May 2011
Volume 52, Issue 6
Free
Retinal Cell Biology  |   May 2011
Suppression of Choroidal Neovascularization and Quantitative and Qualitative Inhibition of VEGF and CCL2 by Heparin
Author Affiliations & Notes
  • Daisuke Tomida
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Koji M. Nishiguchi
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Keiko Kataoka
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Tetsuhiro R. Yasuma
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Eiji Iwata
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Ruka Uetani
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Shu Kachi
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Hiroko Terasaki
    From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan.
  • Corresponding author: Koji M. Nishiguchi, Nagoya University School of Medicine, Department of Ophthalmology, 65 Tsuruma, Showa-ku, Nagoya 466-8550, Japan; kojinish@med.nagoya-u.ac.jp
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science May 2011, Vol.52, 3193-3199. doi:10.1167/iovs.10-6737
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      Daisuke Tomida, Koji M. Nishiguchi, Keiko Kataoka, Tetsuhiro R. Yasuma, Eiji Iwata, Ruka Uetani, Shu Kachi, Hiroko Terasaki; Suppression of Choroidal Neovascularization and Quantitative and Qualitative Inhibition of VEGF and CCL2 by Heparin. Invest. Ophthalmol. Vis. Sci. 2011;52(6):3193-3199. doi: 10.1167/iovs.10-6737.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To study the effect of heparin on the development of laser-induced choroidal neovascularization (CNV) and to assess the underlying molecular mechanisms.

Methods.: Bone marrow transplantation (BMT) was conducted by intravenous injection of green fluorescence protein (GFP)-labeled bone marrow cells (1 × 107 cells) into irradiated (9 Gy) C57BL/6J mice. Laser photocoagulation was applied to induce CNV; subsequently, unfractionated heparin or phosphate-buffered saline was injected into mice that did or did not undergo BMT. The area of CNV, distribution of injected heparin, and quantities of infiltrating cells positive for Griffonia simplicifolia (GS) and GFP inside and outside the CNV were evaluated. Effects of heparin on the secretion of VEGF, CCL2, and TNF-α by ARPE19 cells and on the binding of VEGF, CCL2, TNF-α, and their receptors were analyzed in vitro.

Results.: Intravitreal injection of heparin at higher doses reduced the size of the CNV. Heparin localized at the vascular structures and photoreceptor layers adjacent to the laser scar. Only GS-positive cells infiltrating outside the CNV were reduced significantly, but not those inside the CNV or those expressing GFP. Relative decreases in VEGF and CCL2 levels were observed in media of ARPE19 cells at higher heparin concentrations. In vitro binding assays revealed that heparin and porcine ocular fluid, respectively, suppressed the binding of VEGF to VEGFR2 and CCL2 to CCR2.

Conclusions.: Intravitreal heparin injection inhibited CNV development. Reduced VEGF and CCL2 secretion by RPE cells and suppression of VEGF-VEGFR2 and CCL2-CCR2 interactions at the laser site mediated by heparin may contribute to the pharmacologic effect.

Choroidal neovascularization (CNV), an aberrant invasion of choroidal vascular structures underneath the neural retina, can severely compromise vision in the exudative form of age-related macular degeneration (AMD). Nevertheless, the exact pathomechanisms of CNV development remain largely unknown. Increasing evidence suggests that the presence of inflammation predisposes people to this condition. 1 5 For example, C-reactive protein, an inflammatory marker, is known to be elevated in the blood of patients with the disease, 6,7 whereas a single nucleotide polymorphism in a gene encoding complementary factor H, a modifier of complement pathway, significantly raises the risk for AMD. 8 10  
The laser-induced animal model of CNV shares many important aspects of AMD. For example, laser application is known to result in the rapid accumulation of inflammatory macrophages at the site of injury. 11 This accumulation presumably triggers events that ultimately engender the expression of major angiogenic effector VEGF, which leads to CNV formation. 12,13 These findings are compatible with the accumulation of macrophages noted in histologic sections of patients with AMD 14,15 and the importance of VEGF in CNV development in patients with AMD, demonstrated clearly by the remarkable regression of the pathologic vasculatures by intravitreal injections of anti–VEGF agents. 16 Another example is the inflammatory cytokine TNF-α, which also reportedly promotes laser-induced CNV formation. 17,18 The concept of targeting this cytokine for treating exudative AMD in humans has been justified by the regression of CNV observed in a subset of patients treated with an anti–TNF-α regimen. 19,20  
Heparin and heparan sulfate are glycosaminoglycans (GAGs) that bind to VEGF and their receptors. 21,22 Recently, we demonstrated that intraocular fluid contains a high level of heparan sulfate, which serves as an endogenous inhibitor of pathologic neovascularization in oxygen-induced retinopathy in mice. 22 Inactivation of VEGF, presumably through binding inhibition of VEGF and surface GAGs or VEGF receptor 2 (VEGFR2), the receptor responsible for mediating angiogenic activity, probably contributes to the antiangiogenic effect. 22 Moreover, the administration of heparin and heparan sulfate alone reduced the extent of neovascularization, reflecting the therapeutic potential of these GAGs in the management of intraocular vasoproliferative disorders. 22 Others have also reported similar antiangiogenic effects of heparin-related low molecular compound (5-amino-2-naphthalenesulfonate) for the treatment of the same retinal neovascularization mouse model. 23  
This study revealed that intravitreal heparin injection suppressed the development of laser-induced CNV through close association with the laser site. Reduced numbers of infiltrating monocytes/macrophages around the CNV, mostly from local sources, in heparin-treated eyes suggested that heparin inhibited the recruitment of these cells to the site of laser burn. Consistent with these findings, heparin decreased the expression of VEGF and macrophage/monocyte attractant CCL2 by cultured RPE cells and suppressed the bindings of these proteins and their receptors in vitro. 
