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(Investigative Ophthalmology and Visual Science. 2007;48:5290-5298.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0187
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Fractalkine, a CX3C Chemokine, as a Mediator of Ocular Angiogenesis

Jian-Jang You,1,2 Chang-Hao Yang,2,3 Jen-Shang Huang,3 Muh-Shy Chen,3 and Chung-May Yang3

1From the Department of Ophthalmology, Keelung General Hospital, Department of Health, the Executive Yuan, Keelung, Taiwan; and the 3Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Fractalkine (FKN) is a chemoattractant and adhesion molecule for leukocytes. Angiogenic effect of FKN also has been reported. This study was an investigation of FKN-mediated angiogenesis in vitro and in vivo to determine its role in ocular angiogenic disorders.

METHODS. FKN effects on cultured human umbilical vein endothelial cells (HUVECs) and bovine retinal capillary endothelial cells (BRECs) were evaluated with chemotaxis assay and a synthetic matrix capillary tube formation assay in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were used to detect mRNA and protein expression of FKN and its receptor, CX3CR1, in HUVECs and BRECs. A rabbit corneal neovascularization assay and an oxygen-induced retinopathy (OIR) model of mice were used to test the angiogenic property of FKN in vivo. FKN levels of vitreous samples from patients with proliferative diabetic retinopathy were measured by enzyme-linked immunosorbent assay (ELISA). Immunodepletion of FKN in PDR vitreous samples by anti-FKN polyclonal antibody was observed in endothelial cell chemotaxis assays.

RESULTS. FKN significantly induced migration of HUVECs and BRECs. FKN induced formation of endothelial cell capillary tubes on synthetic matrix. Expression of FKN and CX3CR1 was detected in HUVECs and BRECs by RT-PCR and Western blot analysis. FKN significantly induced more blood vessel growth than did the control in the rabbit corneal pocket neovascularization assay. Intravitreal injection of anti-mouse FKN antibody decreased retinal angiogenesis in the OIR model. The vitreous level of FKN was elevated in patients with PDR compared with control subjects. Immunodepletion of soluble FKN from PDR vitreous samples caused 36.6% less migration of BRECs.

CONCLUSIONS. FKN is an angiogenic mediator in vitro and in vivo. The vitreous level of FKN was elevated in patients with PDR. FKN may play an important role in ocular angiogenic disorders such as PDR.

Angiogenesis is an important aspect of the vasculoproliferation found in tumor growth, wound repair, inflammatory states, and ischemic sequelae in ocular angiogenic diseases.1 Proliferative diabetic retinopathy (PDR), central retinal vein occlusion, and retinopathy of prematurity (ROP) are associated with intraocular neovascularization, which may result in vitreous hemorrhage, tractional retinal detachment, neovascular glaucoma, and blindness. Several mediators are involved in the angiogenic process, including basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-I, vascular endothelial cell growth factor (VEGF),2 3 4 and chemokines.5 6 7 8 9

Chemokines are multifunctional mediators mainly responsible for leukocyte recruitment to inflamed tissues. They are classified by structure into four groups, designated C, CC, CXC, and CX3C, depending on the number and spacing of the cysteine residues in the mature protein.5 6 Although chemokines are generally thought to function as leukocyte attractants, further studies have shown that they also can induce angiogenesis. The CC chemokine monocyte chemotactic protein (MCP)-1 has been identified as an inducer of endothelial cell (EC) chemotaxis in vitro7 and as a mediator of inflammatory angiogenesis in vivo.8 The CXC chemokines containing the ELR motif, consisting of glutamine acid-leucine-arginine preceding the CXC sequence, are chemotactic for neutrophils and are angiogenic.9 These angiogenic effects result from shared expression of specific receptors by leukocytes and endothelial cells.

Fractalkine (FKN), the sole member of the CX3C chemokine family, is named for its fractal geometry. The CX3C motif, with three amino acids between the two terminal cysteines, makes FKN distinct from other chemokines.10 11 The structure of FKN, a membrane-bound glycoprotein with the chemokines domain atop an extended mucin-like stalk, also is unique.12 Membrane-bound FKN can be markedly induced on primary endothelial cells by inflammatory cytokines. This form promotes the robust adhesion of monocytes and T lymphocytes. Soluble FKN can be released by proteolysis at an efficient chemotactic activity level for monocytes and T cells. Thus, FKN is a versatile molecule regulating both cell–cell interactions in its membrane-bound form and directed-cell migration in its soluble form. The receptor of FKN, CXC3R1, is a G-coupled protein13 that expresses T lymphocytes, monocytes, natural killer (NK) cells, microglia, and neurons.14 15 Sulfation of tyrosine enhances the function of CX3CR1 in cell capture and firm adhesion.16

