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Abstract
Background Corneal (lymph) angiogenesis is a predominant risk-factor for immune rejection after transplantation. Techniques to regress pre-existing pathological corneal lymphatic vessels prior to transplantation are missing so far. Therefore we analysed the possibility to regress corneal lymphatic vessels by photodynamic therapy (PDT), after intrastromal verteporfin injection.
Methods Combined hemangiogenesis and lymphangiogenesis was induced in female BALB/c mice using the murine model of suture-induced inflammatory neovascularisation. Thereafter, the treatment group received an intrastromal injection of verteporfin (controls: phosphate buffered saline (PBS)) followed by PDT. Corneas were excised at different time points (1 day, 5 days and 10 days) after PDT and corneal whole mounts were stained with CD31 and LYVE-1 to quantify hemangiogenesis and lymphangiogenesis.
Results Whereas blood vessels showed no significant reduction after PDT, lymphatic vessels could significantly be reduced with PDT after intrastromal verteporfin injection: 1 day after PDT, lymphatic vessels were reduced by 62% (p=0.20). After 5 days and 10 days, lymphatic vessels were reduced by 51% and 48% (p<0.001), respectively.
Conclusions This study for the first time shows that PDT after corneal intrastromal verteporfin injection can selectively regress lymphatic vessels. This may become a new ‘preconditioning strategy’ to reduce pre-existing corneal lymphatic vessels prior to transplantation and thereby reduce allograft rejection in high-risk patients.
- Angiogenesis
- Cornea
- Treatment Lasers
- Neovascularisation
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Introduction
There are only few tissues in the human body that are devoid of blood and lymphatic vessels. The healthy cornea is one of these tissues and actively remains vessel-free due to several antiangiogenic mechanisms. However, several conditions such as chemical burns, infections or trauma can interfere with this (lymph) angiogenic privilege and cause ingrowths of pathological blood and lymphatic vessels from the limbus into the corneal centre.1 Pathological corneal neovascularisation is a worldwide major cause for blindness. These vessels decrease visual acuity and play a crucial role in the development of immune responses after corneal transplantation.2 Therefore, the pathological ingrowths of blood vessels into the cornea is one of the main risk factors for immune mediated allograft rejection after corneal transplantation (keratoplasty) in patients.3 In addition, Dietrich et al found that clinically invisible lymphatic vessels are even more important for the immune mediated allograft rejection after keratoplasty—at least in the murine model of corneal transplantation.2 In addition, Albuquerque et al could show that selective inhibition of lymphangiogenesis promotes corneal allograft survival.4 Removal of draining lymph nodes could also significantly improve graft survival in the murine model.5 This calls for anti(lymph) angiogenic treatment strategies in the cornea especially in vascularised high-risk eyes to promote corneal graft survival.
New therapeutic options to decrease actively outgrowing pathological neovascularisation exist, for example, the off-label application of topical VEGF inhibitors used as eye drops or antisense oligonucleotides eye drops currently tested in phase II and phase III trials.6
For mature corneal blood vessels, fine needle vessel coagulation of existing blood vessels combined with antiangiogenic therapy is an interesting option to precondition such high-risk eyes before transplantation.1 ,7–9
In contrast, so far, there is no preoperative or postoperative treatment known, which specifically induces regression of mature corneal lymphatic vessels to modulate the immune response and to reduce the risk of allograft rejection.
Photodynamic therapy (PDT) using verteporfin has been used for different angiogenic diseases, including certain cancer forms.10 ,11 In ophthalmology it is an established treatment for subfoveal choroidal neovascularisation in patients with age-related macular degeneration, pathological myopia, choroidal haemangioma, polypoidal choroidal vasculopathy and for patients with chronic or recurrent central serous chorioretinopathy.12–15 After intravenous application of the photosensitiser verteporfin, porphyrin derivatives accumulate in actively proliferating active endothelial cells. The activation of the photosensitiser by laser energy releases highly reactive cytotoxic short-lived singlet oxygen and other reactive oxygen radicals.11 This leads to a damage of endothelial cells and vascular leakage, and causes coagulation and induction of thrombocyte aggregation, which triggers vascular occlusion of neovascular vessels.10 Hereby pathological neovascularisation can be reduced.