Methods
Animals
All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines for the Use of Animals at Nagoya University School of Medicine. The research protocol for the use of animals has been approved by the Animal Facility Committee, Nagoya University School of Medicine. The C57BL/6J mice and GFP mice (C57BL/6J strain) 24 used for this study were kept under a 12-hour light/12-hour dark cycle. 
Laser-Induced CNV and Intraocular Injection
Adult C57BL/6J mice (age range, 6–12 weeks) were anesthetized, their pupils were dilated with 1% tropicamide, and four burns of argon laser photocoagulation (75-μm spot size; 0.1-second duration; 100 mW) were delivered to each eye in the 3, 9, 6, and 12 o'clock positions of the posterior pole, each at an equal distance from the optic nerve head. This was followed by intravitreal injection of either unfractionated heparin (0.8, 8, or 80 μg; Sigma-Aldrich Co., St. Louis, MO), FITC-labeled heparin (80 μg; Invitrogen Corp., Carlsbad, CA), or heparinase III (0.01 U; Sigma-Aldrich Co.) dissolved in 1 μL phosphate-buffered saline (PBS; Gibco Invitrogen Co., Grand Island, NY) in one eye and the same amount of PBS or FITC (2.5 ng; Sigma-Aldrich Co.) in the other. 
Histologic Analyses
Flat mount analyses were performed as described previously, with minor modifications. 25 Briefly, the eyecups were fixed in 4% paraformaldehyde for 2 hours and were stained overnight with Griffonia simplicifolia (GS) lectin (a marker for vessels and monocytes/macrophages; 1:100; Sigma-Aldrich Co.). After images of GS-stained CNV were obtained, the files were masked and presented to an independent observer, who excluded unreliable data to make sure a meaningful comparison of the CNV was possible; only CNVs at approximately equal distances from the optic disc and appropriately spaced from each other were included in the analysis. Then the areas of CNV were determined by outlining the border of CNV and measuring the pixels inside the selected area using imaging software (Photoshop CS; Adobe Systems Inc., San Jose, CA). The average area of a CNV within an eye was determined and expressed as an area relative to the average CNV size of all PBS-treated eyes. 
For analyses of histologic sections, the fixed eyecups were incubated in 30% sucrose overnight and frozen in OCT compound (Tissue-Tek; Sakura Finetechnical Co. Ltd., Tokyo, Japan), followed by cryosection (20-μm thick). Sections were permeabilized (10 minutes), blocked (30 minutes), and incubated with GS lectin and diamino-2-phenyl-indol (DAPI; Molecular Probes Inc., Eugene, OR) for 50 minutes with subsequent staining for another 10 minutes with 0.05% toluidine blue (Sigma-Aldrich Co.). 
Bone Marrow Transplantation
Bone marrow transplantation (BMT) was conducted as described previously with modifications. 26 The bone marrow cells were harvested from femurs and tibias of GFP mice 24 (4–8 weeks old); 1 × 107 cells were transplanted intravenously from the tail veins of wild-type mice (4–8 weeks old) after irradiation (9 Gy). After a minimum of 2 weeks after BMT, mice were used for analyses. The animals were perfused with 4% PFA (300 μL/g body weight) through the left ventricle to remove erythrocytes and nonadherent leukocytes from the vessels before enucleation. 
