + cell frequencies and (E) CD3+ cell frequencies of anti-VEGF-C, sVEGFR-3, VEGF-trap, or untreated transplant recipients, presented as fold change relative to naive. Each group consists of n = 5 mice, **P < 0.005, *P < 0.05, error bars represent standard error of the mean (SEM). Data from one experiment of two are shown.

VEGF-Trap Is Most Effective in Improving High-Risk Corneal Graft Survival

We next evaluated the effect of each treatment on long-term graft survival. High-risk transplant recipients received treatment with anti-VEGF-C, sVEGFR3, or VEGF-trap at the time of transplantation then 3, 7, 10, and 14 days after transplantation, and once per week thereafter. Animals were scored once per week to determine graft opacity and create a Kaplan-Meier survival curve (Figure 5). Treatment with VEGF-trap (72% survival, P < 0.005), anti-VEGF-C (25%, P < 0.05), and sVEGFR-3 (11%, P < 0.05) all led to improved allograft survival at 8 weeks after transplantation as compared to the untreated control group (0%). The rate of graft survival in the VEGF-trap–treated group was significantly greater than that seen in the anti-VEGF-C (P < 0.05) and sVEGFR-3 (P < 0.05) groups.

F5-10
FIGURE 5:
Effect of VEGF neutralization on high-risk corneal transplant survival. Animals underwent high-risk allogeneic corneal transplantation and received treatment with anti-VEGF-C, sVEGFR-3, or VEGF-trap at the time of transplantation, and at 3, 7, 10, and 14 days after transplantation, and then once per week for an additional 6 weeks, or they remained untreated. The VEGF-trap treatment group was most effective in increasing allograft survival (72%), though treatment with anti-VEGF-C (25%) and sVEGFR3 (11%) also significantly improved survival compared to the untreated control group. To create the Kaplan-Meier survival curve, graft opacity was evaluated according to an established 0 to 5+ scale by slit-lamp biomicroscopy. Scores greater than or equal to 2+ are considered rejected. Each group consists of n=9–12 mice. **P < 0.005, *P < 0.05, error bars represent standard error of the mean (SEM). Data from one experiment of two are shown.

DISCUSSION

To study the mechanisms of allorejection in transplantation, we and others have used a murine model of high-risk corneal transplantation with near universal graft rejection in which intrastromal corneal sutures are placed before transplantation to induce hemangiogenesis and lymphangiogenesis.17,21 In this model, treatment with VEGF-trap has been shown to decrease graft hemangiogenesis, leading to a modest increase in graft survival.17 Also, by giving antilymphangiogenic therapy at the time of intrastromal suture placement, studies have also demonstrated that transplantation into an inflamed but alymphatic host bed can improve allograft survival, highlighting the importance of the lymphatic vasculature in alloimmunity.21,22 We have evaluated the relative efficacy of targeting hemeangiogenesis versus lymphangiogenesis in high-risk corneal transplantation, where both blood and lymphatic vessels are present at the time of transplantation,21 and treatment begins at the time of surgery.

VEGF-trap is a potent neutralizer of VEGF-A and placental growth factor that has been successfully used to stop pathologic angiogenesis in retinal pathologies, such as neovascular age-related macular degeneration.24,25 In the setting of transplantation, targeting hemangiogenesis represents a strategy for disrupting the effector arm of the immune response and the delivery of immune cells to the graft. We have demonstrated that treatment with VEGF-trap decreased the area of NV after high-risk transplantation. Importantly, VEGF-trap also significantly inhibited immune cell infiltration of the ocular surface, including macrophages and CD3+ T cells, the primary cellular mediators of immune rejection. Although both CD4+ and CD8+ T cells comprise the CD3+ T cell fraction, there is evidence that acute corneal graft rejection is predominantly mediated by CD4+ T cells.6,26–28 In a previous study, treatment with VEGF-trap increased the 8-week graft survival to 23%,17 whereas we observed a survival rate greater than 70%. This may be because of the differences in treatment regimens: we treated mice by subconjunctival injection for the entirety of the 8-week follow-up period, whereas the previous study was treated through day 14 after transplantation by intraperitoneal injection. The increased graft survival seen in the present study suggests that regular and continued treatment with VEGF-trap may be a promising means of improving high-risk corneal transplant survival.