FKN is expressed constitutively in the kidney, heart, lung, and brain. It has been shown to have an important role in central nervous system (CNS) inflammation,17 18 cardiac allograft rejection,19 atherogenesis,20 renal disease,21 and psoriasis22 and in amniotic fluid in pregnancy.23 Silverman et al.24 demonstrated the presence of FKN in normal cultured microvascular endothelial and stromal cells of the iris and retina in vitro. On inflammatory cytokine stimulation, ECs also express FKN and its receptors with FKN secretion in an autocrine manner.25 In addition to EC chemotaxis and capillary tube formation, FKN is an angiogenic mediator in rheumatoid arthritis.26 Therefore, we hypothesize that FKN not only participates in ocular inflammatory reactions, but also plays an important role in ocular angiogenesis. In this study, we investigated the effect of FKN-mediated retina-derived bovine capillary endothelial cell migration and capillary tube formation in vitro. In addition, we examined FKN-mediated angiogenesis in the animal model in vivo and its function as an angiogenic mediator in ocular angiogenic diseases such as PDR.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
All study experiments conducted were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the provisions of the Declaration of Helsinki for research involving human tissue.

Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords as described previously27 and cultured in medium 199 (M199; Invitrogen-Gibco, Carlsbad, CA) and supplemented with 20% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT), 200 µg/mL endothelial growth supplement (EGS; Upstate, Inc., Lake Placid, NY), 88 µg/mL heparin, and antibiotic–antimycotic solution. Bovine eyes were obtained from a local abattoir. Bovine retinal endothelial cells (BRECs) were cultured according to methods described previously.28 Briefly, the eyes were cut circumferentially 3 mm posterior to the limbus. Vitreous humor was removed. Retina was exposed and transferred to phosphate-buffered saline (PBS). Retinas were homogenized by gentle up-and-down strokes in a 15-mL homogenizer (type A pestle; Dounce; Bellco Glass Co., Vineland, NJ). The homogenate was filtered over an 88-µm sieve (Tetko, Inc., Elmsford, NY), and large vessels were removed with forceps from the retentate. The remaining retentate was digested in 0.1% collagenase (142 U/mg, Invitrogen-Gibco) and 0.1% dispase (Gibco- Invitrogen) in 10 mL of PBS for 1 hour at 37°C. The homogenate was subjected to centrifugation (1000 g, 3 minutes). The pellet was resuspended in M199 supplemented with 20% FBS and transferred to culture flasks. After allowing 4 to 6 hours for cell attachment, media were removed and replaced with culture medium. The HUVECs were used in passages 2 and 3, and BRECs were used in passages 5 and 6. Endothelial cell identification was confirmed by an ability to take up acetylated low-density lipoprotein (LDL) by labeling with 1V-dioctadecyl-3,3,3V,3V-tetramethylindocarbocyanine perchlorate-acetylated-low density lipoprotein (Biomedical Technologies, Inc. Stoughton, MA). Cell assays were performed in M199 supplemented with FBS for assay.

Endothelial Cell Chemotaxis Assay
Chemotaxis was performed in 96-well blind-well chemotaxis chambers lined with gelatin-coated polycarbonate membranes with an 8-µm pore size (Neuro Probe, Inc., Gaithersburg, MD).29 30 The HUVECs or BRECs were removed from culture flasks by trypsinization and resuspended at a concentration of 1 x 105 cells/mL in M199 with 2% FBS. One hundred microliters of cell suspension was added to the bottom wells. The chambers were inverted and incubated for 4 hours at 37°C, which allowed EC attachment to the membrane. Human recombinant FKN, VEGF, bFGF, and goat anti-human FKN polyclonal antibody (pAb), were purchased from R&D Systems, Inc. (Minneapolis, MN). FKN (102 nM) was preincubated with 25 µg/mL of either goat anti-human FKN pAb or control goat IgG for 1 hour at 37°C in a 5% CO2 humidified atmosphere. FKN (10–2–102 nM), VEGF (102 nM), an FKN/Ab combination, vehicle PBS as a control, or bFGF (60 nM) as a positive control were added to the top wells followed by incubation of the chambers for 6 hours at 37°C. The membranes were removed, fixed in methanol, and stained with Coomassie blue. The number of cells that had migrated through the filter pores was counted per three high-power fields. Each test group was assayed in quadruplicate.