Cell Culture Assays
ARPE19 cells purchased from the American Type Culture Collection (ATCC; Manassas, VA) were routinely maintained in Dulbecco's modified Eagle's medium and Ham's F12 medium (Sigma-Aldrich Co.) containing 10% FBS. After cells were grown to confluence in 24-well plates, they were cultured in basal medium without FBS for 1 hour. Then various concentrations of heparin were added with and without 10 ng/mL TNF-α (PeproTech Inc., Rocky Hill, NJ). After further incubation for 24 hours, the media were collected, and concentrations of VEGF, CCL2, and TNF-α were measured using ELISA kits (all from R&D Systems Inc., Minneapolis, MN). 
In Vitro Binding Assays
In vitro binding assays were conducted as described previously with minor modifications. 22 The carboxyl terminals of heparin (10 μg/well), VEGFR1 (200 ng/well; R&D Systems Inc.), VEGFR2 (200 ng/well; R&D Systems Inc.), CCR2 (200 ng/well; PeproTech Inc., Rocky Hill, NJ), TNFR1 (200 ng/well; PeproTech Inc.), or TNFR2 (200 ng/well; PeproTech Inc.) dissolved in PBS were covalently attached to the 96-well plate coated with amino acids (Takara Bio Inc., Shiga, Japan) at 4°C for 4 hours using ethyl-carbodiimide hydrochloride (20 mg/mL; Pierce Biotechnology, Rockford, IL) as a cross-linker. After a blocking procedure using gelatin blocking buffer (Sigma-Aldrich Co.), either VEGF, CCL2, or TNF-α (all from PeproTech Inc. at 200 ng/mL) mixed with fractionated (Iduron Co., Manchester, UK) or unfractionated heparin or porcine ocular fluid (mixture of aqueous and vitreous humor containing 63.2 μg/mL heparan sulfate 22 ) was applied to each well (100 μL/well) before further incubation at 4°C overnight. The plate was rinsed with ice-cold PBS, and the amount of each cytokine bound to the plate was measured using components of the ELISA kit specific for each cytokine (all from R&D Systems Inc.) according to the manufacturer's instruction. All procedures were conducted at 4°C until the final washing step was completed. Finally, the plates were analyzed by measuring absorbance at 450 nm (reference at 620 nm) using a plate reader. 
Statistical Analysis
All obtained images were masked and randomized before analyses. The quantities of cells positive for GS or GFP were determined separately for inside and outside the CNV. All cells inside a circular area measuring 0.5 mm2, with its center positioned in the middle of the CNV, were categorized as either inside or outside the CNV/laser scar. Cell counts from four circular areas within the eye were averaged and presented. All the bars in the figures represent the mean, and the error bars indicate the SEM. Paired t-tests were applied to calculate P values. P < 0.05 was considered significant. 
Results
Suppression of CNV by Intraocular Injection of Heparin
To assess whether the administration of exogenous unfractionated heparin was able to alter the development of CNV, we injected three amounts of heparin intravitreally that would result in approximately 100 (low dose), 1000 (middle dose), or 10,000 μg/mL (high dose) in the eyes (Figs. 1A–E). Heparin was chosen over heparan sulfate for injection because we had previously confirmed its greater ability to inhibit pathologic angiogenesis and binding of VEGF to its receptors. 22 Injection of medium doses and high doses, respectively, of heparin caused approximately 48% and 53% decreases in CNV size, though administration of a low dose showed no detectable effect. 
Figure 1.
 
Reduction of CNV by intravitreal heparin injection. (A, B) Representative images of eyes treated with high-dose heparin (Hep; A) and PBS (B). (CE) Relative CNV size in the eyes treated with heparin compared with contralateral eyes treated with PBS. Eyes were treated with either low-dose (n = 8; C), medium-dose (n = 7; D), or high-dose (n = 5; E) heparin. Medium-dose and high-dose heparin reduced CNV size to a similar degree. (F, G) Relative CNV size in eyes treated with heparinase III (HIII) compared with contralateral eyes treated with PBS. Eyes were treated with HIII or PBS 24 hours before (n = 6; F) or immediately after (n = 6; G) laser photocoagulation. No difference was noted in the CNV size between the two conditions. Scale bar, 100 μm.
Figure 1.
 
Reduction of CNV by intravitreal heparin injection. (A, B) Representative images of eyes treated with high-dose heparin (Hep; A) and PBS (B). (CE) Relative CNV size in the eyes treated with heparin compared with contralateral eyes treated with PBS. Eyes were treated with either low-dose (n = 8; C), medium-dose (n = 7; D), or high-dose (n = 5; E) heparin. Medium-dose and high-dose heparin reduced CNV size to a similar degree. (F, G) Relative CNV size in eyes treated with heparinase III (HIII) compared with contralateral eyes treated with PBS. Eyes were treated with HIII or PBS 24 hours before (n = 6; F) or immediately after (n = 6; G) laser photocoagulation. No difference was noted in the CNV size between the two conditions. Scale bar, 100 μm.
