title: Doxycycline inhibits inflammation-induced lymphangiogenesis in mouse cornea by multiple mechanisms.
abstract: Lymphangiogenesis is significantly involved in the pathogenesis of diseases, including graft rejection, cancer metastasis and various inflammatory conditions. The inhibition of lymphangiogenesis has become a new therapeutic target for the treatment of these diseases. Here, we explored the anti-lymphangiogenic effects of doxycycline in inflammation-induced lymphangiogenesis (ILA) in the cornea and the underlying mechanisms. In the present study, mice with ILA of the cornea were treated with topical doxycycline (0.1%) or vehicle control. Lymphangiogenesis was quantified using corneal immunostaining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Human dermal lymphatic endothelial cells (HDLECs) and a murine macrophage cell line (RAW264.7) were used to further explore the underlying mechanisms of doxycycline-mediated anti-lymphangiogenesis in vitro. Our results showed that doxycycline treatment dramatically inhibited ILA in the mouse cornea (p<0.001), with a significant decrease in vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 signalling, macrophage infiltration and inflammatory cytokine expression. Doxycycline also significantly inhibited VEGF-C-induced HDLEC proliferation in vitro by modulating the PI3K/Akt/endothelial nitric oxide (NO) synthase (eNOS) pathway and significantly suppressed interleukin-1β (IL-1β), TNF-α and VEGF-C production in the RAW264.7 cell line by modulating the PI3K/Akt/nuclear factor-kappaB (NF-κB) pathway. Additionally, doxycycline treatment dramatically reduced the phosphorylation of NF-κBp65, Akt and eNOS in ILA and significantly inhibited matrix metalloproteinases (MMPs) activity in vitro and in ILA. In conclusion, doxycycline inhibited ILA, possibly through suppression of VEGF-C signalling, macrophage function and MMPs activity. This observation suggests that doxycycline is a potential therapeutic agent for lymphangiogenesis-related diseases.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; Hebei Provincial Key Laboratory of Ophthalmology, Hebei Provincial Eye institute, Hebei Eye Hospital, Xingtai, China.
authors.forename: Longhui
authors.initials: L
authors.lastname: Han
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Wenru
authors.initials: W
authors.lastname: Su
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Sujuan
authors.initials: S
authors.lastname: Qiu
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liang
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology*; D005260:Female; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D007249:Inflammation / Q000473:pathology; D008070:Lipopolysaccharides / Q000633:toxicity; D042583:Lymphangiogenesis / Q000187:drug effects*; D008213:Lymphocyte Activation / Q000187:drug effects; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D042582:Vascular Endothelial Growth Factor C / Q000378:metabolism / Q000494:pharmacology; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000378:metabolism
chemical_list: D004318:Doxycycline; D006023:Glycoproteins; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D008070:Lipopolysaccharides; D026901:Membrane Transport Proteins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D042582:Vascular Endothelial Growth Factor C; D040321:Vascular Endothelial Growth Factor Receptor-3; C492273:LYVE1 protein, mouse