To disrupt lymphangiogenesis and the affector arm of the alloimmune response, we suppressed VEGFR-3 ligation with a neutralizing anti-VEGF-C antibody or sVEGFR-3, which binds both VEGF-C and VEGF-D. Both treatments significantly reduced the density of graft lymphatic vessels after transplantation, suppressed alloimmune induction, and increased graft survival from 0% in the untreated control group to 25% (anti-VEGF-C) and 11% (sVEGFR3), respectively. In contrast, in the low-risk model of corneal transplantation, treatment with the same agents has been shown to improve graft survival from 50% (the expected low-risk survival rate) to 66% and 87.5%,9,29 respectively. Inflammatory conditions, such as those seen in high-risk corneal transplantation, often demonstrate increased expression of VEGF ligands30 and other inflammatory mediators16 known to influence VEGFR levels and signaling.9,31 In addition, as part of the afferent arm of the immune response, APC migration to the draining lymph nodes depends not only on lymphatic vessels but also a supporting cast of chemokines32 and integrins which are significantly upregulated in high-risk transplantation.33–35 Thus, the lower survival rates seen in the present study, despite treatment with the same antilymphangiogenic agents, likely reflect the increased inflammatory status of high-risk grafts and emphasize the augmented alloimmune response generated in response to high-risk, as compared to low-risk, grafts.

Although treatment with antilymphangiogenic anti-VEGF-C or sVEGFR-3 successfully reduced graft lymphangiogenesis and improved transplant survival, the rate of survival seen was significantly lower than the VEGF-trap group. One reason for this finding may relate to drug dosing because both anti-VEGF-C and sVEGFR-3 inhibit endothelial cell proliferation and lymphangiogenesis in a dose-dependent manner.36, 37 So, even though the highest concentration (39.3 mg/mL) formulations of anti-VEGF-C and sVEGFR-3 available were used in this study, it is possible that had higher concentrations of these agents been used, we may have observed an even greater effect on lymphangiogenesis and possibly long-term graft survival as well. The relatively lower survival in the anti-VEGF-C and sVEGFR-3 groups may also be partially explained by the fact that, in addition to its well-known role in facilitating APC trafficking, the lymphatic vasculature also plays a role in the clearance of inflammatory debris from sites of inflammation.38–40 This raises the question of whether antilymphangiogenic therapies may in some cases act to exacerbate inflammatory conditions by preventing the resolution of inflammation.41,42 Hemangiogenesis has also been shown to play a role in inflammation resolution43,44 and wound healing, including in the corneal epithelium.45 Thus, treatment with VEGF-trap may also act to prolong inflammation and delay healing, particularly at the graft-host junction. However, this did not appear to have an effect on graft survival in the VEGF-trap group. Although the precise contributions of hemangiogenesis and lymphangiogenesis to the resolution of inflammation in corneal transplantation are unknown, going forward, it will be important to consider whether therapies targeting these processes might unintentionally contribute in some manner to sustained inflammation in the high-risk host.

Previous studies using modified models of high-risk transplantation have suggested a relatively more important role for lymphangiogenesis than hemangiogenesis in transplant rejection. Combined VEGFR-3 and integrin blockade in these studies improved graft survival when used prior to transplantation to create an inflamed but alymphatic host bed.21,22 In these animals, lymphatic vessels were not present at the time of transplantation (much like the low-risk corneal transplant setting), meaning that a window of time exists in which anti-lymphangiogenic agents can prevent lymphatic vessels from reaching the graft. In contrast, in the high-risk setting, lymphatic vessels are already extensively present at the time of transplantation17 and allosensitization can begin near immediately,46 with significant frequencies of APCs detectable in the draining lymph nodes as early as 4 hours post transplantation.47 Thus, there is limited opportunity for antilymphangiogenic drugs to modulate the afferent arm of the alloimmune response. This suggests that lymphangiogenic-specific therapies in high-risk transplantation might be best used as part of a conditioning regimen to reduce lymphatic vessel density prior to surgery.

