Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis.
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Ocular infection with HSV may result in the blinding immunoinflammatory lesion stromal keratitis (SK). This represents a CD4+ T cell-mediated immunopathologic lesion in both humans and a mouse model. Early events in the pathogenesis that set the stage for SK are poorly understood. The present study evaluates the role of IL-1 using a transgenic mouse that overexpresses the IL-1 receptor antagonist (IL-1ra) protein. Such transgenic mice were markedly resistant to SK compared with IL-1ra(-/-) and C57BL/6 control animals. The resistance was shown to be the consequence of reduced expression of molecules such as IL-6, macrophage-inflammatory protein-2, and vascular endothelial growth factor, normally up-regulated directly or indirectly by IL-1. A critical event impaired in IL-1ra transgenic mice was vascular endothelial growth factor production with a consequent marked reduction in angiogenesis, an essential step in SK pathogenesis. Targeting IL-1 could prove to be a worthwhile therapeutic approach to control SK, an important cause of human blindness. (+info)
Roles of thrombospondin-1 and -2 in regulating corneal and iris angiogenesis.
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PURPOSE: Thrombospondin (TSP)-1 and -2 are important antiangiogenic factors thought to be involved in maintaining corneal avascularity (angiogenic privilege). This study was undertaken to investigate whether deficiencies of these factors altered developmental and inflammation-induced angiogenesis in the cornea and developmental angiogenesis of the iris of mice. METHODS: Expression of TSP-1 and -2 mRNA and protein was assayed in cornea and iris stroma by RT-PCR and Western blot. Corneas and irides of TSP-1(-/-), TSP-2(-/-), and TSP-1,2(-/-) mice aged 2, 3, and 6 months, and wild-type control mice, were analyzed for spontaneous angiogenesis biomicroscopically, histologically, and with CD31 immunohistochemistry. The mouse model of suture-induced, inflammatory corneal neovascularization was used to evaluate the lack of TSP-1,2 and both TSPs on induced-corneal angiogenesis. Seven days after intrastromal placement of three 11-0 sutures, vascularized areas were analyzed morphometrically on CD31-stained corneal flatmounts. RESULTS: Corneas and irises from normal mouse eyes constitutively expressed TSP-1 and -2 mRNAs and proteins. Corneas of TSP-1(-/-), -2(-/-), and -1,2(-/-) mice displayed no evidence of spontaneous developmental-postnatal angiogenesis, although irises of these mice contained significantly increased iris vessel density compared with wild-type animals (P < 0.01). One week after suturing, corneas of all TSP(-/-) mice had significantly greater corneal angiogenesis than those of control mice (P < 0.05). TSP-1(-/-) had a significantly greater effect on induced corneal neovascularization than did TSP-2(-/-), with the opposite being the case in developmental iris angiogenesis (P < 0.01). CONCLUSIONS: Corneal avascularity during development is redundantly regulated, shown by the fact that lack of the antiangiogenic factors TSP-1 and/or -2 resulted in no spontaneous corneal angiogenesis. By contrast, TSP-1, more than TSP-2, helps to suppress inflammation-induced corneal angiogenesis postnatally, implying that angiogenic privilege in the cornea is actively maintained. (+info)
Lymph node removal enhances corneal graft survival in mice at high risk of rejection.
