Lymphangiogenesis is critically involved in tissue fluid balance, graft rejection, and tumor metastasis. Endogenous regulation of lymphangiogenesis is poorly understood. Herein, we use the lymphatic vessel architecture at the limbal border of the normally avascuLar cornea, a quantitative trait under strong genetic influence, as a model system to identify new candidate genes regulating lymphangiogenesis. Comparing low-lymphangiogenic BALB/cN with high-lymphangiogenic C57BL/6N mice, we performed quantitative trait loci analysis of five phenotypes in a large BALB/cN x C57BL/6N intercross (n = 795) and identified three to eight genome-wide significant loci, the strongest on chromosome 7 containing tyrosinase (Tyr). Tyrosinase-negative mice showed significantly increased limbal lymph vascularized areas, a higher number of lymphatic vessel end points, and branching points and increased inflammation-induced lymphangiogenesis. These findings confirm that tyrosinase is a novel lymphangiogenesis regulator in developmental and inflammatory lymphangiogenesis. Our findings link melanin synthesis with lymphangiogenesis and open new treatment options in lymphangiogenesisrelated diseases.

Background Ophthalmology, principally, is a very successful subdiscipline in medicine. Nonetheless, there are still unmet medical needs which necessitate translational research.Methods The funding instrument of a Research Unit (RU) of the German Research Foundation (DFG) is presented as exemplified by the RU 2240 at the Department of Ophthalmology at the University of Cologne.Results The Research Unit integrates different research groups working on pathologic ocular inflammation, macrophages/microglia and (lymph) angiogenesis to collaborate in a synergistic way. Rotation positions allow young clinicians to rotate into research labs for a defined period of time. A Research Unit is also a powerful strategic tool to strengthen clinical and experimental ophthalmology at individual medical faculties.Conclusions The funding instrument of a Research Unit is highly suitable for fostering translational research in a medical subdiscipline such as ophthalmology, supporting the next generation of (clinician) scientists in ophthalmology and finding new cures for our patients.

The role of IL-10, a primarily anti-inflammatory cytokine, in the regulation of inflammatory lymphangiogenesis is undetermined. Herein, we show that IL-10 modulates corneal Lymphangiogenesis and resolution of inflammation. IL-10 was not expressed in healthy corneas but was up-regulated in inflamed corneas by infiltrating macrophages. Macrophages up-regulated the expression of prolymphangiogenic vascular endothelial growth factor-C upon stimulation with IL-10. Consistently, corneal inflammation resulted in reduced expression of vascular endothelial growth factor-C and decreased corneal Lymphangiogenesis in IL-10 deficient mice (IL-10(-/-)). The effect of IL-10 on Lymphangiogenesis was indirect via macrophages, because IL-10 did not directly affect Lymphatic endothelial cells. The expression of proinflammatory cytokines and the numbers of infiltrating macrophages increased and remained elevated in inflamed corneas of IL-10(-/-) mice, indicating that IL-10 deficiency Led to more severe and prolonged inflammation. The corneal phenotype of IL-10 deficient mice was mimicked in mice with conditional deletion of Stat3 in myeloid cells (Lysozyme M Cre mice Stat(fL/fL) mice), corroborating the critical rote of macrophages in the regulation of lymphangiogenesis. Furthermore, Local treatment with IL-10 promoted Lymphangiogenesis and faster egress of macrophages from inflamed corneas. Taken together, we demonstrate that IL-10 indirectly regulates inflammatory corneal Lymphangiogenesis via macrophages. Reduced Lymphangiogenesis in IL-10(-/-) and lysozyme M Cre Stat3(fL/fL) mice is associated with more severe inflammatory responses, whereas IL-10 treatment results in faster resolution of inflammation. IL-10 might be used therapeutically to terminate pathological inflammation.

