Inhibition of TNF-α reduces laser-induced choroidal neovascularization
Introduction
Choroidal neovascularization (CNV) is known to be the main reason for a severe visual loss in patients with age-related macular degeneration (Green, 1999). The pathogenesis of CNV is multifactorial, involving RPE alterations, ruptures of Bruch's membrane and angiogenic features. Recently, inflammatory mechanisms and immune activation have been hypothesized to play a role in the formation of choroidal neovascularization (Penfold et al., 2001). Macrophages can be co-localized with endothelial cells in choroidal neovascular lesions (Sakurai et al., 2003). The concept of inflammatory mediated neovascularization is further supported by studies showing that a generalized macrophage depletion reduced the size and leakage of laser-induced CNV and was associated with decreased macrophage infiltration and VEGF protein expression (Sakurai et al., 2003). Macrophages in surgically removed choroidal neovascular membranes secrete tumor necrosis factor (TNF-α) and contribute to the development of choroidal neovascularization through triggering VEGF production by RPE cells (Oh et al., 1999). This is in accordance with clinical studies demonstrating that AMD patients with blood monocytes that express high TNF-α mRNA levels demonstrate an almost fivefold increased prevalence of neovascular AMD (Espinosa-Heidmann et al., 2003).
Besides its involvement in inflammatory and apoptotic mechanisms, the inflammatory cytokine TNF plays a role in distinct angiogenic processes. TNF-α is the prototypical member of a family of cytokines that also include FasL, CD40L and TRAIL. These molecules are involved in apoptosis, differentiation and cell activation (Black et al., 1997). TNF-α receptors are expressed on many cell types in the retina and choroid, including retinal pigment epithelial cells (RPE), Müller cells, and choroidal vascular cells (Majka et al., 2002). TNF-α is found in the vitreous and in the endothelial cells of fibrovascular tissue from patients with proliferative diabetic vitreo-retinopathy (Yuuki et al., 2001). We have previously demonstrated that TNF-α is elevated in the diabetic retina and that the TNF receptor inhibitor etanercept suppresses leukocyte adhesion in diabetic retinal arterioles, venules, and capillaries. It further inhibits the blood-retinal barrier breakdown in rat models of inflammation, diabetic retinopathy, and in hypoxia-induced retinal neovascularization (Joussen et al., 2002, Koizumi et al., 2003, Krohne et al., 2003).
Several TNF-inhibitors have been approved by the FDA and the European agency and entered clinical therapy for rheumatoid disease (Feldmann and Maini, 2001). Their application in ophthalmology is currently investigated in clinical trials for uveitis (Benitez-del-Castillo et al., 2005). Etanercept, a soluble TNF receptor, is a fusion protein, composed of the Fc portion of IgG1 and the extracellular domain of the TNF receptor (p75). Infliximab is a chimeric monoclonal antibody, which specifically binds with high affinity to both the soluble and membrane-bound TNF-α.
Although likely to affect the formation of choroidal neovascularization, these substances have not yet been investigated in this context. We demonstrate here that inhibition of TNF-α by either a specific antibody or one of the clinically available inhibitors is able to reduce lesion size and leakiness of laser-induced choroidal neovascularization in vivo. These data identify TNF-α as a key molecule in the pathogenesis of the signature pathologies that characterize neovascular age-related macular degeneration.
Section snippets
Animals
All animal experiments were performed in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Regierungspräsidium Köln, Germany. Female C57BL/6J mice between 4 and 6 weeks of age were used. For all procedures, anesthesia was achieved by intraperitoneal injection of ketamin (10%; Ratiopharm, Germany)/xylazine (2%; Bayer, Germany) (Ratio 16:1) and the pupils were dilated with phenylephrine HCl (0.25%)–tropicamide (0.05%).
Treatment with TNF inhibitiors
C57BL/6J (WT) mice
TNF-α expression after laser photocoagulation
TNF-α was highly expressed in tissue explants of RPE and choroid one week after laser (Fig. 1A, line 1) (Fig. 1A, line 2). Compared to control mice, TNF-α protein expression, normalized to ß-actin expression, increased 4.57-fold after laser injury (Fig. 1B).
Quantitative assessment of fluorescein angiography of laser-induced CNV
Quantitative assessment of the CNV was performed using fluorescein angiography. Mice treated with either etanercept, infliximab, or the TNF-α antibody revealed a reduced leakage after laser compared to untreated control mice (Fig. 2A).
The
Discussion
In this study, we demonstrate that TNF-α is involved in the development of CNV lesions after laser photocoagulation and that inhibition of TNF-α reduces CNV formation in mice. While TNF-α antagonists reduced markedly CNV formation, they were not able to completely prevent this pathological process suggesting that other factors are also involved.
Kobayashi et al. (2005) has first suggested an involvement of TNF-α in the development of CNV in STZ-diabetic rats. We had previously demonstrated the
Acknowledgement
This study was funded by ZMMK TV 76, DFG Jo 324/4-1, DFG Jo 324/6-1 (Emmy-Noether), and DFG Ki 743/6-1 (DFG Priority Program Macular Degeneration) and the Kämpgen Stiftung Köln, and Köln Fortune. X.S. was funded by a scholarship from the DAAD and the RetinoVit Foundation, Cologne.
The authors thank Frank Lacina, Claudia Gavranic and Martina Becker for excellent technical assistance.
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These authors contributed equally to this work.