Formation of Endothelial Cell Tubes In Vitro
An assay on synthetic matrix (Matrigel; BD Bioscience, Franklin Lakes, NJ) was performed according to the method described by Gately et al.31 with minor modification. The matrix was thawed on ice to prevent premature polymerization. Fifty microliters were plated into individual wells of a 96-well chamber, then allowed to polymerize at 37°C for 30 to 60 minutes. The HUVECs or BRECs were removed from culture by trypsinization and resuspended at a concentration of 5 x 104 cells/mL in M199 containing 2% FBS.32 FKN (102 nM) was preincubated with 25 µg/mL of either goat anti-human FKN pAb or control goat IgG for 1 hour at 37°C. One hundred microliters of cell suspension containing FKN (100 nM), VEGF (100 nM), bFGF (60 nM), FKN/Ab combination, or vehicle control PBS were plated in each well and incubated for 8 to 12 hours at 37°C in a 5% CO2 humidified atmosphere. Each chamber was photographed at a final magnification of 100x. The number of tube branches was quantitated by a blinded observer, according the methods described by Gately et al.31 Each concentration of control or test substance was assayed in triplicate.

Reverse Transcription–Polymerase Chain Reaction Amplification of EC FKN and Receptor
Total RNA (1 µg) was prepared from HUVECs and BRECs, and first-strand cDNAs were synthesized with an oligo dT-primed Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen-Gibco). A primer pair for a constitutively expressed gene, glyceraldehyde 3'-phosphate dehydrogenase (GAPDH), was included in each assay as an internal control. Nuclease-free water was included as a negative control. The primer sequences used (Integrated DNA Technologies, Inc., Coralville, IA) for HUVECs were as follows: FKN sense, 5'-ATGCTGCCCTGTGAGTACTAC-3', and antisense, 5'-GGTCCAAAGACAAGTTAGTCC-3', 534-bp amplicon; CX3CR1 sense, 5'ATGCTTGGCTTCTCATACGTC-3', and antisense, 5'-CATTATTACAATTGTTTTCGAGC-3', 710-bp amplicon. The primer sequences used for BRECs were as follows: FKN sense, 5'-ATTCTGTGCTGACCCAAAGG-3', and antisense, 5'-AGCCTCGTTGAAAAGCTCAA-3', 439-bp amplicon; CX3CR1 sense, 5'-CCATGAACAACCGGACCG-3', and antisense, 5'-ATGGCTAAATGCAACCGT-3', 445-bp amplicon. The GAPDH sense, 5'-CCACCCATGGCAATTCCATGGCA-3', and antisense, 5'-TCTAGACGGCAGGTCAGGTCCACC-3', 597-bp amplicon, were used for the primer sequence of the internal control. The polymerase chain reaction cycling conditions were 95°C for 5 minutes followed by 30 cycles of 95°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes, ending with a 10-minute extension at 72°C. Amplification products were characterized by size fractionation on 1% agarose gels.

Western Blot Analysis for EC Expression of FKN and Receptor
The HUVECs and BRECs were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, and protease inhibitors (Complete Mini; Roche Diagnostics Corp., Indianapolis, IN). Cell lysates were mixed 1:1 with Laemmli’s sample buffer and boiled for 5 minutes. Sample (100 µg) was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred from the gel onto polyvinylidene (PVDF) membranes (Immobilon-P; Millipore Corp., Billerica, MA) using alkaline-phosphatase buffer containing 200 mM/L NaCl, 200 mM/L Tris-base, and 10 mM/L MgCl2 [pH 9.5]. To block nonspecific binding, membranes were incubated with 5% milk in PBS containing 0.1% phosphate-buffered saline Tween-20 (PBST) for 1 hour at room temperature. The blots were incubated with goat anti-human FKN Ab or rabbit anti-human CX3CR1 (ProSci Inc., Poway, CA) diluted 1:1000 in PBST and 5% milk at 4°C overnight. After washing with PBST, the blots were incubated with horseradish peroxidase-conjugated rabbit anti-goat or goat anti-rabbit IgG (diluted 1:5000) for 1 hour at room temperature. An enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ) was used to detect FKN and CX3CR1 bands.