Background: In the murine cornea, which is an established model for analyzing pathologic lymphatic vessel growth, phenotypic heterogeneity of the endogenous lymphatic vessels in the limbus of the cornea was previously described. In this study, the cornea of BALB/c, C57BL/6, and FVB mice with different limbal lymphangiogenic phenotypes was analyzed to identify novel candidates potentially influencing lymphatic vessel growth. Methods and Results: Pathway specific expression analysis of the cornea was performed to identify novel candidate genes. Corneal protein expression of the respective candidates was analyzed by fluorescent immunohistochemistry. The effect of the candidates on proliferation of human dermal lymphatic endothelial cells (HDLECs) was analyzed by BrdU proliferation ELISA. Thirteen genes were differentially regulated in corneas of mouse strains with more endogenous limbal lymphatic vessels (high-lymphangiogenic) (C57BL/6) compared to mouse strains with less endogenous limbal lymphatic vessels (low-lymphangiogenic) (BALB/c, FVB). Two candidates, Tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10/Trail) and Plasminogen activator, tissue (Plat/tPA) were expressed in the cornea of BALB/c and C57BL/6 mice on the protein level. In vitro, Trail and recombinant tPA inhibited the proliferation of human dermal lymphatic endothelial cells. Conclusion: Molecular analysis of the naive cornea in mouse strains with different limbal lymphatic phenotypes is a valuable model to identify novel endogenous regulators of lymphangiogenesis.

There is currently considerable controversy about existence and classification of "lymphatic vessels" in the eye. Some of the confusion is certainly caused by inappropriate use (or nonuse) of the correct immunohistochemical markers. Many experts in the field expressed the need for a consensus statement, and, in this perspective, authors offer arguments and solutions to reliably continue with immunohistochemical ocular lymphatic research.

LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a homolog of the hyaluronan receptor CD44, and one of the most widely used markers of lymphatic endothelial cells in normal and tumor tissues. However, the physiologic role of LYVE-1 in the lymphatic system still remains unclear. It is well established that fibroblast growth factor 2 (FGF2) induces lymphangiogenesis. Based on the known interaction between FGF2 and CD44 and based on the structural similarity of CD44 and LYVE-1, we investigated whether FGF2 might interact with LYVE-1. We found that FGF2 is able to bind LYVE-1 using AlphaScreen, or after surface-immobilization or in solution. FGF2 binds to LYVE-1 with a higher affinity than any other known LYVE-1–binding molecules, such as hyaluronan or PDGF-BB. Glycosylation of LYVE-1 is important for FGF2 binding. Furthermore, FGF2 interacts with LYVE-1 when overexpressed in CHO cells. Soluble LYVE-1 and knockdown of LYVE-1 in lymphatic endothelial cells impaired FGF2 signaling and functions. In addition, FGF2 but not VEGF-C-induced in vivo lymphangiogenesis, was also inhibited. Conversely, FGF2 also modulates LYVE-1 expression in cells and ex vivo. Thus, our data demonstrate a functional relationship to the interaction between FGF2 and LYVE-1.

PURPOSE:Tumor-derived VEGF-A, apart from expediting sufficient vascularization, subsequent tumor growth, and metastatic spread, can act on malignant cells themselves provided that VEGF receptors 1 or 2 (VEGF-R1, -R2) are co-expressed. The study goal was to investigate whether such autocrine VEGF-A signaling exists in uveal melanoma (UM).METHODS:Primary (MEL-270, OM-431) and metastatic (OMM-2.3, OMM-2.5) UM cell lines were analyzed for VEGF-A, VEGF-R1, and VEGF-R2 expression by RT-PCR, ELISA (VEGF-A protein), and immunocytochemistry (VEGF receptors). Proliferation of UM cells incubated with neutralizing anti-VEGF-A antibody bevacizumab (≤ 2.5 mg/mL), or VEGF-A (≤ 100 ng/mL) was assessed by bromodeoxyuridine (BrdU) ELISA. It was measured by real-time PCR, whether VEGF-A (100 ng/mL) modulated the expression ratio of VEGF-A itself and its antiangiogenic antagonist pigment epithelium-derived factor (PEDF).RESULTS:All UM cells expressed VEGF-A, VEGF-R1, VEGF-R2 mRNA, and protein. In each cell line, the proliferation was stimulated by VEGF-A or inhibited by blocking VEGF-A, or both: bevacizumab significantly decreased the proliferation in MEL-270 (P = 0.005), OMM-2.3 (P = 0.001), and OMM-2.5 (P = 0.011). Increased VEGF-A signaling significantly raised the proliferation in MEL-270, OM-431 (P < 0.001, respectively), and OMM-2.3 (P = 0.043) in a dose-dependent manner but did not significantly change the VEGF-A/PEDF mRNA expression ratio.CONCLUSIONS:Autocrine VEGF-A signaling seems to be present in UM, sustaining the proliferation of both primary and metastatic UM cells. Apparently, VEGF-A signaling in UM cells neither acts retroactively on VEGF-A expression, in the sense of a feedback loop, nor contributes to a pro-angiogenic shift of the VEGF-A/PEDF ratio.