Next, to assess the role of endogenous heparan sulfate in the development of CNV, we first injected heparinase III, which specifically degrades heparan sulfates. This enzyme treatment leads to 80% to 90% reductions in the levels of heparan sulfate in the ocular fluid in vitro and in vivo. 22 No significant difference in CNV size was detected when the enzyme was injected 24 hours before or immediately after laser treatment, followed by the evaluation of the eyes 7 days later (Figs. 1F, 1G). 
Close Association of Injected Heparin with the Laser Site
In an oxygen-induced retinopathy model, heparin treatment specifically inhibited the extraretinal extension of the vessels into the vitreous but showed no effect on the extent of intraretinal vascularization. 22 To investigate whether heparin influences subretinal CNV development through close association with the lesion or by indirect mechanisms, FITC-labeled heparin was injected into the eyes and was evaluated 3, 12, or 48 hours later. Exogenous FITC-heparin associated with the surface retina, specifically at the GS-positive retinal vasculatures 3 hours after injection (Figs. 2A–C), was comparable to only modest or no vascular staining with control FITC injection (Figs. 2D–F). However, the most intense accumulation of FITC-heparin was observed in a granular pattern at the outer nuclear layer immediately adjacent to the laser scar. This was also true when the eyes were examined at 12 or 48 hours after administration (data not shown). Meanwhile, the accumulation of FITC-heparin in the subretinal space observed was considered nonsignificant because injections of FITC alone in the control eyes revealed an indistinguishable distribution. 
Figure 2.
 
Distribution of FITC-labeled heparin after intravitreal injection. (AC) Distribution of injected heparin-FITC (green) 3 hours after laser treatment and GS staining (red). Injected heparin-FITC (arrowhead) was accumulated at the outer nuclear layer immediately adjacent to the laser injury (A; black arrow) and the GS-positive retinal vessels (B, C; white arrow). (DF) Distribution of injected control FITC. FITC colocalized modestly with GS-positive retinal vessels (white arrow). Note the control FITC was evenly distributed throughout all retinal layers. (A, D) Toluidine blue–stained retinal section was overlain with FITC staining. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; C, choroid; S, sclera. Scale bar, 100 μm.
Figure 2.
 
Distribution of FITC-labeled heparin after intravitreal injection. (AC) Distribution of injected heparin-FITC (green) 3 hours after laser treatment and GS staining (red). Injected heparin-FITC (arrowhead) was accumulated at the outer nuclear layer immediately adjacent to the laser injury (A; black arrow) and the GS-positive retinal vessels (B, C; white arrow). (DF) Distribution of injected control FITC. FITC colocalized modestly with GS-positive retinal vessels (white arrow). Note the control FITC was evenly distributed throughout all retinal layers. (A, D) Toluidine blue–stained retinal section was overlain with FITC staining. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; C, choroid; S, sclera. Scale bar, 100 μm.
Reduced Monocyte/Macrophage Infiltration in the RPE Layer Surrounding the CNV in Heparin-Treated Eyes
While quantifying CNV size, we noticed fewer GS-positive cells at the level of RPE layer in heparin-treated eyes than in PBS-treated eyes (Figs. 3A, 3B). These cells were round or oval, had no discernible processes, and were presumed to be monocytes/macrophages from the circulation or from the local choroid or subretinal space. Distribution of these cells, most of which were concentrated around the CNV/laser scar, combined with the invariable migratory nature of inflammatory cells in injury implied that these cells were moving toward the CNV/laser scar. To assess whether this observation was statistically significant, we quantified the number of GS-positive cells inside and outside the CNV/laser scar 24 hours after laser application and subsequent injection of high-dose heparin or PBS; retinal macrophages are known to migrate toward a laser burn by 6 hours. 10 At this time, we were not yet able to confirm the presence of aberrant subretinal invasion of choroidal vasculature in histologic sections and measured no detectable difference in the areas of the GS-positive structures, presumably reflecting laser scar, between the eyes treated with heparin and those treated with PBS (data not shown). GS-positive cells in the RPE layer surrounding the CNV/laser scar were markedly less prevalent in the eyes treated with heparin than in those injected with PBS (Fig. 3D), whereas those residing inside the CNV/laser scar showed no clear difference in number (Fig. 3C). 
Figure 3.