Rabbit Corneal Neovascularization Assay
Rabbit corneal neovascularization assay was modified and performed according to the method described by Kenyon et al.32 A hydron polymer (polyHEME; Sigma-Aldrich, St. Louis, MO) was dissolved in absolute ethanol (12% w/v) in a rotator at 37°C overnight, then stored at room temperature before pellet making. Each pellet for the corneal pocket assay contained 90 ng of bFGF, 500 or 1000 ng of VEGF, or 500 or 1000 ng of FKN and 20 µg of sucralfate in 3 µL of casting gel, which was constituted as a 50:50 (vol/vol) mixture of hydron gel and factor-sucralfate-PBS. The casting gel was promptly pipetted onto an autoclaved, sterilized 20 x 20-mm piece of nylon mesh with an approximate pore size of 2 x 2 mm. The pellets were prepared the day before corneal surgery in a laminar flow hood under sterile conditions. Subsequently, the fibers of the mesh were pulled apart, and uniformly sized pellets of 2 x 2 x 0.4 mm were selected for implantation. All procedures were performed in sterile conditions. Such pellets can be stored frozen at –20°C for several days without loss of bioactivity. Each group contained six eyes. New Zealand White male rabbits (2 kg) were anesthetized with ketamine, eyes were topically anesthetized with 0.5% proparacaine (Alcain; Alcon Laboratories, Inc., Fort Worth, TX). Using an operative microscope, we performed a central intrastromal linear keratotomy (~2.5 mm in length) with a surgical knife at the 12 o’clock position. A lamellar micropocket was dissected to 2 mm near the limbus. The pellet was advanced to the end of the pocket. Antibiotic ointment (erythromycin) was applied once to the surgical eye to prevent infection and to decrease irritation of the irregular ocular surface. On postoperative days 3, 7, 10, and 14 after pellet implantation, the rabbits were anesthetized with ketamine. The eyes were exposed, and the maximum vessel length (VL) of the neovascularization zone, extending from the base of the limbal vascular plexus toward the pellet, was measured. Photographs were taken. The contiguous circumferential zone of neovascularization (CN) was measured in clock hours with a 360° reticule (where 30° of arc equals 1 clock hour).

Oxygen-Induced Retinopathy Model of Mice
Pregnant female C57BL/6 mice were obtained from the Animal Resource Centre at National Taiwan University Medical College. The nine mouse pups were treated with goat anti-mouse FKN antibody (R&D Systems, Inc. Minneapolis, MN) in the right eye and were treated with goat IgG in the left eye. The oxygen-induced retinopathy (OIR) model in mice followed a previously published method.33 Seven-day-old pups and their mothers were housed in sealed chambers that contained 75% ± 5% O2 and 2% CO2, using an O2-producing machine. Gas levels in the chamber were monitored daily with a gas analyzer and chart recorder. Mice remained in the chamber for 5 days (hyperoxic period, postnatal day [P]7–P12), then were housed in room air for another 5 days (hypoxic-induced angiogenic period P12-P17). The mice were deeply anesthetized with ketamine for all procedures at the 12th day. The lid fissure was opened with a no. 11 scalpel blade, and the eye was proptosed. Intravitreal injections were performed by first entering the eye with a 8-0 suture needle (Ethicon, Inc., Somerville, NJ) at the posterior limbus. A 32-gauge needle and syringe (Hamilton Co., Reno, NV) were used to deliver 1 µL of antibody solution at the concentration of 200 µL/mL through the existing entrance site in the right eye, and the same volume of concentration of IgG in the left eye. The eye was then repositioned and the lids were approximated over the cornea. Erythromycin ointment was applied after surgery. During the experiment, mothers were provided with water and standard mouse chow and were exposed to normal 12-hour light–dark cycles. Pups received nutrition from their mothers. Experimental procedures were consistent with the guidelines by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Quantitation of neovascularization on P19 was performed by using a technique described by Smith et al.,33 with minor modification. Briefly, 6-µm-thick serial sections, each separated by at least 40 µm, were taken from around the region of the optic nerve. The hematoxylin-and-eosin–stained sections were examined in masked fashion for the presence of neovascular tufts projecting into vitreous from the retina. The neovascular score was defined as the mean number of neovascular tufts per section found in 16 sections per eye.

Immunohistochemical Staining of FKN
Formalin fixed, 6-µm, paraffin-embedded mouse eye tissue sections were placed on slides, deparaffinized in xylenes, and rehydrated through graded ethanol into PBS. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. Then the sections were treated with 5% normal rat serum and incubated overnight with rabbit anti-mouse FKN immunoglobulin G (BioVision, Mountain View, CA) at 4°C. Thereafter, a biotinylated horse secondary antibody against rabbit IgG and an avidin-biotinylated peroxidase complex (Santa Cruz Biotechnology) were used with 3,3'diaminobenzidine as a peroxidase substrate. Sections were counterstained with hematoxylin, dehydrated, and mounted. Rabbit IgG (BioVision) were used as primary antibody in the sections of negative control.