The cornea is one of the few tissues which actively maintain an avascular state, i.e. the absence of blood and lymphatic vessels (corneal [lymph]angiogenic privilege). Nonetheless do several diseases interfere with this privilege and cause pathologic corneal hem- and lymphangiogenesis. The ingrowths of pathologic blood and lymphatic vessels into the cornea not only reduce transparency and thereby visual acuity up to blindness, but also significantly increases the rate of graft rejections after subsequent corneal transplantation. Therefore great interest exists in new strategies to target pathologic corneal (lymph)angiogenesis to promote graft survival. This review gives an overview on the vascular anatomy of the normal ocular surface, on the molecular mechanisms contributing to the corneal (lymph)angiogenic privilege and on the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea. In addition we summarize the current preclinical and clinical evidence for three novel treatment strategies against ocular surface diseases based on targeting pathologic (lymph)angiogenesis: (a) modulation of the immune responses after (corneal) transplantation by targeting pathologic (lymph)angiogenesis prior to and after transplantation, (b) novel concepts against metastasis and recurrence of ocular surface tumors such as malignant melanoma of the conjunctiva by anti(lymph)angiogenic therapy and (c) new ideas on how to target ocular surface inflammatory diseases such as dry eye by targeting conjunctival and corneal lymphatic vessels. Based on compelling preclinical evidence and early data from clinical trials the novel therapeutic concepts of promoting graft survival, inhibiting tumor metastasis and dampening ocular surface inflammation and dry eye disease by targeting (lymph)angiogenesis are on their way to translation into the clinic.

Lacrimal gland inflammation during autoimmune Sjögren's syndrome (SS) leads to ocular surface inflammation - Keratoconjunctivitis sicca (KCS). This condition afflicts both the cornea and conjunctiva that form the ocular surface. Thrombospondin-1 (TSP-1) deficiency in mice results in lacrimal gland and corneal inflammation that resembles the human disease. In this study we report conjunctival pathology in this mouse model of SS. We found that TSP-1 null mice develop inflammation in the conjunctiva and associated loss of goblet cell function similar to that seen in patients with SS. Increased expression of Th1 (IFN-γ, TNF-α) and Th17 (IL-6, IL-17A) inflammatory cytokines and related transcription factors (Tbet and RORγt) were detected in TSP-1 null conjunctiva as well as their draining lymph nodes (LNs). The conjunctival inflammation was also accompanied by an increase in local lymphatic vessels. Interestingly, migration of antigen-bearing dendritic cells (DCs) from the ocular surface to the LNs was dependent on the TSP-1 available in the tissue. These results not only reveal potential immunopathogenic mechanisms underlying KCS in SS but also highlight the therapeutic potential of TSP-1.

PURPOSE:Ranibizumab (Lucentis(®) ) is a Fab-Fragment of a recombinant, humanized, monoclonal VEGF (anti-vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied.METHODS:The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme-linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE-1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F.RESULTS:An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea.CONCLUSION:Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications.