 
Effect of heparin on monocyte/macrophage infiltration. (A, B) Representative images of GS staining of CNV/laser scar and infiltrating monocytes/macrophages 24 hours after photocoagulation and intraocular injection. The number of GS-positive cells (some represented by filled arrowhead) outside the CNV/laser scar was reduced in the eyes treated with heparin (A) compared with the contralateral eyes treated with PBS (B). (C, D) Quantification of GS-positive cells inside and outside of CNV/laser scar (n = 9). The number of GS-positive cells was reduced outside the CNV/laser scar in the heparin-treated eyes. (E, F) Representative images of CNV/laser scar and GFP-positive cells (open arrowhead) 24 hours after photocoagulation and intraocular injection in mice that underwent BMT. (G, H) Quantities of GFP-positive cells inside and outside the CNV/laser scar. There were no differences between heparin-treated and PBS-treated eyes (n = 9). (A, B, E, F) Weak autofluorescence marked the boundaries of the CNV/laser scar. Scale bar, 100 μm.
Figure 3.
 
Effect of heparin on monocyte/macrophage infiltration. (A, B) Representative images of GS staining of CNV/laser scar and infiltrating monocytes/macrophages 24 hours after photocoagulation and intraocular injection. The number of GS-positive cells (some represented by filled arrowhead) outside the CNV/laser scar was reduced in the eyes treated with heparin (A) compared with the contralateral eyes treated with PBS (B). (C, D) Quantification of GS-positive cells inside and outside of CNV/laser scar (n = 9). The number of GS-positive cells was reduced outside the CNV/laser scar in the heparin-treated eyes. (E, F) Representative images of CNV/laser scar and GFP-positive cells (open arrowhead) 24 hours after photocoagulation and intraocular injection in mice that underwent BMT. (G, H) Quantities of GFP-positive cells inside and outside the CNV/laser scar. There were no differences between heparin-treated and PBS-treated eyes (n = 9). (A, B, E, F) Weak autofluorescence marked the boundaries of the CNV/laser scar. Scale bar, 100 μm.
Next, to address the origin of these infiltrating cells (i.e., resident versus circulating cells), we conducted BMT using cells derived from GFP mice. Four weeks after BMT was conducted (estimated engraftment rate, ∼77%), 26 photocoagulation and intraocular injection of heparin or PBS were performed, and eyes were evaluated 24 hours later. GFP-positive cells were found primarily inside the laser/CNV scar and rarely outside the lesion. Quantification of GFP-positive cells, for those inside and those outside the CNV/laser scar, showed nearly equal quantities of cells treated with the two agents (Figs. 3E–H). 
Heparin-Mediated Suppression of VEGF and CCL2 Production by RPE Cells
Although VEGF plays a key role in CNV formation in both humans and mice, the exact sources of this cytokine are unclear. Nevertheless, excessive production of VEGF by RPE contributes to CNV development in mice. 27 29 Therefore, we studied the effect of heparin on VEGF production by a cultured RPE cell line, ARPE19 (Fig. 4A). Results showed that lower concentrations of heparin increased VEGF levels in the medium. However, further increasing the heparin concentration caused a relative decrease in VEGF level dose dependently. At the highest heparin concentration (10,000 μg/mL), the VEGF level was 37.2% lower than the lowest heparin concentration (10 μg/mL). When exogenous TNF-α was added to the medium with and without different amounts of heparin, TNF-α administration constantly, but minimally, increased the production of VEGF by APRE19 by 1.0% to 13.7%, showing a pattern similar to that of conditions without exogenous TNF-α (Fig. 4A). 
Figure 4.
 
Decreased ARPE19-derived VEGF and CCL2 in the culture media by soluble heparin. (A, B) Measurements of VEGF and CCL2 secretion by cultured human RPE. Addition of lower concentrations of heparin increased APRE19-derived VEGF in the culture media compared with conditions without heparin (A). Further increasing the heparin concentration caused relative decreases in VEGF levels at higher doses. Meanwhile, exogenous heparin decreased the CCL2 level in the media in a dose-dependent manner (B). Supplementation of the media with TNF-α minimally elevated VEGF levels while increasing CCL2 concentrations to a much greater degree. No clear inhibitory effects on TNF-α–mediated release of VEGF and CCL2 by ARPE19 cells were noted. The experiment was conducted in triplicate and repeated.
Figure 4.
 
Decreased ARPE19-derived VEGF and CCL2 in the culture media by soluble heparin. (A, B) Measurements of VEGF and CCL2 secretion by cultured human RPE. Addition of lower concentrations of heparin increased APRE19-derived VEGF in the culture media compared with conditions without heparin (A). Further increasing the heparin concentration caused relative decreases in VEGF levels at higher doses. Meanwhile, exogenous heparin decreased the CCL2 level in the media in a dose-dependent manner (B). Supplementation of the media with TNF-α minimally elevated VEGF levels while increasing CCL2 concentrations to a much greater degree. No clear inhibitory effects on TNF-α–mediated release of VEGF and CCL2 by ARPE19 cells were noted. The experiment was conducted in triplicate and repeated.