Vitreous Levels of FKN in Proliferative Diabetic Retinopathy
Thirty-two subjects with active PDR and 32 control subjects, including 10 idiopathic epiretinal membranes and 22 idiopathic macular holes, were enrolled. Undiluted vitreous samples were obtained at the time of vitreoretinal surgery after informed consent was obtained from each subject by using a syringe attached to an automated vitrector, immediately placed on ice, and maintained in a freezer at –80°C until analysis. All the patients with PDR enrolled in this study had never undergone panretinal photocoagulation (PRP) laser surgery. The new vessels were active if they were perfused, multibranching iridic, or preretinal capillaries. The patients with active PDR were enrolled in the study group. FKN and VEGF levels in vitreous samples were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Inc.).

Immunodepletion of FKN in PDR Vitreous Samples for BREC Chemotaxis Assays
Vitreous samples were isolated from 10 patients with PDR during pars plana vitrectomy. The vitreous sample was preincubated with 25 µg/mL of goat anti-human FKN antibody or goat IgG control for 1 hour at 37°C. On completion of this neutralization period, the PDR vitreous sample/Ab combination was assayed in the BREC chemotaxis assay as described earlier.

Statistical Analysis
Data in the text and figure legends are expressed as the mean ± SD. Differences between the means of experimental and respective control groups were calculated by Mann-Whitney test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
FKN-Induced EC Chemotaxis In Vitro
FKN was assayed for its ability to induce HUVEC and BREC chemotaxis in vitro. Results of a representative experiment of four are shown in Figure 1A . FKN induced EC chemotaxis in a concentration-dependent manner compared with the negative PBS control (P < 0.05). There were no significant differences among the FKN, VEGF, and bFGF groups in the EC chemotaxis study (Fig. 1B) . When incubated with 25 µg/mL of antibody specific for CX3C chemokine domain of FKN, the FKN solution significantly inhibited EC migration (P < 0.05).


Figure 1
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  		FIGURE 1. FKN induced BREC migration. Results represent the mean number of cells/well ± SE of one representative assay of four. (A) FKN induced chemotaxis in a concentration-dependent manner. (B) FKN, bFGF, and VEGF induced more significant EC chemotaxis than the control. *P < 0.05, significantly different from PBS control. Anti-FKN inhibited FKN-induced EC migration. {dagger}P < 0.05, significantly different from goat IgG control.

 
FKN-Induced EC Capillary Tube Formation In Vitro
The ability of FKN to induce endothelial cell capillary tube formation, one facet of angiogenic response, was assayed in vitro by testing the tube formation of HUVECs or BRECs plated on synthetic matrix (Matrigel; BD Bioscience). The results of a representative experiment of four are shown in Figure 2 . Photomicrographs of BREC and HUVEC tube formation induced by FKN are shown in Figures 2A and 2B , respectively. In contrast, PBS did not induce EC tube formation (Fig. 2C) . Figure 2D shows EC tube counts for FKN-induced tube formation and tube counts in the positive controls (VEGF and bFGF groups) as well as the vehicle controls (PBS group). FKN induced significantly more EC tube formation than did PBS (28 ± 2.8 vs. 9 ± 1.6 tubes/well; P < 0.05, n = 5 in BRECs; 32.8 ± 1.7 vs. 6.5 ± 4.0 tubes/well; P < 0.05, n = 5 in HUVECs). Similar results were obtained from VEGF (30.8 ± 3.1 vs. 9 ± 1.6 tubes/well; P < 0.05, n = 5 in BREC; 38.8 ± 1.7 vs, 6.5 ± 4.0 tubes/well; P < 0.05, n = 5 in HUVECs) and bFGF (32.8 ± 1.3 vs. 9 ± 1.6 tubes/well; P < 0.05, n = 5 in BRECs; 38 ± 2.2 vs. 6.5 ± 4.0 tubes/well; P < 0.05, n = 5 in HUVECs). There was no significant difference among the FKN, VEGF, and bFGF groups. Anti-CX3C domain antibody significantly inhibited FKN-induced HUVEC and BREC capillary tube formation (P < 0.05).


Figure 2
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  		FIGURE 2. FKN induces EC tube formation in vitro. Representative assay showing FKN-induced EC tube formation and phophate-buffered saline (PBS) control. (A, B) Individual capillary tubes are shown in FKN-treated BRECs/well (A) and HUVECs/well (B). (C) Less EC capillary tube formation was noted in a PBS-treated well. (D) FKN, bFGF, and VEGF induced EC tube formation relative to their negative controls. Data represent the mean number of EC tube branches/well ± SE. *P < 0.05, significantly different from PBS control. {dagger}P < 0.05, significantly different from goat IgG control. Original magnification, x100.

