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

Preparation of patient samples

This clinical study investigated the levels of various proteins in the aqueous humor of patients with CNV secondary to AMD and in normal control cataract patients. This study was approved by the Institutional Review Board of Kim’s Eye Hospital, Seoul, Korea. Informed consent was obtained from all participants. Patients were enrolled from the Retina Center at Kim’s Eye Hospital from March 2010 to August 2010. Patients (male: 22, female: 13) over 50 years old with active CNV secondary to AMD identified with fluorescein angiogram who underwent injections of 0.5 mg of ranibizumab with paracentesis of the aqueous humor before injection were included in this study. Exclusion criteria were as follows: (1) ocular disease apart from AMD and cataract; (2) previous ocular surgery apart from cataract surgery, photodynamic therapy with verteporfin, and intravitreal triamcinolone injection; and (3) cataract surgery, intravitreal triamcinolone injection, and photodynamic therapy within 6 months before entry in the study. Undiluted aqueous humor samples (100–150 μl) were obtained through anterior chamber paracentesis from 30 eyes in 35 patients with AMD. All patients did not have significant systemic diseases. Undiluted aqueous humor (100–150 μl) was also obtained as a control sample from 24 eyes immediately before cataract surgery. Samples were stored at −80 °C until analysis. No significant statistical difference was noted in the mean age of the age-related macular degeneration group (67.9±8.7 years) versus the control group (64.4±10.7 years).

Membrane-based human antibody array

In this study, we examined the differences in protein expression levels between the aqueous humor of controls and patients with AMD by profiling the proteins using RayBio^® Biotin Label-based Human Antibody Array I (Catalog No: AAA-BLM-1–2; Ray Biotech, Inc., Norcross, GA). The array membrane is spotted with 507 specific antibodies toward cytokines, chemokines, adipokines, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules, and other proteins in culture supernatant and serum. Biotinylated proteins of samples can be captured by these specific antibodies and horseradish peroxidase (HRP)-conjugated streptavidin in a sandwich format. After enhanced chemiluminescence (ECL) solution was added, the signals can be visualized with chemiluminescence. The assay for antibody arrays was performed according to the manufacturer’s instructions. Briefly, to biotinylate the samples, 50 μl of each undiluted aqueous humor sample (control or patients with AMD, 24 cases each) was mixed and dialyzed with phosphate buffer saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4), and an internal control was added to the dialyzed samples. After 1.5 μl of labeling reagent was added to the samples and they were incubated at room temperature (RT) for 30 min, the reaction was stopped with 5 μl of stop solution. Free biotin was removed with the spin column, and the biotin-labeled samples were diluted with 7 ml of blocking buffer. Array membranes were blocked with 8 ml of 1× blocking buffer at RT for 1 h, and then incubated with the biotin-labeled samples at 4 °C overnight. Then the membranes were washed three times with 20 ml of wash buffer I and II, followed by incubation with 8 ml of 500-fold diluted HRP-conjugated streptavidin for 2 h at RT. After the membranes were washed, mixed detection buffer was loaded onto the membranes to cover the entire surface, and they were incubated for 2 min. Finally, spot signals were visualized by exposing the membranes to radiographic film (Kodak X-Omat^TM; Kodak, Rochester, NY) for variable times. To select the target molecules showing significantly different spot signal intensities between controls and patients with AMD, results from multiple films were compared by three persons in a blinded manner. Biotinylated and non-biotinylated proteins placed on the membranes were used as positive and negative controls, and internal controls were used to monitor the whole process including biotin-labeling.

Protein quantitation

The levels of angiopoietin-2 and insulin-like growth factor binding protein 7 (IGFBP-7) were measured using sandwich enzyme-linked immunosorbent assay (ELISA) according to the respective manufacturers’ protocols. To measure the level of angiopoietin-2, 30 μl of undiluted aqueous humor of controls or patients with AMD (18 cases for control and 13 cases for patients with AMD) were prediluted with 70 μl of standard diluent buffer supplied with the Human Angiopoietin-2 ELISA kit (Invitrogen, Camarillo, CA). One hundred μl of prediluted samples were added to each well filled with 25 μl of incubation buffer and then incubated with 50 μl of biotinylated anti-Ang-2 solution at RT for 2 h. After the wells were aspirated and washed four times, 100 μl of streptavidin-HRP working solution was added to each well and incubated at RT for 30 min. Then the wells were washed four times and incubated with 100 μl of stabilized chromogen at RT for 30 min in the dark. The reaction was stopped with 100 μl of stop solution, and then the absorbance was read at 450 nm. To assay the level of IGFBP-7, the Human IGF-BP7 “SUPER X” ELISA kit was purchased from Antigenix America (Huntington, NY). In brief, 5 μl of each undiluted aqueous humor sample (30 cases for control and 23 cases for patients with AMD) was diluted with 95 μl of PBS containing 0.05% Tween-20 and 0.1% BSA, loaded into each precoated well, and incubated at RT for 2 h. After the wells were aspirated and washed four times, 100 μl of 0.1 μg/ml of biotin-labeled tracer antibody was added and incubated at RT for 1 h. After the solution was decanted, each well was washed four times, 100 μl of streptavidin-HRP conjugate (1:2,000 in diluent) was added, and the well was incubated at RT for 30 min. TMB substrate solutions A and B were mixed (1:1) just before being added and loaded into each well, which was washed four times and then incubated at RT for 10 min. Color development was stopped by adding 100 μl of stop solution, and the absorbance was read at 450 nm.