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BACKGROUND: As shown previously, the submandibular (SM) lymph node (LN) is required for priming the immune response during corneal graft rejection. In this study, we wished to determine whether corneal grafts at "high-risk" of rejection were also protected after selective SM LN removal and if so to investigate whether this improved corneal graft survival was due to induction of specific regulatory/suppressor cells or was due to immunological "ignorance". METHODS: Two sets of experiments were performed. (1) Adoptive transfer of possible regulatory splenocytes from mice with long-term accepted corneal graft after SM LN removal. (2) SM LN removal and corneal grafts in "high-risk" hosts, which had been (A) subjected to corneal trauma with vascularization or (B) allosensitized by previous corneal graft or (C) allosensitized by previous skin graft. RESULTS: Adoptive transfer of splenocytes from tolerant mice after SM LN removal did not enhance corneal graft survival in naive recipients (p > 0.05). SM LN removal in mice with corneal vascularization enhanced corneal allograft survival compared to grafted controls with/without vascularization (p < 0.0001). The removal of the SM LN in mice, who had already been allosensitized by a previous corneal graft, did not statistically prolong corneal graft survival (p > 0.05). SM LN removal procedure did not delay rejection of corneal grafts in mice allosensitized by a previous skin transplant with the same strain combination (p > 0.05). CONCLUSION: The results suggest that removal of the SM LN in "high-risk" mice prevents rejection by mechanisms involving immune "ignorance", since prior allosensitization prevents graft acceptance after LN removal. In allosensitized recipients the stronger the allosensitization (skin- vs. corneal graft-presensitization) the greater the possibility of priming for rejection at alternative draining LN sites. (+info)
Activin a in the regulation of corneal neovascularization and vascular endothelial growth factor expression.
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Activin A, a dimeric glycoprotein that belongs to the transforming growth factor-beta superfamily, governs cellular differentiation in a wide variety of models and has been implicated in the regulation of angiogenesis. We examined the role of activin A and its downstream signaling pathway in a murine model of inflammatory corneal neovascularization induced by mechanical injury (debridement), and in vitro in corneal epithelial cells. Activin A expression increased steadily from day 2 until day 8 after mechanical debridement in vivo, paralleling vascular endothelial growth factor (VEGF) expression. Administration of recombinant activin A in mice increased the area of neovascularization, VEGF expression, and the kinase activities of p38 and p42/44 MAPKs after mechanical debridement. Systemic inhibition of activin A in vivo with a neutralizing antibody reduced the area of neovascularization, VEGF expression, and p38 and p42/44 MAPK activity, whereas administration of an isotype-matched control antibody had no effect. In vitro treatment with activin A increased VEGF secretion, as well as p38 and p42/44 MAPK activity in corneal epithelial cells, whereas concurrent administration of specific inhibitors of p38 or p42/44 MAPK abolished the stimulatory effect of activin A on VEGF production. We conclude that activin A stimulates inflammatory corneal angiogenesis by increasing VEGF levels through a p38 and p42/44 MAPK-dependent mechanism. (+info)
VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.
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Lymphangiogenesis, an important initial step in tumor metastasis and transplant sensitization, is mediated by the action of VEGF-C and -D on VEGFR3. In contrast, VEGF-A binds VEGFR1 and VEGFR2 and is an essential hemangiogenic factor. We re-evaluated the potential role of VEGF-A in lymphangiogenesis using a novel model in which both lymphangiogenesis and hemangiogenesis are induced in the normally avascular cornea. Administration of VEGF Trap, a receptor-based fusion protein that binds and neutralizes VEGF-A but not VEGF-C or -D, completely inhibited both hemangiogenesis and the outgrowth of LYVE-1(+) lymphatic vessels following injury. Furthermore, both lymphangiogenesis and hemangiogenesis were significantly reduced in mice transgenic for VEGF-A(164/164) or VEGF-A(188/188) (each of which expresses only one of the three principle VEGF-A isoforms). Because VEGF-A is chemotactic for macrophages and we demonstrate here that macrophages in inflamed corneas release lymphangiogenic VEGF-C/VEGF-D, we evaluated the possibility that macrophage recruitment plays a role in VEGF-A-mediated lymphangiogenesis. Either systemic depletion of all bone marrow-derived cells (by irradiation) or local depletion of macrophages in the cornea (using clodronate liposomes) prior to injury significantly inhibited both hemangiogenesis and lymphangiogenesis. We conclude that VEGF-A recruitment of monocytes/macrophages plays a crucial role in inducing inflammatory neovascularization by supplying/amplifying signals essential for pathological hemangiogenesis and lymphangiogenesis. (+info)
Interleukin-12 inhibits tumor growth in a novel angiogenesis canine hemangiosarcoma xenograft model.