Besides choroidal neovascularisation, PDT after systemic intravenous injection of verteporfin was shown to be an effective method to reduce corneal blood vessels.16–18 However, thus far, there is no therapeutic option to reduce mature existing pathological lymph vessels in the cornea to lower the risk of immune-mediated allograft rejection before and after corneal transplantation. To our knowledge there is no study which analysed the effect of corneal PDT on lymphangiogenesis. We hypothesised that the locally applied photosensitiser is drained by the blind ending open lymphatics and applied the photosensitiser by intrastromal corneal injection. Therefore we analysed whether it is possible to selectively regress existing lymphatic vessels in the cornea. This manuscript shows for the first time the lymphangioregressive effect of photodynamic therapy after local application of verteporfin. This may provide a new model to better characterise the specific role of lymphatic vessels in graft rejection and also might be a new strategy to precondition vascularised high-risk corneas prior to transplantation.
Material and methods
Animals and anaesthesia
All animal protocols were approved by the local animal care committee and were in accordance with the The Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were anaesthetised with an intraperitoneal injection of a combination of 8 mg/kg ketamine (Ketanest S; Godecke AG, Berlin, Germany) and 0.1 ml/kg xylazine (Rompun; Bayer, Leverkusen, Germany). For the suture-induced inflammatory corneal neovascularisation assay 6–8 week-old female BALB/c mice were used.
Mouse model of suture induced inflammatory corneal neovascularisation
The mouse model of suture-induced inflammatory corneal neovascularisation was used as previously described.19 Before corneal neovascularisation, each animal was deeply anaesthetised. Three 11–0 nylon sutures (Serag Wiessner, Naila, Germany) were then placed intrastromally with two stromal incursions (figure 1). The outer point of suture placement was chosen near the limbus and the inner suture point was chosen near the corneal centre equidistant from the limbus to obtain standardised angiogenic responses. Sutures were left in place for 14 days (figure 1).
Intrastromal sutures inducing neovascularisation into the cornea. (A) Three 11-0 nylon sutures are placed into the corneal stroma (white arrows). (B) After 14 days, blood and clinically invisible lymphatic vessels grow from the limbus into the corneal centre (black arrow).
PDT after intrastromal injection of verteporfin
Two weeks after suture placement mice were anaesthetised and all sutures were removed. The treatment group received an injection of 2.5 µL verteporfin (4 µg/mL) (Visudyne; Novartis AG, Basel, Switzerland) into the centre of the corneal stroma using a 33 gauge Hamilton needle (Hamilton Messtechnik GmbH, Höchst, Germany). Control mice received an intrastromal injection of 2.5 µl phosphate buffered saline (PBS). After 2 h of dispersion, laser treatment of the corneas with the Opal Photoactivator PDT laser (Lumenis GmbH, Dieburg, Germany) was performed on all mice (t=40 s, P=46 mW, Ø 2 mm).
Morphological analysis of corneal hemangiogenesis and lymphangiogenesis
Thirty mice were used for the assessment of hemangiogenesis and lymphangiogenesis (treatment group with verteporfin n=15; control group with PBS n=15). Five corneas of each group were excised 1 day, 5 days and 10 days after laser treatment. Corneal blood and lymphatic vessels were stained in whole mounts with CD31-fluorescein isothiocyanate (FITC) (Acris Antibodies GmbH, Hiddenhausen, Germany) as a pan-endothelial marker and LYVE-1 (AngioBio, DelMar, USA) as a specific marker for lymphatic endothelial cells as described previously.2 ,19–24 LYVE-1 was detected with a Cy3-conjugated goat antirabbit secondary antibody (Dianova GmbH, Hamburg, Germany). Isotype control was assured with a FITC-conjugated normal rat2A IgG for CD31 and with a normal rabbit IgG for LYVE-1 (both Santa Cruz Biotechnology, Santa Cruz, California, USA).