Although VEGF-trap and anti-VEGF-C/sVEGFR3 are intended to be antihemeangiogenic and lymphangiogenic, respectively, there is promiscuity in VEGF ligand-receptor binding and overlap in VEGFR function depending on various factors, such as age, stage of development, and the local environmental milieu.48 This is illustrated by studies demonstrating a role for VEGF-A in mediating lymphangiogenesis49,50 and roles for VEGF-C and VEGF-D in mediating hemangiogenesis.51–54 In particular, VEGFR-2 is known to play an important role in mediating both hemangiogenesis and lymphangiogenesis, making VEGF-trap an extremely effective agent in high-risk transplantation because of its ability to directly inhibit both of these processes.55–57 In addition, VEGF ligands are chemotactic for macrophages, which can secrete VEGF-A, VEGF-C, and VEGF-D, making them capable of promoting both hemeangiogenesis and lymphangiogenesis.58,59 Thus, neutralization of VEGF-A, a classically hemangiogenic ligand, will decrease macrophage recruitment and the concomitant increase in VEGF-C and D, also leading to a decrease in lymphangiogenesis. This is consistent with our data indicating that VEGF-trap not only decreases hemangiogenesis but also has a modest effect on lymphangiogenesis and Th1 cell induction. Similarly, neutralization of VEGF-C or VEGF-D will decrease lymphangiogenesis and limit the prohemangiogenic effects of recruited macrophages. In line with this, we observed that anti-VEGF-C and sVEGFR3 both had modest effects on hemangiogenesis, which led to decreases in the frequency of graft-infiltrating immune cells. This may contribute to the increase in graft survival seen in these groups. Although hemeangiogenesis and lymphangiogenesis are closely intertwined and all three agents used had an effect on both processes, we did observe zthat VEGF-trap was relatively more effective than anti-VEGF-C and sVEGFR-3 in limiting hemangiogenesis (leading to a significant decrease in graft-infiltrating immune cells), and anti-VEGF-C and sVEGFR-3 were relatively more effective in limiting lymphangiogenesis as compared to VEGF-trap.

In summary, our findings suggest that in a clinically relevant model of high-risk corneal transplantation, VEGF-trap was most effective at reducing immune cell infiltration of the corneal graft and improving transplant survival. Our data emphasize the potential value of VEGF-modifying agents in modulating the induction and expression of alloimmunity after high-risk corneal transplantation and raise the questions of what role anti-VEGF agents may have in directly modulating T cell immunity and what the translational potential of combined antihemangiogenic and antilymphangiogenic therapy might be.

MATERIALS AND METHODS

Animals

Male BALB/c and C57Bl/6 mice at 6 to 8 weeks of age were obtained from Charles River Laboratories (Wilmington, MA) and treated according to the Statement for the Use of Animals in Ophthalmic and Visual Research (Association for Research in Vision and Ophthalmology).60 Experiments were approved by the Institutional Animal Care and Use Committee.

High-Risk Allogeneic Corneal Transplantation

To create high-risk host beds, the central 1.5 mm of the host cornea was demarcated by trephine, and three figure-of-eight suture knots consisting of two intrastromal incursions extending from above the limbus to the trephine demarcation were placed using 11-0 nylon suture (Sharpoint, Reading, PA).8,61,62 Sutures were left in place for 2 weeks, and corneal transplantation (C57BL/6 to BALB/C) performed as previously described.63 Neovascularization was evaluated according to a 0 to 8+ scale based on NV per corneal quadrant.23 Graft rejection was evaluated according to a 0 to 5+ scale,23 with a score of 2+ or greater indicating graft rejection. To exclude primary failure grafts, only grafts with scores less than 2+ at 10 days after transplantation were included in our studies. To follow long-term graft survival, mice were scored once per week for 8 weeks.