 
Expression of FKN Receptor by HUVECs and BRECs
RT-PCR products were synthesized using specific human CX3C and CX3CR1 primers that amplify 354- and 710-bp fragments in HUVECs and 439- and 445-bp fragments in BRECs (Fig. 3A) . Western blot analysis performed with goat polyclonal anti-human FKN antibody revealed a band of the correct size (95 kDa) in HUVECs and BRECs. Western blot analysis performed with rabbit polyclonal anti-human CX3CR1 antibody revealed a 50-kDa band in HUVECs and BRECs (Fig. 3B) .


Figure 3
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  		FIGURE 3. The BRECs and HUVECs expressed CX3CR1. (A) Agarose gel shows 534-bp CX3C and 710-bp CX3CR1 RT-PCR products from HUVECs and 439-bp CX3C and 445-bp CX3CR1 RT-PCR products from BRECs. (B) Western blot showing 95-kDa CX3C band and 50-kDa CX3CR1 band in both HUVECs and BRECs.

 
FKN-Induced Corneal Neovascularization in a Rabbit Corneal Pocket Assay
Pellets containing the slow-release polymer hydron with sucralfate-PBS alone, FKN plus sucralfate, or VEGF plus sucralfate were implanted in rabbit corneas. Pellets containing sucralfate alone (n = 6 eyes in all groups) did not induce neovascularization (Fig. 4A) . Pellets containing 500 ng of VEGF induced neovascularization on postoperative day 7 with a vessel length (VL) of 1.85 ± 0.08 mm and CN of 1.62 ± 0.08 clock hours. Pellets containing 1000 ng VEGF further stimulated increased VL and CN on postoperative day 7, with a VL of 1.98 ± 0.02 mm and a CN of 2.38 ± 0.17 clock hours (Fig. 4C) . The same neovascular response occurred in implanted cornea with 1000 ng of FKN plus sucralfate on day 7 with a VL of 1.61 ± 0.12 mm and CN of 1.57 ± 0.12 clock hours (Fig. 4B) . Pellets containing 500 ng FKN stimulated corneal neovascularization with a similar CN (1.17 ± 0.14 clock hours), but decreased vessel length with a VL of 1.14 ± 0.09 mm. A comparison of the severity of corneal neovascularization is shown as VL and CN in Figure 4D .


Figure 4
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  		FIGURE 4. Photographs of rabbit corneas on day 7 after implantation with hydron pellets (*) containing both sucralfate (45 µg) and VEGF (1000 ng) (A), sucralfate (45 µg) and FKN (1000 ng) (B), and sucralfate (45 µg) and PBS (C). (D) Corneal neovascularization response in Lewis rabbit cornea after implantation of the various concentrations of FKN or VEGF is expressed as the length of central extension (in millimeters from the limbus and as the sectoral circumference, in clock hours). *P < 0.05, significantly different from vehicle control.

 
Suppression of Retinal Neovascularization in Oxygen-Induced Retinopathy of Mice by Anti-FKN Antibody
A mural model of OIR was used to evaluate the angiogenic effect of FKN on the development of retinal neovascularization in vivo. The results are shown in Figure 5 . Intraocular injection of 1 µL (200 µg/mL) of goat anti-mouse FKN antibody or IgG, performed when the mice were returned to room air (P12), achieved a 38.7% ± 8.5% decrease in histologically evident retinal neovascularization at P17 (P < 0.001) compared with an equivalent injection of control Ig G in the contralateral eye (n = 9). The ability of anti-FKN antibody to suppress neovascular response was obvious by histologic examination of paraffin-embedded ocular cross sections. No retinal toxicity or inflammation was noted by light microscopy.


Figure 5
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  		FIGURE 5. In an OIR model, an intraocular injection of 1 µL (200 µg/mL) anti-FKN antibody or IgG, performed as mice were returned to room air, decreased histologically evident retinal neovascularization (arrow) at P17 in 39% of the eyes compared with an equivalent injection of control IgG in the contralateral eye (P < 0.01; n = 9). Original magnification, x200. Scale bar, 75 µm.

 
Immunohistochemical Staining of FKN
For immunochemical staining of the retina, the mice with OIR were found to have strong FKN staining in the nerve fiber layer and the outer plexiform layer and faint staining in the nuclear layers. Intense FKN expression was observed in the neovascular vessels of mice with OIR. FKN expression was also stronger in vascular endothelial cells in the sensory retina in OIR mice compared with normal mice (Figs. 6A 6B , respectively). Treatment with anti-mouse FKN antibody led to a reduction not only of neovascular vessels, but also of FKN expression in the retina (Fig. 6C) . Control sections showed only background staining (Fig. 6D) .


