Abstract
Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.
Highlights
Corneal neovascularization and lymphangiogenesis can be induced by various triggers including limbal insufficiency, inflammation, trauma or surgical manipulations [1,2]
Tumour necrosis factor α (TNFα)-/- mice showed more prominent central stromal neovascularization, accompanied by increased expression of transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF)-A compared with wild-type mice [2]
Comparable to the human tissue, no co-localization was found between the receptors and the F4/80 positive macrophages (Fig 1B and 1C). mRNA expression of both receptors Tumor necrosis factor (TNF)-Rp55 and TNF-Rp75 can be detected in the wildtype as well as in the TNF-Rp55d and TNF-Rp75d mice
Summary
Corneal neovascularization and lymphangiogenesis can be induced by various triggers including limbal insufficiency, inflammation, trauma or surgical manipulations [1,2]. Cytokines and growth factors orchestrate the cells involved in the development of new blood and lymph vessels Major regulators of both inflammation-driven neovascularization and lymphangiogenesis are growth factors of the vascular endothelial growth factor (VEGF) family (VEGF A, C and D) [1,5]. TNFα-/- mice showed more prominent central stromal neovascularization, accompanied by increased expression of transforming growth factor (TGF)-β1 and VEGF-A compared with wild-type mice [2]. Concerning the cornea, Lu et al demonstrated in the alkali-burn model that TNF-Rp55d exhibited impaired corneal neovascularization through reduced expression of VEGF-A and iNOS by infiltrating macrophages [7]. In the present study we assessed the role of TNF-Rp55 and TNF-Rp75 in the model of suture-induced inflammatory corneal neovascularization and lymphangiogenesis using TNF-Rp55 deficient mice (TNF-Rp55d) and TNF-Rp75 deficient mice (TNF-Rp75d). We compared blood- and lymph vessel growth as well as TNFα expression
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