VEGF Neutralization

Treated mice received (i) anti-VEGF-C (VGX-100, 39.3 mg/ml, Circadian Technologies Ltd./Opthea Pty Ltd., Australia), a human monoclonal antibody that neutralizes VEGF-C; (ii) sVEGFR-3 (VGX-300, 39.3 mg/mL, Circadian Technologies Ltd/Opthea Pty Ltd, Australia), a soluble VEGFR-3 receptor consisting of the extracellular region of human VEGFR-3 fused to the Fc portion of human immunoglobulin G1, which neutralizes VEGF-C and VEGF-D; or iii) VEGF-trap (Eylea, 40 mg/mL, Regeneron Pharmaceuticals Inc., Tarrytown, NY), consisting of the extracellular domains of human VEGF receptors 1 and 2 fused with the Fc portion of human immunoglobulin G1. Eylea acts as a soluble decoy receptor for VEGF-A and placental growth factor. Animals received a 10-μL subconjunctival injection (400 μg of agent, approximately 20 mg/kg body weight) at the time of corneal transplantation and at 4, 7, and 10 days after transplantation. Although these are human agents, all three drugs are known to be cross-reactive and effective in mice.9,64,65 Untreated transplants served as the control group, as performed previously.8 In vitro studies were performed 14 days after transplantation (n=10 per group), or treatment was continued (one injection/week) and the mice were followed up for 8 weeks (n=9–12 per group). Injections were performed by anesthetizing mice with 2% to 4% isoflurane in 100% oxygen, and injecting by Hamilton syringe (Hamilton Company, Reno, NV) while holding the temporal conjunctiva with jeweler’s forceps (Katena Products, Denville, NJ).66

Flow Cytometry

Single cell suspensions of ipsilateral cervical and submandibular lymph nodes were created and stimulated with phorbol 12-myristate 13-acetate and ionomycin (Sigma Aldrich, St. Louis, MO) in the presence of Golgistop (BD Pharmingen, San Jose, CA). Grafts and host corneal beds were collected 14 days after transplantation and digested with Dnase (0.2 mg/mL; Roche, Basel, Switzerland) and collagenase (0.4 mg/mL, Roche) to create single cell suspensions, but were not stimulated with phorbol 12-myristate 13-acetate and ionomycin. All cell suspensions were incubated with an Fc-blocking agent (R&D Systems, Minneapolis, MN). Corneal cells were stained with PE/Cy5 anti-CD45 (eBioscience, San Diego, CA), FITC anti-CD11b (BD Pharmingen) and PE anti-CD3 antibodies (BD Pharmingen). Lymph node cells were stained with Violet-421 anti-CD4 antibody (Biolegend, San Diego, CA), and, following fixation and permeabilization, FITC anti-IFN-γ antibody (eBioscience). Cells were analyzed using a LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ) and Summit v4.3 Software (DAKO Colorado Inc., Fort Collins, CO).

Immunohistochemistry

Grafts plus host corneal bed were incubated with 2 mM ethylenediaminetetraacetic acid to remove corneal epithelium. Corneas were fixed in acetone and blocked with 2% bovine serum albumin, then stained with FITC anti-CD31 (Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti–lymphatic vessel endothelial hyaluronan receptor-1 antibodies (R&D Systems). Cy3 donkey anti-goat antibody (Jackson Immunoresearch Laboratories, West Grove, PA) was used as secondary. Whole-mounts were prepared with 4′,6-diamidino-2-phenylindole mounting medium, and pictures taken at 4× magnification with an E800 epifluorescent microscope (Nikon Corporation, Tokyo, Japan). The area of vessel invasion of the donor graft, including the graft-host junction, was calculated using ImageJ software (National Institutes of Health, Bethesda, MD).

Statistics

Data normality was evaluated using the D’Agostino-Pearson test (Prism GraphPad Software, Inc., La Jolla, CA). Analysis was performed using Microsoft Excel (Microsoft Corporation, Redmond, WA) and the paired two-tailed Student t test for comparisons of means the and Ekuseru-Toukei add-in for survival analysis. Data are reported as means, with P less than 0.05 considered significant and error bars representing standard error of the mean (SEM).

ACKNOWLEDGMENTS

The authors thank Randy Huang and Donald Pottle for their technical help and support, Dr. Dean Eliott and Dr. Demetrios Vavvas along with the Retina Service of the Massachusetts Eye and Ear Infirmary for VEGF-trap, and Dr. Susanne Eiglmeier for her intellectual input and assistance in the preparation of the article.

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