Anti-sphingosine-1-phosphate monoclonal antibodies inhibit angiogenesis and sub-retinal fibrosis in a murine model of laser-induced choroidal neovascularization

https://doi.org/10.1016/j.exer.2008.07.012Get rights and content

Abstract

The efficacy of novel monoclonal antibodies that neutralize the pro-angiogenic mediator, sphingosine-1-phosphate (S1P), were tested using in vitro and in vivo angiogenesis models, including choroidal neovascularization (CNV) induced by laser disruption of Bruch's membrane. S1P receptor levels in human brain choroid plexus endothelial cells (CPEC), human lung microvascular endothelial cells, human retinal vascular endothelial cells, and circulating endothelial progenitor cells were examined by semi-quantitative PCR. The ability of murine or humanized anti-S1P monoclonal antibodies (mAbs) to inhibit S1P-mediated microvessel tube formation by CPEC on Matrigel was evaluated and capillary density in subcutaneous growth factor-loaded Matrigel plugs was determined following anti-S1P treatment. S1P promoted in vitro capillary tube formation in CPEC consistent with the presence of cognate S1P1–5 receptor expression by these cells and the S1P antibody induced a dose-dependent reduction in microvessel tube formation. In a murine model of laser-induced rupture of Bruch's membrane, S1P was detected in posterior cups of mice receiving laser injury, but not in uninjured controls. Intravitreous injection of anti-S1P mAbs dramatically inhibited CNV formation and sub-retinal collagen deposition in all treatment groups (p < 0.05 compared to controls), thereby identifying S1P as a previously unrecognized mediator of angiogenesis and subretinal fibrosis in this model. These findings suggest that neutralizing S1P with anti-S1P mAbs may be a novel method of treating patients with exudative age-related macular degeneration by reducing angiogenesis and sub-retinal fibrosis, which are responsible for visual acuity loss in this disease.

Introduction

Age-related macular degeneration (AMD), the most common cause of adult blindness in developed countries, is complicated by choroidal neovascularization (CNV) and subsequent bleeding and exudation beneath the macula, which lead to severe vision loss and, ultimately, blindness. Both genetic and environmental factors influence the development of CNV. Although the exact pathogenesis of CNV is not known, chronic inflammation adjacent to the retinal pigmented epithelium (RPE), Bruch's membrane, and choriocapillaris appears to play a crucial role in CNV (Augustin and Offermann, 2006, Zarbin, 2007). Current clinical strategies for treating CNV are primarily aimed at reducing VEGF levels in the eye. However, other angiogenic and inflammatory mediators contribute to CNV either directly via activation of their cognate receptors, or indirectly via crosstalk with vascular endothelial growth factor (VEGF) and other signaling pathways. Thus, new therapies that interfere with VEGF, as well as other signaling pathways that promote angiogenesis, vascular leakage and inflammation would represent a significant advance in disease treatment.

Current therapies focus exclusively on inhibition of VEGF with two recently marketed anti-VEGF agents, pegaptanib (Macugen, OSI/Pfizer) (Ferrara et al., 2006) and ranibizumab (Lucentis, Genentech) (Ferrara et al., 2006, Rosenfeld et al., 2006), demonstrating efficacy as treatments for AMD. Despite the real advance that Lucentis represents, mean visual acuity improved only seven letters in the MARINA and 11 letters in the ANCHOR trials. Baseline acuity for MARINA was 20/80 and for ANCHOR 20/125; this translates to about a 20/70+ acuity at 24 months in MARINA and 20/80+ in ANCHOR. Therefore, the average patient who had a successful anti-angiogenic response to treatment did not regain driving vision or the ability to read without low-vision aids in the treated eye. In addition, although about 40% of all patients were 20/40 at the conclusion of the study, 60% of these patients did not have normal driving or normal reading vision (Ferrara et al., 2006). Thus, an unmet medical need for the development of novel therapeutic modalities for the treatment of exudative AMD remains and development of agents targeting signaling pathways distinct from VEGF or acting synergistically with anti-VEGF-based therapies could represent a more effective treatment strategy.

The bioactive lipid signaling mediator, sphingosine-1-phosphate (S1P) may represent just such a novel therapeutic target (Gardell et al., 2006). S1P is a small (<400 Da) extracellular, sphingolipid, signaling growth factor with pleiotropic actions mediated by a complement of five high-affinity G protein-coupled receptors (S1P1–5, also known as EDG1–5) specific for the ligand (Anliker and Chun, 2004, Gardell et al., 2006).

S1P is involved in the regulation of a variety of cellular processes, including proliferation, migration, survival, cytoskeletal organization, adherens junction assembly, and morphogenesis in multiple cell lineages, including endothelial cells (Ozaki et al., 2003), fibroblasts (Watterson et al., 2007), and immune cells (Cyster, 2005, Olivera and Rivera, 2005). As a consequence, S1P is pro-angiogenic, pro-inflammatory and pro-fibrotic.

