Critical Role of TNF-alpha-Induced Macrophage VEGF and iNOS Production in the Experimental Corneal Neovascularization
ABSTRACT We evaluated the roles of tumor necrosis factor (TNF)-α in alkali-induced corneal neovascularization (CNV).
CNV was induced by alkali injury and compared in wild-type (WT) BALB/c mice, and TNF receptor 1-deficient (TNF-Rp55 KO) counterparts, or in mice treated with TNF-α antagonist and recombinant TNF-α. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by real-time PCR and immunohistochemical analysis, respectively.
Alkali injury augmented the intraocular mRNA expression of TNF-α and its receptor, together with a transient macrophage and neutrophil infiltration. Compared to WT mice, TNF-Rp55 KO mice exhibited reduced CNV. Intraocular F4/80-positive macrophages and Ly-6G-positive neutrophils infiltration did not change in KO mice compared to WT mice after the injury. Alkali injury induced a massively increased intraocular mRNA expression of angiogenic factors, including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, E-selectin, and intercellular adhesion molecule (ICAM)-1 in WT mice, whereas these increments were retarded severely in KO mice. Immunofluorescence analysis demonstrated that F4/80-positive cells expressed VEGF and iNOS. Moreover, TNF-α enhanced VEGF and iNOS expression by peritoneal macrophage from WT, but not KO mice. Topical application of TNF-α antagonist reduced CNV, while topical application of recombinant TNF-α enhanced it.
TNF-Rp55-KO mice exhibited impaired alkali-induced CNV through reduced intracorneal infiltrating macrophage VEGF and iNOS expression.
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ABSTRACT: Histone deacetylases (HDACs) regulate gene transcription by modifying the acetylation of histone and nonhistone proteins. Deregulated expression of HDACs has been implicated in tumorigenesis and angiogenesis. In this study, we examined the effect of suberoylanilide hydroxamic acid (SAHA), a potent inhibitor of HDACs, on inflammatory corneal angiogenesis. In a mouse model of alkali-induced corneal neovascularization (CNV), topical application of SAHA to the injured corneas attenuated CNV. In addition, in vivo treatment with SAHA downregulated the expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGFβ1), and epidermal growth factor (EGF), but upregulated the expression of the anti-angiogenic factors thrombospondin (TSP)-1, TSP-2, and ADAMTS-1 in the injured corneas. Furthermore, SAHA inhibited the expression of pro-angiogenic factors, migration, proliferation, and tube formation by human microvascular endothelial cells (HEMC-1) in vitro. These data indicate that SAHA has therapeutic potential for CNV.Canadian Journal of Physiology and Pharmacology 10/2014; 92(10):879-885. DOI:10.1139/cjpp-2014-0117 · 1.56 Impact Factor
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ABSTRACT: HSV-1 is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously identified HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of VEGFA dependent upon expression of VEGFR2 during acute infection. We hypothesized other factors may be involved in lymphangiogenesis with pro-inflammatory cytokines as the leading candidates. In the absence of infection or inflammation, intrastromal administration of TNF-α coupled with VEGFA elicited lymphatic vessel genesis significantly above either factor alone as well as vehicle control. Consistent with this observation, anti-TNF-α Ab blocked HSV-1-mediated corneal lymphangiogenesis within the first five days post infection. However, TNF-α deficient (TNF-α(-/-)) mice displayed a similar level of corneal vessel growth as wild type (WT) controls. To investigate the likely redundant nature of cytokines, PCR array analysis of HSV-1-infected TNF-α(-/-) mice revealed several factors elevated above that found in HSV-1-infected WT mice including IL-1β, platelet-derived growth factor, angiopoietin 2, insulin-like growth factor 2, and IL-6. Subconjunctival administration of neutralizing Ab to IL-6 blocked lymphangiogenesis in TNF-α(-/-) mice. Whereas the cornea levels of IL-6 were significantly reduced, there was no appreciable change in the level of IL-1β or other pro-angiogenic factors analyzed. Collectively, the results suggest in addition to VEGFA, TNF-α and IL-6 promotes and likely synergize with VEGFA in corneal lymphangiogenesis during acute HSV-1 infection.
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ABSTRACT: Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-α, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-α, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-α at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-α, CCL2, CCL3, CXCL2, or DLL4.Experimental Eye Research 01/2014; 125:118–127. · 3.02 Impact Factor