Whole mounts were analysed with a fluorescence microscope (BX53, Olympus Optical, Hamburg, Germany) and digital pictures were taken with a digital camera (XM10, Olympus, Hamburg, Germany). The areas covered with blood or lymphatic vessels were detected with an algorithm established in the image analysing programme Cell^F (Olympus, Hamburg, Germany) as described previously.19 ,21 Briefly, 9 to 12 images at a 100× magnification were taken of the corneas and automatically assembled to one whole image. Then, an algorithm was used to detect the areas covered with blood or lymphatic vessels in an image-analysis programme (analySIS^B; Soft Imaging System). Briefly, different filters modified grey scale images of whole mounts before analysis. Afterwards, the total area of the cornea was defined along the limbus. The area covered by blood and lymphatic vessels was determined by setting a threshold including the bright vessels and excluding the dark background in the measurements. These areas covered vessels correlated with the total area of the cornea (vessel ratio).
Morphological analysis of corneal CD45 cells
Ten corneas were excised one day after photodynamic therapy of the cornea, 2 weeks after suture placement. Corneal whole mounts of both groups (treatment group with verteporfin n=5; control group with PBS n=5) were fixed with acetone for 15 min, thereafter washed with PBS, followed by a blocking step with a Fc-Block rat antimouse (BD Bioscience Pharmingen, Heidelberg, Germany). Staining with rabbit antimouse LYVE-1 (AngioBio, DelMar, USA) and CD45 rat antimouse (BD Bioscience Pharmingen, Heidelberg, Germany) was performed overnight at 4°C in the dark. After another washing step, samples were incubated with Cy3-conjugated secondary antibody, goat antirabbit (Jackson Immuno Research, Newmarket, UK) to detect CD45 cells; CD45 is a marker, which is expressed by all leucocytes.22 4',6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. Three pictures of each whole mount were taken with the Zeiss Axio Imager fluorescence microscope equipped with ApoTome 2 at the level of lymphatic vessels. Cells were manually counted using ImageJ and the Plugin Cell_Counter (http://rsbweb.nih.gov/ij/download.html; accessed 20 Oct 2013).
Statistical analysis
Statistical analysis was done by Microsoft Excel 2003 and graphs were drawn using Prism6, V.6.02 (GraphPad Software, San Diego, California, USA). A two-tailed unpaired t test was used to detect the difference between the groups.
Results
Effect of PDT after intrastromal injection of verteporfin on corneal lymphangiogenesis and hemangiogenesis
Existing mature lymphatic vessels in the cornea, induced by the inflammatory neovascularisation assay, can significantly be reduced by photodynamic therapy after intrastromal injection of verteporfin (figure 2). Corneas treated with verteporfin, excised one day after PDT, showed 62% regression of lymphatic vessels (p=0.20; treatment group n=5, control group n=5) (figure 2B). Five days after PDT, lymphatic vessels were significantly reduced by 51% (p=0.027; treatment group n=5, control group n=5) (figure 2D) and 10 days after PDT, lymphatic vessel area was significantly reduced by 48% (p=0.0014; treatment group n=5, control group n=5) (figure 2F). In contrast, PDT after intrastromal verteporfin injection had no effect on mature corneal blood vessels (figure 2). Corneal blood vessels were not reduced significantly at any analysed time point (reduction one day after PDT: 22% (p=0.27; treatment group n=5, control group n=5); reduction after 5 days: 24% (p=0.32; treatment group n=5, control group n=5); increase after 10 days: 12% (p=0.36; treatment group n=5, control group n=5). Our results show for the first time a technique to specifically ablate mature lymphatic vessels in the cornea, with no effect on blood vessels (figure 3).
Lymphatic vessels can significantly be regressed without effecting blood vessels by photodynamic therapy (PDT) after intrastromal corneal injection of verteporfin. In all groups blood vessels (A, C, E) showed no significant reduction after PDT. One day after photodynamic therapy lymphatic vessels showed a reduction of 62% (p=0.20; treatment group n=5, control group n=5), after 5 days 51% (p=0.027; treatment group n=5, control group n=5) and after 10 days 48% (p=0.0014; treatment group n=5, control group n=5) when compared with control mice. Data expressed as mean+SEM.