A critical component of S1P's biosynthetic pathway is sphingosine kinase type 1 (SphK1) which phosphorylates sphingosine to produce S1P. This isoform of the kinase is thought to be responsible for the release of S1P into the extracellular compartment. Recent evidence demonstrates that SPHK is expressed in retinal endothelial cells (Maines et al., 2006). Furthermore, S1P receptors are expressed by vascular endothelial cells (Chae et al., 2004, Maines et al., 2006, Sanchez and Hla, 2004, Sanchez et al., 2007) and, in particular, murine choroidal vasculature.(Sanchez et al., 2007). A knock out of S1P2 receptors has been shown to mitigate vascular growth and permeability in the murine model of oxygen-induced retinopathy (Skoura et al., 2007). These data suggest that S1P may not only promote CNV lesion growth but also permeability. Following the finding that SphK1 is expressed in retinal endothelial cells, Maines et al. (2006) established that intraperitoneal administration of SphK1 inhibitors could decrease vascular leakage in a rat model of streptozotocin-induced diabetic retinopathy. This is not surprising since a role for S1P has been well-established in non-retinal endothelial cell systems. For example, S1P promotes vascular leakage (Sanchez et al., 2007), proteolytic degradation of the extracellular matrix (Bayless and Davis, 2003, Bernatchez et al., 2003, Langlois et al., 2004, Lee et al., 2000), endothelial cell differentiation (Augustin and Offermann, 2006) and proliferation (Ferrara et al., 2006, Kee et al., 2005, Lee et al., 2000, Radeff-Huang et al., 2004), cell survival from migration-induced stress (Ferrara et al., 2006, Kee et al., 2005, Langlois et al., 2004, Limaye et al., 2005), and finally, assembly, alignment and adhesion to form the tubular structures of new vessels (Garcia et al., 2001, Li et al., 2004). As an angiogenic mediator, S1P is comparable to basic fibroblast growth factor (FGF-2) and VEGF in promoting vessel growth, and synergizes with these factors to promote development of vascular network in vivo (Ferrara et al., 2006, Igarashi et al., 2003, Spiegel and Milstien, 2003).

Thus, the S1P signaling system represents an attractive target for therapeutic intervention in ocular disease models where inappropriate angiogenesis and/or vascular leakage are involved. Inactivation of S1P signaling might provide additional therapeutic benefit by addressing the inflammatory and fibrotic component of this disease process. An anti-S1P murine mAb, designated LT1002, was recently developed with high binding affinity and specificity to S1P (Visentin et al., 2006). This anti-S1P mAb demonstrated significant anti-neovascularization effects using in vitro and in vivo assays.(Sabbadini, 2006, Visentin et al., 2006) Furthermore, LT1002 slowed tumor progression and reduced tumor vascularization in several murine models of human cancer. The murine mAb has recently been humanized (designated LT1009) and is currently in Phase 1 clinical trials for cancer as an anti-angiogenic agent.

In this report we show, for the first time, that laser rupture of Bruch's membrane increases local expression of S1P in the sub-retinal space. Intravitreous administration of either murine or humanized anti-S1P monoclonal antibodies (mAbs) potently attenuated CNV lesion volumes. Human endothelial cell from various vascular beds, including brain choroidal plexus (CPEC), lung microvascular (LMEC), retinal vascular (HREC) endothelial cells, and circulating endothelial precursor cells express S1P1–5 receptors, and CPEC respond to anti-S1P mAbs with reduced angiogenesis in the matrigel tube formation assay. Thus, neutralization of S1P with anti-S1P mAbs may provide an effective, novel treatment of AMD and related ocular disorders where S1P is involved in disease progression.

Section snippets

Anti-S1P antibodies

Murine and humanize IgGk1 mAb against S1P were obtained from Lpath, Inc. (San Diego, CA). These high affinity mAbs have been shown to be highly specific for S1P (Visentin et al., 2006); in the same study, the murine anti-S1P mAb (designated LT1002) showed efficacy as an anti-angiogenic agent in multiple in vivo models (Visentin et al., 2006). The humanized mAb (LT1009) showed similar specificity and affinity for S1P as the target when compared to the murine mAb (data not shown). For control

Ocular S1P levels

Table 1 shows that S1P can be detected within the vitreous fluid of healthy rabbits. As measured by a competitive ELISA assay, the concentration of S1P in the vitreous was substantial (120 nM) and exceeds the Kd for S1P receptors (∼8–70 nM), but not as high as the levels found in rabbit plasma (∼1000 nM). Rabbit vitreous was used because of the limited amount of vitreous fluid that can be obtained from mouse eyes, which would preclude making these measurements from mouse vitreous fluid.

S1P receptor gene expression

When

Discussion

The present study reveals, for the first time, that S1P is an important bioactive mediator that may have pleiotropic roles in a well-established animal model of exudative AMD. We demonstrate that S1P is expressed in the milieu surrounding choroidal neovascular tissue and that its expression is increased following laser disruption of Bruch's membrane. Enhanced expression of this very potent pro-angiogenic, pro-inflammatory and pro-fibrotic growth factor suggests that it may be an attractive

Acknowledgements

The authors thank Lynn Shaw, University of Florida, for important help initiating the CNV studies and help with preparation of the manuscript; Steve Searle, Sanger Centre, Cambridge for use of the sequence analysis program SR7.6; and Dr. H. Brad Cunningham for providing the rabbit eyes and plasma for the examination of vitreous and plasma studies of S1P.

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