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We established a canine hemangiosarcoma cell line derived from malignant endothelial cells comprising a spontaneous tumor in a dog to provide a renewable source of endothelial cells for studies of angiogenesis in malignancy. Pieces of the hemangiosarcoma biopsy were engrafted subcutaneously in a bg/nu/XID mouse allowing the tumor cells to expand in vivo. A cell line, SB-HSA, was derived from the xenograft. SB-HSA cells expressed vascular endothelial growth factor (VEGF) receptors 1 and 2, CD31, CD146, and alpha(v)beta(3) integrin, and produced several growth factors and cytokines, including VEGF, basic fibroblast growth factor, and interleukin (IL)-8 that are stimulatory to endothelial cell growth. These results indicated that the cells recapitulated features of mitotically activated endothelia. In vivo, SB-HSA cells stimulated robust angiogenic responses in mice and formed tumor masses composed of aberrant vascular channels in immunocompromised mice providing novel opportunities for investigating the effectiveness of antiangiogenic agents. Using this model, we determined that IL-12, a cytokine with both immunostimulatory and antiangiogenic effects, suppressed angiogenesis induced by, and tumor growth of, SB-HSA cells. The endothelial cell model we have described offers unique opportunities to pursue further investigations with IL-12, as well as other antiangiogenic approaches in cancer therapy. (+info)
Application of femtosecond ultrashort pulse laser to photodynamic therapy mediated by indocyanine green.
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BACKGROUND/AIMS: To evaluate treatment with high peak power pulse energy by femtosecond ultrashort pulse laser (titanium sapphire laser) delivered at an 800 nm wavelength for corneal neovascularisation using photodynamic therapy (PDT) mediated by indocyanine green (ICG). METHODS: Using a gelatin solid as an in vitro corneal model, the safety of laser power was studied to determine if it degenerated gelatin with or without ICG. The authors then induced corneal neovascularisation in rabbit eyes by an intracorneal suturing technique. Fluorescein angiography was used to evaluate occlusion before PDT and 0, 1, 3, and 10 days after PDT. The authors performed light microscopy with haematoxylin eosin staining and transmission electron microscopy to determine thrombosis formation in the neovascular regions. RESULTS: The threshold of peak laser power density ranged from 39 to 53 W/cm(2). Laser irradiation was started 30 seconds after a 10 mg/kg ICG injection, and all irradiated segments were occluded at 0, 1, 3, and 10 days at 3.8 J/cm(2). Light and electron microscopy documented thrombosis formation in the neovascular region. CONCLUSION: Femtosecond pulse laser enhanced by ICG can be used for PDT. Because of effective closure of corneal neovascularisation at a low energy level, the high peak power pulse energy of the femtosecond pulse laser might be more efficacious than continuous wave laser for use with PDT. (+info)
Suppression of corneal neovascularization by PEDF release from human amniotic membranes.
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PURPOSE: Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM. METHODS: The effects of HAM protein on proliferation of vascular endothelial cells and corneal epithelial cells were determined by quantifying viable cells using the MTT assay. The presence of pigment epithelium-derived factor (PEDF) in HAM was demonstrated at the protein level by Western blot analysis and immunohistochemistry using a monoclonal antibody specific to human PEDF. The PEDF concentration was measured by a specific ELISA. The expression of PEDF in HAM was confirmed at the RNA level by RT-PCR and DNA sequencing. RESULTS: Soluble proteins from HAM inhibited proliferation of human umbilical vein endothelial cells and bovine retinal capillary endothelial cells (BRCECs) while promoting proliferation of bovine cornea epithelial cells. Moreover, the HAM-induced inhibition of BRCECs was neutralized by a specific anti-PEDF antibody. PEDF protein was identified with an abundance of 103.84 +/- 33.21 ng/mg of soluble proteins, which is comparable to that in the retina, a PEDF-rich tissue. PEDF expression was predominantly localized in the basement membrane of HAM. RT-PCR using specific PEDF primers amplified a single product from HAM RNA. The PCR product has a sequence identical with that of human PEDF. CONCLUSION: HAM specifically inhibits endothelial cell growth and thus suppresses neovascularization in the cornea. PEDF in HAM has a major role in eliciting this antiangiogenic activity. (+info)