The TCCR level was measured with western blot. Briefly, 15 μl of undiluted aqueous humor of controls or patients with AMD was heated in sodium dodecyl sulfate (SDS) sample buffer (Laemmli) for 5 min at 94 °C, and subjected to SDS–PAGE based on the Tris-Aspartate buffer system and then transferred to nitrocellulose membrane. To minimize non-specific binding, the membrane was blocked for 1 h at RT with 5% (w/v) skimmed milk in TBS-T buffer (25 mM Tris-HCl, 150 mM NaCl [pH 7.5], and 0.05% Tween-20) and then incubated overnight at 4 °C with primary antibody to TCCR (1:200; R&D Systems; Minneapolis, MN) in TBS-T containing 5% skimmed milk. Then the membrane was washed in TBS-T and probed for 1 h at RT with horseradish peroxidase-conjugated antigoat secondary antibody (1:5,000; Vector Laboratories; Burlingame, CA) in TBS-T containing 5% skimmed milk. After being washed in TBS-T, the immunoreactive bands were visualized with ECL detection reagent (Pierce; Rockford, IL).

To assay the expression levels and phosphorylation status of p44/42 MAPK and β-actin, HUVECs were cultured in 12-well plates coated with 1% gelatin and then treated with 100 pM recombinant human TCCR/WSX-1/Fc chimera protein or human immunoglobulin G (IgG) Fc protein for 2, 5, or 30 min. After 100 μl of SDS sample buffer was added, the samples were subjected to western blot using antiphospho-p44/42 MAPK (p-ERK, 1:1,000; Cell Signaling Technology; Danvers, MA), p44/42 MAPK (ERK, 1:2,000; Cell signaling), or β-actin (1:1,000; Santa Cruz Biotechnology; Santa Cruz, CA) antibodies.

Cell culture

HUVECs were obtained from ATCC (Manassas, VA) and grown (37 °C, 5% CO₂) in M199 (Thermo Fisher Scientific; Waltham, MA) with 20% fetal bovine serum (Thermo Fisher Scientific) and 3 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich; St. Louis, MO). The cells were studied between passages 4 and 7.

Tube formation assay

Tube formation was performed by placing ice-cold growth factor-reduced Matrigel matrix (8.2 mg/ml; BD Biosciences; Franklin Lakes, NJ) in a prechilled 96-well plate (50 μl/well) and incubating it at 37 °C for 30 min to allow polymerization. Thereafter, HUVECs (1×10⁴ cells/well) were seeded on a Matrigel-coated 96-well plate and incubated in the M199 media for 8 h in the presence or absence of 20% fetal bovine serum, different concentrations (10, 100, 1,000 pM) of recombinant human TCCR/WSX-1/Fc chimera protein (R&D Systems), or human IgG Fc protein (R&D Systems). Tube-like structures were observed after 8 h and photographed under phase contrast microscopy using an inverted Olympus microscope (Tokyo, Japan) at 50× magnification. Quantification of the images was performed in a blinded manner by counting total tubes per five fields. The experiment was performed three times in triplicate wells.

Analysis of secretory proteins in aqueous humor

To identify novel secretory proteins for which the number changed in patients with AMD, we analyzed aqueous humor by using a membrane-based human antibody array kit. For convenience, all 30 samples were mixed and subjected to antibody-array analysis. Using an array designed to detect 507 proteins (Figure 1A), we found that the number of several proteins were changed in the aqueous humor of patients with AMD compared to the control (Figure 1B). Angiopoietin-2 (Ang-2), C-C motif chemokine receptor 7 (CCR7), CCR8, CCR9, ciliary neurotrophic factor (CNTF), C-X-C motif chemokine ligand 14 (CXCL14/BRAK), C-X-C motif chemokine receptor 1 (CXCR1/IL-8 RA), CXCR2 (IL-8 RB), EGF-TM7 latrophilin-related protein (ETL), insulin-like growth factor binding protein-related protein 1 (IGFBP-rp1/IGFBP-7), and insulysin (insulin-degrading enzyme/IDE) were decreased in the aqueous humor of patients with AMD. On the other hand, the number of TCCR/WSX-1, transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1/Tomoregulin-1), and u-plasminogen activator (uPA) were increased in patients with AMD (Figure 1C).

Protein quantification

To verify each sample, we next used quantitative analysis. Among the candidate proteins shown in Figure 1B, Ang-2, IGFBP-7, and uPA are available for ELISA analysis. As shown in Figure 2A, Ang-2 was decreased by about 20% in patients with AMD compared with the control group (p=0.074). IGFBP-7, however, did not appear to be different between the groups (Figure 2B), and the level of uPA was not in the detectable range (data not shown).

We also verified the number of proteins by using western blot. Twenty-one samples in the control and AMD patient groups were used for western blot using a specific antibody against TCCR (Figure 3A). Densitometry data showed that TCCR was increased significantly in the aqueous humor of patients with AMD compared to the control group (p=0.0013) (Figure 3B).

TCCR promotes angiogenesis of human umbilical vein endothelial cells in vitro

To test whether TCCR could induce angiogenesis in endothelial cells, tube formation was measured using HUVECs in vitro. We found that exogenous human TCCR/WSX-1/Fc chimera protein promoted tube formation in HUVECs (Figure 4A,B). However, the control IgG did not show a similar outcome. ERK activation has been known to be closely associated with tube formation of HUVECs. As shown in Figure 4C, phosphorylation of ERK was increased transiently after treatment with the TCCR protein. Taken together, these data show that TCCR activates ERK and stimulates tube formation in endothelial cells.