Deniz Hos

Cutting Edge: Lymphatic Vessels, Not Blood Vessels, Primarily Mediate Immune Rejections After Transplantation

The purpose of this study was to determine the relative importance of blood vessels (hemangiogenesis) versus lymphatic vessels (lymphangiogenesis) in mediating immunological responses after transplantation. Using the murine model of corneal transplantation, graft survival was compared in differentially prevascularized and avascular recipient beds. Donor corneas (C57BL/6) were transplanted into uninflamed or inflamed avascular, prehemvascularized only or prehemvascularized and prelymphvascularized recipient murine eyes (BALB/C). Selective inhibition of lymphangiogenesis was achieved using antivascular endothelial growth factor receptor 3 Abs and anti-integrin α5 small molecules. Grafts placed into only prehemvascularized recipient beds had a similarly good graft survival compared with grafts placed into completely avascular, normal recipients, whereas the pre-existence of lymphatic vessels significantly deteriorated corneal graft survival (p < 0.05). Lymphatic vessels seem to contribute significantly to graft rejection after (corneal) transplantation. That may allow for selective, temporary, perioperative antilymphangiogenic treatment to promote graft survival without affecting blood vessels, even after solid organ transplantation.

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.

Improved semiautomatic method for morphometry of angiogenesis and lymphangiogenesis in corneal flatmounts

Purpose of the study was to describe a novel semiautomatic, quantitative image analysis method based on threshold analysis for morphometry of corneal (lymph)angiogenesis and to test its validity, reliability and objectivity. Murine corneas were vascularized by using a suture-induced neovascularization assay. For immunohistochemistry, flatmounts of the vascularized corneas were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry of corneal hem and lymphangiogenesis was performed semi-automatically on digital images using image analysis software. Data were analyzed by a paired t-test, intraclass-correlation and systemic difference analysis compared to a manual method. The semiautomatic method based on threshold analysis was more valid in measuring the area covered by blood or lymphatic vessels. Both methods had a good reproducibility with respect to both vessel types (blood vessels: manual: 0.969, semiautomatic: 0.982; lymphatic vessels: manual: 0.951, semiautomatic: 0.966), whereas the systemic difference was significant for both groups measuring lymphatic vessels (manual: p<0.003; semiautomatic: p<0.035) and for the manual method measuring blood vessels (manual: p<0.0001; semiautomatic: p<0.419). The new semiautomatic morphometry method based on threshold analysis provides higher accuracy, is more valid than and at least as reproducible and objective as the manual outlining method. Therefore the semiautomatic method can be used to detect even small effects on hem and lymphangiogenesis in murine corneal flatmounts with greater precision.

Transient postoperative vascular endothelial growth factor (VEGF)-neutralisation improves graft survival in corneas with partly regressed inflammatory neovascularisation

Background: High-risk keratoplasties are usually performed after an uninflamed and quiescent interval in corneas with partly regressed blood and lymphatic vessels. We analysed whether the inhibition of post-keratoplasty revascularisation in mice with partly regressed corneal vessels (“intermediate-risk”) improves graft survival. Methods: Three interrupted stromal sutures (11-0) in corneas of Balb/c mice (6–8 weeks old) were placed for 6 weeks. Six months after suture removal, penetrating keratoplasty was performed with C57BL/6 donors. The treatment group received a vascular endothelial growth factor-A specific cytokine trap (VEGF Trap) intraperitoneally at days 0, 4, 7 and 14 after keratoplasty (25 mg/kg per mouse; controls received equal amounts of Fc protein). Pathological haemangiogenesis and lymphangiogenesis prior to as well as 3 days or 8 weeks after keratoplasty and graft survival were analysed. Results: Three days after keratoplasty corneal revascularisation was sufficiently reduced by VEGF Trap (haem-vascularised areas 42.7% reduction; lymph-vascularised areas 54.7% reduction). Survival proportions 8 weeks after keratoplasty were 36% in the treatment group compared with 9% in the control group (n = 11; p<0.05). At that time no differences in haemangiogenesis or lymphangiogenesis were observed between the two groups. Conclusion: Early transient postoperative induction of haemangiogenesis and lymphangiogenesis and reformation of regressed corneal blood and lymphatic vessels are important for transplant rejections after “intermediate-risk” corneal transplantation.

Split Cornea Transplantation : Relationship between Storage Time of Split Donor Tissue and Outcome