Figure 6
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  		FIGURE 6. Immunohistochemical staining for FKN expression in retinas of C57BL/6 mice. (A) Mice with OIR were found to have FKN staining in the nerve fiber, outer plexiform, and nuclear layers. Stronger FKN expression was observed in vascular endothelial cells in the sensory retina and in the neovascular vessels of mice with OIR (arrow). (B) The staining patterns of FKN in normal rats. (C) Treatment with anti-mouse FKN antibody led to a reduction not only in neovascular vessels, but also in FKN expression in retina. (D) Negative control with goat IgG as the primary antibody showed only background staining. Original magnification, x200. Bar, 75 µm.

 
Vitreous Levels of FKN in Proliferative Diabetic Retinopathy
The vitreous levels of FKN in the PDR group (5.01 ± 3.04 ng/mL) were significantly higher than in the control group (2.55 ± 1.02 ng/mL; P < 0.01). The VEGF concentration in vitreous samples also was elevated significantly in the PDR group (1.83 ± 0.76 ng/mL vs. 0.52 ± 0.41 ng/mL; P < 0.01) and correlated with the vitreous level of FKN (Fig. 7) .


Figure 7
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  		FIGURE 7. Vitreous levels of FKN and VEGF in patients are shown. The vitreous level of FKN was significantly elevated in the patients with PDR compared with control (5.01 ± 3.04 ng/mL vs. 2.55 ± 1.02 ng/mL). *P < 0.05, significantly different from vehicle control.

 
Effect of Immunodepletion of FKN on Chemotactic Activity in BRECs in Vitreous Samples from PDR
To determine whether FKN has biological relevance in a disease characterized by angiogenesis, vitreous samples from 10 patients with PDR were immunodepleted of FKN and assayed for their BREC chemotactic activity. Results of the immunodepletion experiments are shown in Table 1 . Although the PDR vitreous sample was potently chemotactic for BRECs, immunodepletion of FKN resulted in its significantly decreased (39.3% ± 6.6%) chemotactic ability for BREC relative to the immunodepletion with isotype control antibody (P < 0.05).


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  		TABLE 1. BREC Migration in PCR Vitreous Samples Incubated with and without Anti-FKN Antibody

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that FKN could induce angiogenesis with both in vivo and in vitro experimental models. Results from RT-PCR and Western blot analysis showed that HUVECs and BRECs expressed mRNA and protein of FKN and its receptor CX3CR1. FKN induced HUVEC chemotaxis in a concentration-dependent manner from 10–2 to 102 nM. FKN (100 nM) induced ECs to form capillary tubes in synthetic matrix (Matrigel; BD Bioscience) with the same efficiency as the angiogenic mediators, bFGF (60 nM) and VEGF (100 nM). The effect of FKN to induce EC migration and capillary tube formation on the matrix were mainly mediated by its chemokine domain, since an antibody specific for the chemokine domain significantly inhibited FKN-induced EC migration and capillary tube formation. A rabbit corneal pocket assay showed that FKN as well as bFGF and VEGF could induce neovascularization in the rabbit cornea. Furthermore, immunohistochemical studies showed that expression of FKN in the neovascular tufts of retina in the mouse OIR model and the angiogenic effect were inhibited by intravitreal injection of anti-FKN antibody; vitreous sample from patients with PDR revealed higher FKN concentrations compared with the control. These experimental results strongly indicate that FKN plays an important role in ocular angiogenesis.

Chemokines are small chemoattractant cytokines that induce leukocyte accumulation at inflammatory sites and modulate inflammatory activities via the recruited cells.6 According to NH2-terminal cysteine motifs, chemokines can be grouped into four families: C, CC, CXC, and CX3C. Studies have shown that chemokines not only function as leukocyte attractants, but also act as angiogenesis inducers.5 The CC chemokine MCP-1 and macrophage inflammatory protein (MIP)-1 have been identified in aqueous and vitreous samples from patients with PDR.34 35 36 The CXC chemokine, including interleukin (IL)-8),37 interferon-induced protein (IP)-10,38 and stromal-derived factor (SDF)-139 have also been reported to participate in the pathogenesis of ocular neovascularization. FKN is unique in that it is the sole member of the CX3C chemokine family and its angiogenic property has been reported recently. While Volin et al.26 have proposed that FKN mediates angiogenesis in rheumatoid arthritis, the angiogenic effect of FKN in ocular diseases has not been reported. Silverman et al.24 found that iris and retina explants constitutively express FKN in microvascular ECs and also in several stromal cell types. Fang et al.40 described the sequential expression of FKN and its receptor CX3CR1 in the course of experimental autoimmune anterior uveitis, indicating that FKN can serve as an inflammatory mediator in the ocular tissues. Our study further established the role of FKN as an angiogenic mediator in ocular disorders.

