Evidence for enhanced tissue factor expression in age-related macular degeneration.

Age-related macular degeneration (AMD) represents a leading cause of irreversible blindness in the elderly.1, 2 Advanced AMD is generally subclassified into geographic atrophy (dry) and neovascular/exudative (wet) AMD. Dry AMD progresses relatively slowly and is characterized by the accumulation of drusen deposits, degeneration and atrophy of both retinal pigment epithelium (RPE) and photoreceptors. Wet AMD leads to sudden and severe vision loss and is characterized by choroidal neovascularization (CNV) - the growth of new blood vessels from the choroid into Bruch's membrane or the subretinal space and retina. Tissue factor (TF), a transmembrane cell-surface receptor for plasma coagulation factor VII (FVII) and its activated form FVIIa, is the primary initiator of mammalian blood coagulation. Normal endothelium lacks detectable TF expression, whereas vascular subendothelial cells constitutively express TF.3 Upon vascular injury, FVII binds to TF to form the TF/VIIa complex, which initiates the coagulation cascade by activating downstream coagulation factors. In addition to hemostasis, the TF/VIIa complex can also mediate intracellular signaling through protease-activated-receptors, and promote inflammation4, 5 and angiogenesis.6–8 Inflammation and angiogenesis have been implicated in the pathogenesis of AMD. Drusen formation initiates inflammation by stimulating the production of cytokines and reactive oxygen species (ROS), and leads to further RPE and photoreceptor damage. Studies have found an association of AMD with single-nucleotide polymorphisms of the genes coding for complement factor H, factor B, C2, and C3.1, 9 Of interest, coagulation factor X and fibrinogen, which are downstream mediators of blood coagulation closely related to the expression of TF activity, have been detected in AMD lesions as well.10–14 Inflammation elicited by lipopolysaccharide (LPS), TNF-α, IL-1, IL-6, C5a, and many other factors can result in increased cellular expression of TF. Enhanced TF expression may in turn induce expression of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and macrophage inflammatory protein-2α (MIP-2α/CXCL2α).4, 15 Analogous to the process of tumor angiogenesis, in the later stages of AMD, CNV serves to actually reduce the blood supply to the retina, with diminished transport of macromolecules such as oxygen, thereby creating a hypoxic environment. Hypoxia subsequently induces the expression of vascular endothelial growth factor (VEGF) and promotes further CNV formation.16 In cancer and inflammation, a positive feedback loop exists between VEGF and TF; TF can induce angiogenesis by upregulating VEGF,17–19 and enhanced VEGF can in turn increase TF expression.20 Recent studies have suggested the possibility that TF may be implicated in the pathogenesis of AMD.14 Immunostaining revealed TF expression in RPE cells and macrophages in post-mortem eyes with CNV and in surgically excised CNV specimens,21 TF was expressed strongly in macrophages and variably in RPE cells. In addition, TF staining was observed to be increased in “inflammatory-active” versus “inflammatory-inactive” CNV. Belting and colleagues reported that TF phosphorylation was associated with pathological neovascular vessels but not with normal vessels in human diabetic retina,6 indicating a potential role of TF in CNV formation. Furthermore, targeting TF has been proposed as a potential immunotherapy for treating CNV. Administration of a FVII-Fc chimeric antibody, which targets TF, selectively obliterated CNV without any side effects in laser-induced mouse and pig models, which simulate neovascular AMD.22, 23 Taken together, these findings suggest that aberrant expression or activity of intraocular TF may play a role in AMD pathogenesis and that TF may serve as an effective therapeutic target for pathological neovascular lesions in AMD retina. TF expression in normal and diseased eyes as well as its association with increasing age has not been previously demonstrated. In addition, it still remains unclear whether TF can serve as a target or marker in early and dry AMD. In the current study, we examined TF expression in human AMD retina, as well as in the eyes of the murine model of AMD, Ccl2−/−/Cx3cr1−/− double-knockout (DKO) mice, which spontaneously develop focal, progressive retinal lesions and elevated A2E levels as early as 4–6 weeks of age.24 TF expression under the control of inflammatory and oxidative stress was also evaluated in vitro in ARPE-19 cells after stimulation with LPS and H2O2.