Representative sections of corneal flat mounts after photodynamic therapy (PDT) with intrastromal injection of verteporfin or PBS (control) (magnification ×100). (B, F, J) There is no difference in the presence of blood vessels after intrastromal injection of verteporfin and PDT when compared with control (A, E, I) in all time points. (D, H, L) After treatment with verteporfin corneal lymphatic vessels disappear compared with control mice (C, G, K).
Effect of PDT after intrastromal injection of verteporfin on corneal immune cells
Both groups showed enrichment of CD45 haematopoietic cells in the corneal stroma after corneal suture placement and PDT (figure 4). Cell counts measured in both groups showed no significant difference (verteporfin treated mice: 89.93±16.06 cells/visual field; control mice: 105.80±28.45 cells/visual field; p=0.322) (figure 5).
Representative sections of corneal flat mounts one day after photodynamic therapy (PDT) after intrastromal verteporfin injection shows no significant effect on inflammatory cells (magnification ×200). (A) Control mice after intrastromal injection of PBS showed numerous CD45 cells. (B) Treatment group showed no significant reduction of CD45 cells.
Photodynamic therapy after intrastromal injection of verteporfin has no effect on corneal CD45 cells. One day after photodynamic therapy (PDT) control group treated with PBS (n=5) showed same amount of CD45 cells as group treated with verteporfin (n=5). Data expressed as mean+SEM.
Discussion
Our experiments show for the first time that corneal PDT after an intrastromal injection of the photosensitiser verteporfin can selectively induce regression of corneal lymphatic vessels without affecting blood vessels. This has two important implications:
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PDT after intrastromal injection is the first therapeutic approach that has a specific angioregressive effect on pre-existing mature pathological corneal lymph vessels and does not affect blood vessels in the cornea. As lymphatic vessels play a major role in the induction of immune responses towards inflammatory stimuli2 this could be a promising new minimally invasive preconditioning technique to reduce allograft rejection in high-risk patients.3 The intrastromal photodynamic therapy of the cornea can induce regression of mature lymphatic vessels without the need of an intravenous injection of the photosensitiser and potential severe systemic side effects.25–28
Pathological lymphatic vessels in the cornea drain particles and fluids from the centre to the limbus and from there into venous vessels. As a liposome verteporfin has the ideal molecule size for a specific uptake into the lymphatic system.29–31 Tammela et al could demonstrate that verteporfin is selectively taken up by lymphatic vessels when injected into the skin.11 In accordance with that, we could not observe an effect on pre-existing blood vessels in the cornea in this study.
Thinking about a clinical translation of this concept, a combination of PDT therapy to reduce lymphatics for example, with thermal cautery of clinically visible blood vessels might be feasible.8
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So far, several studies have shown the feasibility to selectively inhibit progressive corneal lymphatics without affecting blood vessels for example, by the use of a small molecule integrin inhibitor, VEGFR3 antibody or sVEGFR2.4 ,32 ,33 Selective pharmacological blockade of corneal lymphangiogenesis has been shown to promote corneal allograft survival. However, immune cells such as antigen presenting cells or macrophages also express receptors for adhesion molecules like integrins, VEGFR2 or VEGFR3. Therefore, the possible beneficial effect of these used lymphangiogenesis inhibitors on corneal allograft survival could be at least partially due to the modulation of immune cells. The specific destruction of corneal lymphatic vessels by PDT without affecting blood vessels and immune cell numbers and potentially their activation status could be a promising model to gain deeper insight into the role of lymphatic vessels in transplantation immunology.
In summary, PDT after intrastromal injection of verteporfin is a novel technique to specifically induce regression of corneal lymphatics without affecting blood vessels.
References
Footnotes
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Contributors FBu undertook the laboratory work and research. YB helped with the study design and protocols. UG helped with imaging of immunohistochemical stainings. DH supervised the laboratory work and helped with the animal model. CC and FBo main-reviewed the article and supervised the research.
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Funding EU (FP7 ‘STRONG’; FP7 COST BM1302 ‘Joining forces in corneal regeneration’), DFG (Cu 47/4-1; Cu 47/6-1).
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Competing interests FBu: none; YB: none; UG: none; DH: none; CC: Gene Signal; FBo: none.
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Provenance and peer review Not commissioned; externally peer reviewed.