In laser-induced CNV models, pathologic angiogenesis takes place in concert with the direct and indirect contribution of inflammatory cells and RPE, partially through the production of various cytokines. Other than VEGF, evidence suggests that CCL2, a chemoattractant of macrophages/monocytes, 30 and TNF-α, a potent proinflammatory cytokine, also contribute to the pathogenesis of CNV. 17,18,31,32 Functions of both molecules can be affected by heparin. 33,34 Therefore, we also measured CCL2 and TNF-α levels in the culture medium of ARPE19 cells. Heparin dose dependently decreased the level of CCL2 in the culture medium (Fig. 4B). In fact, CCL2 was reduced by 46.7% between the highest heparin concentration (10,000 μg/mL) and the lowest (10 μg/mL). As reported previously, 35 TNF-α administration caused robust increases in the concentrations of CCL2 by approximately fourfold, although further addition of heparin failed to inhibit the TNF-α–mediated release of CCL2 by ARPE19 cells. Meanwhile, endogenous TNF-α in the medium was at undetectable levels for all conditions (data not shown). 
Heparin Size Is Positively Correlated with the Extent of Interference on VEGF Surface Heparin Binding
We showed previously that soluble heparin and porcine ocular fluid inhibit the binding of VEGF and human umbilical vascular endothelial cells, surface GAGs, and VEGFR2 in cell-based assays or in vitro binding assays. 22 First, to confirm the result of our previous work, VEGF was added together with various concentrations of heparin to heparin-coated, VEGFR2-coated, and VEGFR1-coated wells (Figs. 5A, 5B). Soluble heparin inhibited the binding of VEGF and surface heparin or VEGFR2 dose dependently, as reported. 22 Conversely, heparin posed a minimal influence on VEGF-VEGFR1 association. To extend these observations, the influence of heparin size on the interference between VEGF and surface heparin was explored using variously fractionated heparin (average molecular weights: 1330, 3270, 6620, >9000; Fig. 5C). The efficiency as an inhibitor of VEGF-surface heparin binding was enhanced with increased heparin size up to molecular weight of 6620. However, heparin with molecular weight of 6620 and that with molecular weight over 9000 showed no difference. 
Figure 5.
 
Binding inhibition of VEGF and VEGFR2 or CCL2 and CCR2 by heparin or porcine ocular fluid in vitro. (A, B) Inhibition of interaction between VEGF and surface heparin or VEGFR2 by soluble heparin or porcine ocular fluid. Binding of VEGF and VEGFR1 was unaffected. (C) Molecular size effect of heparin on the VEGF-surface heparin interaction. Function of heparin as an inhibitor of VEGF-surface heparin binding was positively correlated with heparin size. (D, E) Inhibition of interaction between CCL2 and surface heparin or CCR2 by soluble heparin or ocular fluid. (F, G) No clear inhibition of interaction between TNF-α and surface heparin or TNFRs by soluble heparin or ocular fluid. Data are presented as averages of three measurements for each experimental condition. The experiment was repeated. OF, porcine ocular fluid; mw, molecular weight.
Figure 5.
 
Binding inhibition of VEGF and VEGFR2 or CCL2 and CCR2 by heparin or porcine ocular fluid in vitro. (A, B) Inhibition of interaction between VEGF and surface heparin or VEGFR2 by soluble heparin or porcine ocular fluid. Binding of VEGF and VEGFR1 was unaffected. (C) Molecular size effect of heparin on the VEGF-surface heparin interaction. Function of heparin as an inhibitor of VEGF-surface heparin binding was positively correlated with heparin size. (D, E) Inhibition of interaction between CCL2 and surface heparin or CCR2 by soluble heparin or ocular fluid. (F, G) No clear inhibition of interaction between TNF-α and surface heparin or TNFRs by soluble heparin or ocular fluid. Data are presented as averages of three measurements for each experimental condition. The experiment was repeated. OF, porcine ocular fluid; mw, molecular weight.
Heparin and Porcine Ocular Fluid Inhibit the Binding of CCL2 and CCR2
Next, we tested whether heparin or porcine ocular fluid interferes with molecular interactions of CCL2 or TNF-α in vitro (Figs. 5D–G). Binding of CCL2 to surface heparin was inhibited competitively and dose dependently by soluble heparin (Fig. 5D), consistent with CCL2 being a heparin-binding protein. 33 Ocular fluid exhibited a similar effect. Meanwhile, the TNF-α-surface heparin interaction was not suppressed by soluble heparin or ocular fluid (Figs. 5F). We further assessed the effect of heparin and ocular fluid on ligand-receptor bindings (Figs. 5E, 5G). Binding of CCL2 and CCR2 was inhibited by the presence of higher concentrations of heparin (> 100 μg/mL) or ocular fluid (Fig. 5E). This result contrasted with the results for binding of TNF-α and its receptors, showing little or no inhibition (Fig. 5G). 
