Birgit Regenfuss

Novel anti(lymph)angiogenic treatment strategies for corneal and ocular surface diseases

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.

Suppression of Inflammatory Corneal Lymphangiogenesis by Application of Topical Corticosteroids

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).

Evidence for the interaction of fibroblast growth factor-2 with the lymphatic endothelial cell marker LYVE-1

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.

Sufficient Evidence for Lymphatics in the Developing and Adult Human Choroid

We read with interest the recent article by Koina et al.1 suggesting evidence for the presence of lymphatic vessels in the developing and adult human choroid. However, this study does not meet the recently published consensus criteria on the immunohistochemical detection of ocular lymphatic vessels,2 and therefore, in our opinion, requires critical revision. First, appropriate positive and unequivocal negative controls are not presented in the study of Koina et al. In particular, when describing novel anatomical structures for the first time, and in order to change an existing dogma, a detailed documentation of blood and lymphatic vessel detection in the control tissue is mandatory. The provided supplementary data do not fulfill these criteria. Second, the immunohistochemical marker panel used is critical. Endomucin does not represent an established lymphatic marker,3,4 but is rather expressed by “endothelial cells along the whole vascular tree including lymphatic vessels.”5 Thus, an unequivocal discrimination between blood and lymphatic vessels is impossible with this marker. A further discrepancy is the use of the transcription factor prospero-related homebox gene-1 (Prox-1) as an extranuclear lymphatic endothelial precursor marker. Although reports of the extranuclear presence of PROX-1 in cell types other than lymphatic endothelium exist,6–8 PROX-1 clearly shows a nuclear expression in lymphatic endothelia in human,9 as well as mouse10 and avian,11 embryos, retaining its nuclear localization into adulthood.12–14 On the other hand, it is not clear why lymphatic endothelial surface markers, such as podoplanin, lymphatic vascular endothelial-specific hyaluronic acid receptor-1 (LYVE-1), and the vascular endothelial marker CD34 display nuclear expression in this study. Additionally, the only lymphatic endothelial cell marker used in whole mounts is VEGFR-3, which is also expressed in fenestrated blood vessels, and, as such, also in the choriocapillaris.15,16 Morphologically, the supposed lymphatic VEGFR-3–positive vessels are indistinguishable from the honeycomb-like lobular pattern of the choriocapillaris.17 Furthermore, the study of Koina et al. includes a blatant inconsistency in the use and documentation of immunohistochemical markers between fetal and adult eyes. Although one has to acknowledge that certain lymphatic markers might be expressed during embryogenesis, this pattern easily changes during maturation.18 Therefore, such an approach would require extensive comparison of the same markers in different ages, thus representing an extensive survey in its own right. However, this is not the case in the study of Koina et al. Third, the ultrastructural study would be greatly strengthened by immunoelectron microscopy. Indeed, anchoring filaments with a diameter of 40 to 100 A—becoming readily identifiable only at magnifications of 40,000× to 50,000×—are present in lymphatics,19 but could be easily present in the choroid as well without any association to lymphatic vessels,20–22 particularly in aged eyes with typical alterations of the extracellular matrix. For this purpose, as well as for ruling out Weibel-Palade bodies, serial ultrathin sectioning with appropriate labeling would be necessary. Despite possible postmortem tissue alterations, numerous previous studies successfully applied different detection systems for ultrastructural investigations using ocular human donor tissue.23–29 A limited use of immunomarkers for these investigations, as claimed, seems therefore not justified. In regard to the above-mentioned criticisms, the evidence presented in the study of Koina et al. does not justify the hypothesized paradigm shift that functional lymphatic vessels are present in the human choroid. Rather, the findings of Koina et al. confirm previous reports of net-like structures with a “pseudo-vessel” appearance in the human choroid endowed with lymphatic vascular precursor cells (represented as LYVE-1+ macrophages).25 Those “atypical” lymphatic-like cells (i.e., endothelial cells with divergent or uncommon immunohistochemical phenotypes) may also exist in other parts of the eye. For example, the endothelial cells of Schlemms canal display many, but not all, features of terminally differentiated lymphatic endothelial cells, including responsiveness to VEGF-C–induced lymphangiogenesis.30 In closing, we acknowledge that the work of Koina et al. is a further contribution to our understanding of the choroid, but although the existence of lymphatics in the human choroid cannot be ruled out per se, because of the aforementioned points and the sheer volume of evidence to date, we maintain that the inner human eye and in particular the choroid should still be considered an immune-privileged site devoid of lymphatic vessels. Further unequivocal evidence of “typical lymphatic vessels” in the human choroid is still missing.

Neue Therapieansätze bei entzündlichen Augenerkrankungen durch Modulation von Lymphangiogenese und zellulärer Immunität: Die DFG-Forschergruppe 2240 stellt sich vor

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.