TCCR/WSX-1 is a novel angiogenic factor in age-related macular degeneration

Abstract

Purpose: Age-related macular degeneration (AMD) is the major cause of blindness among persons aged 60 years and older. The current approved therapies for AMD are exclusively limited to inhibiting vascular endothelial growth factor. However, substantial improvement in vision occurs in only one-third of patients treated with vascular endothelial growth factor antagonists, and one-sixth of treated patients still progress to legal blindness. Therefore, more specific targets are needed to treat AMD. Our goal was to find secretory proteins that change in number in the aqueous humor and that cause exudative AMD disease.

Methods: The number of molecules changed in the aqueous humor of patients with AMD compared to the control group was determined using antibody array analysis. The levels of angiopoietin-2 and insulin-like growth factor binding protein-related protein 7 were measured using enzyme-linked immunosorbent assay. The levels of T-cell cytokine receptor (TCCR/WSX-1) were determined using western blot. Potential TCCR/WSX-1-mediated effects on tube formation as well as phosphorylation of extracellular signal-regulated kinase in human umbilical vein endothelial cells were determined.

Results: We found that the numbers of several molecules were changed in the aqueous humor of patients with AMD compared to the control group. Among them, angiopoietin-2 was reduced by 20% and TCCR/WSX-1 was increased twofold. Moreover, exogenous TCCR protein induced tube formation and phosphorylation of extracellular signal-regulated kinase in human umbilical vein endothelial cells.

Conclusions: Our study suggests that TCCR/WSX-1 is closely associated with angiogenesis and could serve as a novel therapeutic target in patients with AMD.

Figure 1

Expression levels of 507 proteins in aqueous humor of control or patients with age-related macular degeneration (AMD). A: The list of human antibodies. B: The mixed aqueous humor samples were assayed directly with the RayBio^® Biotin Label-based Human Antibody Array kit. C: The following proteins were changed in the aqueous humor of patients with AMD compared to control: Ang-2, CCR7, CCR8, CCR9, CNTF, CXCL14, CXCR1, CXCR2, ETL, IGFBP-7, and Insulysin (down-regulated); TCCR, TMEFF1, and uPA (upregulated). Negative control (15) and positive control (16) represent internal controls.

Figure 2

Enzyme-linked immunosorbent assay (ELISA) data of Ang-2 and IGFBP-7. The protein levels of Ang-2 (A) and IGFBP-7 (B) in aqueous humor samples were measured using the ELISA kit (* p=0.074).

Figure 3

Western blot of T-cell cytokine receptor (TCCR). A: Twenty-one aqueous humor samples of the control and the age-related macular degeneration (AMD) patient groups were assessed with western blot using a specific antibody against TCCR. Upper numbers indicate the value by densitometry. B: The western blot data were quantified with a densitometer (** p=0.0013). The y-axis represents arbitrary scanning units.

Figure 4

Effects of exogenous T-cell cytokine receptor (TCCR) on human umbilical vein endothelial cells (HUVECs). A: After HUVECs were treated with recombinant human TCCR/WSX-1/Fc chimera protein for 8 h, tube formation was detected with phase contrast microscopy. Twenty percent fetal bovine serum (FBS) was used as a control. B: The number of tubes was quantified by counting the total tubes per five fields in a blinded manner. C: western blot using antibody against ERK, phosphorylated extracellular signal-regulated kinase (ERK), and β-actin from HUVECs that were treated with 100 pM recombinant TCCR or control IgG for the indicated times.