Elsevier

Experimental Eye Research

Volume 83, Issue 6, December 2006, Pages 1325-1334
Experimental Eye Research

Inhibition of TNF-α reduces laser-induced choroidal neovascularization

https://doi.org/10.1016/j.exer.2006.07.007Get rights and content

Abstract

To investigate the role of the TNF-α in the development of laser-induced choroidal neovascularization (CNV) in a mouse model.

Four separate laser burns were applied to induce ruptures of Bruch's membrane and subsequent choroidal neovascularization in C57BL/6J mice. TNF-α protein expression was semiquantitatively assessed by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. To investigate the effect of TNF-α inhibition on CNV formation, animals were treated for 7 days via intraperitonealy implanted osmotic pumps either 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept), a chimeric monoclonal antibody (infliximab), or a purified rat anti-mouse/rat TNF monoclonal antibody (TNF-mAb), respectively. Fluorescein angiography, flat-mount preparations, and histopathology were performed at day 7, 10, or 14 after laser treatment. Western blotting demonstrated that TNF-α expression was 4.57-fold higher in the choroid and RPE one week after laser injury compared to control mice without laser. When evaluated one and two weeks after laser injury, etanercept and infliximab given from the 3rd day before laser-damage significantly reduced CNV size and pathological fluorescein leakage compared to the control group after laser treatment only. The inhibitory effect of the monoclonal TNF-α antibody on CNV formation was evident two weeks after photocoagulation but not after one week. Only etanercept administered 3 days after laser injury still reduced significantly the development of CNV lesions. Histopathology confirmed that CNV lesions in treated mice were smaller in size compared to the control animals without TNF inhibitor treatment. In conclusion, anti-TNF-α treatment with different inhibitors reduces both the size and the leakage of laser-induced CNV. These results suggest the involvement of TNF-α in the development of laser-induced CNV and its potential use as a therapeutic agent in the age-related macular degeneration.

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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