Tyrosinase Is a Novel Endogenous Regulator of Developmental and Inflammatory Lymphangiogenesis.

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 × 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 lymphangiogenesis-related diseases.

IL-10 Indirectly Regulates Corneal Lymphangiogenesis and Resolution of Inflammation via Macrophages.

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 Stat3(fl/fl) mice), corroborating the critical role 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.

The Naïve Murine Cornea as a Model System to Identify Novel Endogenous Regulators of Lymphangiogenesis: TRAIL and rtPA.

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.