PURPOSE To analyze the relationship between storage time of split donor tissue and outcomes after deep anterior lamellar keratoplasty (DALK) and Descemets membrane endothelial keratoplasty (DMEK). DESIGN Retrospective analysis of a nonrandomized, consecutive, interventional case series. PARTICIPANTS One hundred ten eyes with anterior stromal disease suitable for DALK and 110 eyes with endothelial disease suitable for DMEK underwent surgically successful split cornea transplantation combining both procedures within 7 days after splitting. METHODS Split donor storage times (splitting to grafting) and total storage times (death to grafting) were correlated with the 1-year functional and morphologic outcomes after DALK and DMEK surgery using a Spearman correlation coefficient and a Mann-Whitney U test. MAIN OUTCOME MEASURES Best spectacle-corrected visual acuity (BSCVA), endothelial cell density, and complication rates within 12 months of follow-up. RESULTS The mean split donor storage time was 35 ± 47 hours (range, 0-162 hours) after splitting for anterior donor grafts and 21 ± 40 hours (range, 0-158 hours) for posterior grafts. The mean total storage time was 352 ± 108 hours (range, 108-678 hours) for anterior lamellas and 339 ± 109 hours (range, 96-630 hours) for posterior lamellas. One year after DALK, the mean BSCVA was 20/30 (range, 20/50-20/20), endothelial cell loss was 8% (range, 2%-16%), and the complication rate (Descemets folds, epitheliopathy, loose sutures) was 18%. One year after DMEK, the mean BSCVA was 20/25 (range, 20/40-20/16), endothelial cell loss was 41% (range, 17%-63%), and the complication rate (partial graft detachment) was 62%. For DALK and DMEK, no significant association was observed between split donor storage time as well as total storage time and BSCVA (P ≥ 0.409), endothelial cell loss (P≥0.236), or complication rate (P ≥ 0.647) within 1 year of follow-up. CONCLUSIONS Anterior and posterior donor tissue may be stored safely for up to 1 week in organ culture before use in DALK and DMEK surgery. This simplifies the clinical feasibility of split cornea transplantation to reduce donor shortage and cost in corneal transplantation in the future. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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).

Stem cell factor Sox2 and its close relative Sox3 have differentiation functions in oligodendrocytes

Neural precursor cells of the ventricular zone give rise to all neurons and glia of the central nervous system and rely for maintenance of their precursor characteristics on the closely related SoxB1 transcription factors Sox1, Sox2 and Sox3. We show in mouse spinal cord that, whereas SoxB1 proteins are usually downregulated upon neuronal specification, they continue to be expressed in glial precursors. In the oligodendrocyte lineage, Sox2 and Sox3 remain present into the early phases of terminal differentiation. Surprisingly, their deletion does not alter precursor characteristics but interferes with proper differentiation. Although a direct influence on myelin gene expression may be part of their function, we provide evidence for another mode of action. SoxB1 proteins promote oligodendrocyte differentiation in part by negatively controlling miR145 and thereby preventing this microRNA from inhibiting several pro-differentiation factors. This study presents one of the few cases in which SoxB1 proteins, including the stem cell factor Sox2, are associated with differentiation rather than precursor functions.

Inflammatory corneal (lymph)angiogenesis is blocked by VEGFR-tyrosine kinase inhibitor ZK 261991, resulting in improved graft survival after corneal transplantation.

PURPOSE To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P < 0.001; ZK991 by 53%, P < 0.001) and lymphangiogenesis (PTK/ZK by 70%, P < 0.001; ZK991 by 71%, P < 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P < 0.001; ZK991, P < 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P < 0.02). CONCLUSIONS Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation.

Immunreaktionen nach DMEK, DSAEK und DALK

PURPOSE The aim of this study is to describe incidence, diagnosis and therapy for endothelial immune reactions after modern lamellar corneal transplantat surgery (DMEK, DSAEK, DALK). METHODS A PubMed-based literature review and our own clinical and experimental data are evaluated. RESULTS There is no longer an endothelial immune reaction after DALK for keratoconus. DMEK significantly reduces the risk for endothelial immune reactions after surgery for Fuchs dystrophy. CONCLUSIONS Modern lamellar corneal transplant techniques such as DALK and DMEK have nearly abolished the risk for endothelial immune reactions in the avascular recipient bed.

Antilymphangiogenic therapy to promote transplant survival and to reduce cancer metastasis: what can we learn from the eye?

The lymphatic vasculature is - amongst other tasks - essentially involved in inflammation, (auto)immunity, graft rejection and cancer metastasis. The eye is mainly devoid of lymphatic vessels except for its adnexa, the conjunctiva and the limbus. However, several pathologic conditions can result in the secondary ingrowth of lymphatic vessels into physiologically alymphatic parts of the eye such as the cornea or the inner eye. Therefore, the cornea has served as an excellent in vivo model system to study lymphangiogenesis, and findings from such studies have substantially contributed to the understanding of central principles of lymphangiogenesis also with relevance outside the eye. Grafting experiments at the cornea have been extensively used to analyze the role of lymphangiogenesis in transplant immunology. In this regard, we recently demonstrated the crucial role of lymphatic vessels in mediating corneal allograft rejection and could show that antilymphangiogenic therapy increases graft survival. In the field of cancer research, we recently detected tumor-associated lymphangiogenesis in the most common malignant tumors of the eye, such as conjunctival carcinoma and melanoma, and ciliochoroidal melanoma with extraocular extension. These neolymphatics correlate with an increased risk of local recurrence, metastasis and tumor related death, and may offer potential therapeutic targets for the treatment of these tumors. This review will focus on corneal and tumor-associated ocular lymphangiogenesis. First, we will describe common experimentally used corneal lymphangiogenesis models and will recapitulate recent findings regarding the involvement of lymphatic vessels in corneal diseases and transplant immunology. The second part of this article will summarize findings about the participation of tumor-associated lymphangiogenesis in ocular malignancies and their implications for the development of future therapeutic strategies.