Objectives To analyze whether topical application of corticosteroids inhibits inflammatory corneal lymphangiogenesis and to study the potential underlying antilymphangiogenic mechanisms. Methods Inflammatory corneal neovascularization was induced by suture placement, and the corneas were then treated with topical fluorometholone, prednisolone acetate, or dexamethasone sodium phosphate. After 1 week, the corneas were stained with lymphatic vessel endothelial hyaluronan receptor 1 for detection of pathological corneal lymphangiogenesis. The effect of these corticosteroids on macrophage recruitment was assessed via fluorescence-activated cell sorting analysis. The effect of these corticosteroids on proinflammatory cytokine expression by peritoneal exudate cells was tested via real-time polymerase chain reaction. Furthermore, the effect of steroid treatment on the proliferation of lymphatic endothelial cells was assessed via enzyme-linked immunosorbent assay. Results Treatment with corticosteroids resulted in a significant reduction of inflammatory corneal lymphangiogenesis. The antilymphangiogenic effect of fluorometholone was significantly weaker than that of prednisolone and dexamethasone. Corneal macrophage recruitment was also significantly inhibited by the application of topical steroids. Treatment of peritoneal exudate cells with corticosteroids led to a significant downregulation of the RNA expression levels of tumor necrosis factor and interleukin 1β. Additionally, proliferation of lymphatic endothelial cells was also inhibited. Conclusions Corticosteroids are strong inhibitors of inflammatory corneal lymphangiogenesis, with significant differences between various corticosteroids in terms of their antilymphangiogenic potency. The main mechanism of the antilymphangiogenic effect seems to be through the suppression of macrophage infiltration, proinflammatory cytokine expression, and direct inhibition of proliferation of lymphatic endothelial cells. Clinical Relevance Steroids block corneal lymphangiogenesis, the main risk factor for immune rejections after corneal transplantation. The different antilymphangiogenic potency of these drugs should be taken into account when using steroids in clinical practice (eg, after keratoplasty).

Purpose.: To analyze whether insulin receptor substrate (IRS-1) is involved in lymphatic vessel development and whether IRS-1 blockade can inhibit lymphangiogenesis in vivo. Methods.: The impact of IRS-1 blockade by GS-101 (Aganirsen), an antisense oligonucleotide against IRS-1, on lymphatic endothelial cell (LEC) proliferation was assessed by ELISA. Furthermore, the effect of IRS-1 blockade on prolymphangiogenic growth factor expression by LECs and macrophages (peritoneal exudate cells) was tested by real-time PCR. The mouse model of inflammatory corneal neovascularization was used to analyze the effect of IRS-1 blockade in vivo: after corneal suture placement, mice were treated with GS-101 eye drops (twice daily afterwards for 1 week, 5 μL per drop; 50, 100, or 200 μM). Afterward, corneal wholemounts were prepared and stained for blood and lymphatic vessels. Results.: Blockade of IRS-1 by GS-101 inhibited LEC proliferation dose dependently. GS-101 led to decreased VEGF-A expression levels in LECs, whereas VEGF-C, VEGF-D, and VEGFR3 showed no significant change. In macrophages, VEGF-A expression levels were also inhibited by IRS-1 blockade. Additionally, GS-101 strongly inhibited macrophage-derived VEGF-C, VEGF-D, and VEGFR3 expression. In vivo, corneal hemangiogenesis was significantly inhibited when used at a concentration of 200 μM (by 17%; P < 0.01). Corneal lymphangiogenesis was significantly inhibited when used at a dose of 100 μM (by 21%; P < 0.01), and the highest used dose (200 μM) showed an even stronger inhibition (by 28%; P < 0.001). Conclusions.: Blockade of IRS-1 inhibits not only hemangiogenesis but also lymphangiogenesis. To the authors' knowledge, this is the first evidence that IRS-1 is involved in the molecular pathway leading to lymphangiogenesis.