Chemokines have a pivotal role in the control of inflammation and angiogenesis, as a result of the shared expression of their specific receptors by leukocytes and endothelial cells.5 At present, only one known FKN receptor, CX3CR1, has been reported.14 Originally, CX3CR1 is known to be expressed on the surface of monocytes, NK cells, and T lymphocytes. In this study, we demonstrated that HUVECs and BRECs expressed CX3CR1 mRNA and protein. It has been reported that FKN stimulated by nuclear factor {kappa}B-dependent inflammatory signals induces aortic smooth muscle cell proliferation through an autocrine pathway.41 Since CX3CR1 is the only known receptor of FKN, it is possible that FKN-induced EC migration and capillary tube formation was mediated through the interaction of FKN and its EC receptor CX3CR1 in an autocrine manner.

The mechanism of FKN expression and its angiogenic effect are different from those of VEGF. Our cornea pocket assay showed that the potency of FKN was weaker than VEGF. Whereas ischemia induces strong expression of VEGF, hypoxia actually inhibits the expression of FKN.42 Recent evidence strongly suggests that inflammation of vessels and neural tissue occurs early in experimental and human diabetic retinopathy.43 44 Angiogenic factors and inflammatory mediators produced by ocular tissues can induce expression of adhesion molecules, which promote the leukostasis of neutrophils on vascular endothelium and induce the extravasation of inflammatory cells.45 46 The coordination of angiogenesis and inflammation is achieved by the ability of both endothelial cells and leukocytes to respond to common stimuli, such as chemokines. Because FKN can serve as an adhesion molecule as well as a chemoattractant for leukocytes, it is one molecule capable of fulfilling the criteria. FKN can be induced by inflammatory mediators such as tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1, and lipopolysaccharide (LPS), and its expression is reported to be dependent on the activation of transcription factor NF-{kappa}B.41 Expression of FKN in EC participates in an amplification circuit of Th1 responses.47 The leukocytes actively tether themselves to the endothelial cell lining via adhesion molecules, including intercellular adhesion molecule (ICAM)-1 on the vasculature48 and ß2 integrins on the leukocytes.49 The induction of FKN expression produces more secreted-form FKN and recruits more monocytes and Th1 cells through the interaction of FKN with its receptor CX3CR1. These inflammatory cells produce more cytokine and induce more expression of FKN on ECs. Thus, a positive-feedback mechanism develops. Through the interaction of FKN with its EC receptor CX3CR1 in an autocrine manner, FKN induced EC migration and capillary tube formation, and then further angiogenesis developed. Therefore, in diabetic retinopathy, FKN may mediate retinal angiogenesis via inflammatory mechanisms.

Studies demonstrated that vitreous levels of VEGF and several kinds of chemokine with potent angiogenic activity are significantly elevated in PDR groups.50 51 In our study, elevated vitreous levels of FKN were also noted in eyes with PDR and were compatible with those of VEGF. Immunodepletion with anti-FKN antibody of vitreous samples from PDR patients has shown significantly reduced chemotactic activity of ECs and significantly reduced angiogenic activity. The reasons for the profound reduction in PDR vitreous samples ability to induce migration after anti-FKN antibody treatment may be that a complex sharing of chemokine receptors and signaling molecules among different chemokines exists or different angiogenic mediators act in a synergetic manner. If these are the actual mechanisms, immunodepletion of an individual factor may have a major impact on the total angiogenic response.

In summary, FKN, the sole member of the CX3C chemokine family, induces EC chemotaxis, EC tube formation, and corneal neovascularization. Retinal endothelium cells can express FKN and its receptor. The concentration of FKN is elevated in the vitreous of patients with PDR. Our results suggest that FKN plays an important role in ocular angiogenic diseases and serves as a new therapeutic target for treatment of these diseases.


   Footnotes
 
2 Contributed equally to the work and therefore should be considered equivalent authors. Back

Supported by Grant 93003 from the Department of Health, Executive Yuan, Republic of China (JJY); Grant NTUH 93S093 from the National Taiwan University Hospital (CHY); and Grant NSC 93-2314-B-002-093 from the National Science Council, Executive Yuan, Republic of China (CMY).

Submitted for publication February 14, 2007; revised June 7 and July 15, 2007; accepted September 10, 2007.

Disclosure: J.-J. You, None; C.-H. Yang, None; J.-S. Huang, None; M.-S. Chen, None; C.-M. Yang, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Chung-May Yang, Department of Ophthalmology, National Taiwan University Hospital, Chung-Shan S Rd, No. 7, Taipei, Taiwan; chungmay{at}ha.mc.ntu.edu.tw.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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