Discussion
The potential role of heparin GAG as an inhibitor of ocular inflammation and angiogenesis was demonstrated using a murine laser-induced CNV model. Results show that the addition of exogenous heparin caused a reduction in CNV size in vivo. The injected heparin accumulated in the retinal vasculatures and deep retinal layers just above the laser site, suggesting that it exerts its effect through close, or perhaps direct, association with the injury. Although porcine ocular fluid inhibited VEGF-VEGFR2 and CCL2-CCR2 bindings in vitro, the heparinase III-mediated enzymatic breakdown of endogenous heparan sulfate in vivo showed no detectable effect on CNV formation. Because the enzyme only incompletely degrades intraocular heparan sulfate by 80% to 90%, 22 we were unable to draw a definitive conclusion from the current experiment about the role of endogenous heparan sulfate on CNV development. 
Using the same mouse model, a few groups have implied the role of inflammation, specifically monocytes/macrophages, 12,13,31,32 in CNV development. We have observed reduced quantities of monocytes/macrophages in the RPE layers surrounding the CNV/laser scar in the eyes treated with heparin. Labeling the peripheral leukocytes through BMT, we showed that these cells were probably not from the peripheral circulation but were primarily local cells, presumably from the choroid or the subretinal space, based on the lack of labeling and the round to oval cell morphology; macrophages in the retina are primarily ramified and have a distinct polymorphic appearance. Meanwhile, some BMT-derived leukocytes were observed inside laser scars, with no detectable differences in their numbers between eyes treated with heparin and eyes treated with PBS. Taken together, it appears that intravitreal heparin acts primarily on local inflammatory cells within the eyes, which corroborates the finding of limited distribution of injected heparin that maintains effective concentrations extrapolated from the results of in vitro binding assays. Meanwhile, CCL2 is a known chemoattractant that is reportedly involved in the subretinal invasion of monocytes/macrophages and in CNV formation. 31,32 Therefore, the reduced infiltration of monocytes/macrophages in heparin-treated eyes prompted us to assess the direct and indirect effects of heparin on the expression and molecular interaction of CCL2, also a heparin-binding protein. 33 Results showed that the secretion of CCL2 by RPE cells was inhibited in a dose-dependent manner by heparin in cell culture. Results of in vitro binding assays indicated that heparin can block the binding of CCL2 to surface GAGs and its receptor, CCR2. Taken together, the outcomes were consistent with quantitative and qualitative inhibition of CCL2 signaling by heparin GAG, possibly leading to the reduced recruitment of inflammatory cells and the growth of abnormal choroidal vasculatures. Meanwhile, the resistance of TNF-α–mediated secretion of CCL2 by RPE to heparin implied a potential additive treatment effect of anti–TNF-α agents when combined with heparin/GAGs for the management of inflammatory conditions in which elevated TNF-α plays a role in the pathogenesis. Because other cytokines have overlapping roles in inflammation, the exact molecular mechanisms that engender reduced monocytes/macrophage infiltration remain unclear. 
Irrespective of the underlying mechanisms, VEGF appears to contribute to many forms of intraocular pathologic angiogenesis in human eye diseases, including those induced by inflammation. In mice, the two most widely explored models of intraocular neovascularization—laser-induced CNV and oxygen-induced retinopathy—also respond to anti–VEGF treatment. 36 We previously showed the potential role of heparin in reducing migration and tube formation by cultured vascular endothelial cells and inhibiting the binding of VEGF and VEGFR2, a receptor responsible for the proangiogenic effect of this growth factor. 22 This process might also account for the therapeutic effect of heparin on CNV formation. In addition to confirming the results of earlier in vitro binding assays, 22 the present study added a novel aspect of heparin in reducing the secretion of VEGF by RPE cells only at higher concentrations compared with the lower concentrations through unknown mechanisms. Given that RPE is deemed a potential source of VEGF that underlies the development of CNV, 27 29 it seems plausible that heparin also suppresses VEGF signaling in CNV through both quantitative and qualitative mechanisms. Meanwhile, results of the in vitro binding assay showing almost no binding inhibition of TNF-α to surface GAGs and TNFRs by heparin indicated that it had little effect on TNF-α signaling. 
In conclusion, our study shows that intraocular heparin can serve as an inhibitor of multiple heparin-binding growth factors, such as VEGF and CCL2, through pleiotropic actions. The results raise the possibility of using soluble GAGs to regulate intraocular inflammatory diseases and vasoproliferative pathologies, including AMD. 
