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Anna-Karina B. Maier, Sabrina Klein, Norbert Kociok, Aline I. Riechardt, Enken Gundlach, Nadine Reichhart, Olaf Strauß, Antonia M. Joussen; Netrin-4 Mediates Corneal Hemangiogenesis but Not Lymphangiogenesis in the Mouse-Model of Suture-Induced Neovascularization. Invest. Ophthalmol. Vis. Sci. 2017;58(3):1387-1396. doi: https://doi.org/10.1167/iovs.16-19249.
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Netrin-4, a secreted protein, is found in the basement membrane of blood vessels and acts as a key regulator of angiogenesis. Here we investigated the role of Netrin-4 in the mouse-model of suture-induced corneal hem- and lymphangiogenesis.
Corneal hem- and lymphangiogenesis were induced in Netrin-4-deficient (Ntn4−/−) and wild-type (WT) mice by placing three 11-0 nylon sutures intrastromally. Fourteen days after suturing, the vascularized area was analyzed via corneal flat mount immunohistochemistry. Messenger RNA levels for VEGF-A, VEGF-C, Lyve-1, Netrin-4, Unc5H2, “deleted in colon cancer” receptor, and Neogenin in treated and nontreated mouse corneas, cultured human corneal keratocytes (HCK) and epithelial cells (HCEC+HCET) were analyzed by quantitative PCR.
In wild-type mice, Netrin-4 mRNA expression in the cornea decreased in growing corneal neovascularization after suturing. Correspondingly, Ntn4−/− mice showed an increased vascularized area compared to that in WT mice. Expression of VEGF-A mRNA was higher in Ntn4−/− versus WT mice. There was no Netrin-4 expression in lymphatic vessels and the area of lymphatic vascularization did not differ between Ntn4−/− and WT mice, nor did expression of VEGF-C and Lyve-1 mRNA. Human corneal epithelial cells showed mainly Netrin-4 mRNA expression, which increased after stimulation, while HCK demonstrated Unc5H2 mRNA expression. Expression of VEGF-A, Netrin-4, Unc5H2, and Neogenin mRNA in HCEC and HCK did not differ significantly between the serum-free condition and VEGF-A or Netrin-4 stimulation.
Absence of Netrin-4 increased corneal hemangiogenesis but not lymphangiogenesis in the mouse-model of suture-induced neovascularization. Netrin-4 acted as an antiangiogenic factor in the cornea, with which the healthy cornea is enriched via its expression by corneal epithelial cells.
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