Footnotes
 Supported by Grant-in-Aid for Young Scientists B21791676.
Footnotes
 Disclosure: D. Tomida, None; K.M. Nishiguchi, P; K. Kataok, None; T.R Yasuma, None; E. Iwata, None; R. Uetani, None; S. Kachi, None; H. Terasaki, None
The GFP mouse strain (RBRC00267) was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. 
References
Ambati J Ambati BK Yoo SH Ianchulev S Adamis AP . Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Surv Ophthalmol. 2003;48:257–293. [CrossRef] [PubMed]
Penfold PL Madigan MC Gillies MC Provis JM . Immunological and aetiological aspects of macular degeneration. Prog Retin Eye Res. 2001;20:385–414. [CrossRef] [PubMed]
Hageman GS Luthert PJ Victor Chong NH Johnson LV Anderson DH Mullins RF . An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res. 2001;20:705–732. [CrossRef] [PubMed]
Donoso LA Kim D Frost A Callahan A Hageman G . The role of inflammation in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2006;51:137–152. [CrossRef] [PubMed]
Hollyfield JG Bonilha VL Rayborn ME . Oxidative damage-induced inflammation initiates age-related macular degeneration. Nat Med. 2008;14:194–198. [CrossRef] [PubMed]
Kikuchi M Nakamura M Ishikawa K . Elevated C-reactive protein levels in patients with polypoidal choroidal vasculopathy and patients with neovascular age-related macular degeneration. Ophthalmology. 2007;114:1722–1727. [CrossRef] [PubMed]
Seddon JM Gensler G Milton RC Klein ML Rifai N . Association between C-reactive protein and age-related macular degeneration. JAMA. 2004;291:704–710. [CrossRef] [PubMed]
Klein RJ Zeiss C Chew EY . Complement factor H polymorphism in age-related macular degeneration. Science. 2005;308:385–389. [CrossRef] [PubMed]
Edwards AO Ritter RIII Abel KJ Manning A Panhuysen C Farrer LA . Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308:421–424. [CrossRef] [PubMed]
Haines JL Hauser MA Schmidt S . Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005;308:419–421. [CrossRef] [PubMed]
Humphrey MF Moore SR . Microglial responses to focal lesions of the rabbit retina: correlation with neural and macroglial reactions. Glia. 1996;16:325–341. [CrossRef] [PubMed]
Yi X Ogata N Komada M . Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol. 1997;235:313–319. [CrossRef] [PubMed]
Sakurai E Anand A Ambati BK van RN Ambati J . Macrophage depletion inhibits experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003;44:3578–3585. [CrossRef] [PubMed]
Kvanta A Algvere PV Berglin L Seregard S . Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci. 1996;37:1929–1934. [PubMed]
Grossniklaus HE Ling JX Wallace TM . Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis. 2002;8:119–126. [PubMed]
Ferrara N Mass RD Campa C Kim R . Targeting VEGF-A to treat cancer and age-related macular degeneration. Annu Rev Med. 2007;58:491–504. [CrossRef] [PubMed]
Shi X Semkova I Muther PS Dell S Kociok N Joussen AM . Inhibition of TNF-alpha reduces laser-induced choroidal neovascularization. Exp Eye Res. 2006;83:1325–1334. [CrossRef] [PubMed]
Lichtlen P Lam TT Nork TM Streit T Urech DM . Relative contribution of VEGF and TNF-α in the cynomolgus laser-induced CNV model: comparing the efficacy of bevacizumab, adalimumab, and ESBA105. Invest Ophthalmol Vis Sci. 2010;51:4738–4745. [CrossRef] [PubMed]
Markomichelakis NN Theodossiadis PG Sfikakis PP . Regression of neovascular age-related macular degeneration following infliximab therapy. Am J Ophthalmol. 2005;139:537–540. [CrossRef] [PubMed]
Theodossiadis PG Liarakos VS Sfikakis PP Vergados IA Theodossiadis GP . Intravitreal administration of the anti-tumor necrosis factor agent infliximab for neovascular age-related macular degeneration. Am J Ophthalmol. 2009;147:825–830. [CrossRef] [PubMed]
Stringer SE . The role of heparan sulphate proteoglycans in angiogenesis. Biochem Soc Trans. 2006;34:451–453. [CrossRef] [PubMed]
Nishiguchi KM Kataoka K Kachi S Komeima K Terasaki H . Regulation of pathologic retinal angiogenesis and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate. PLoS ONE. 2010;5:e13493. [CrossRef] [PubMed]
Lange C Ehlken C Martin G . Intravitreal injection of the heparin analog 5-amino-2-naphthalenesulfonate reduces retinal neovascularization in mice. Exp Eye Res. 2007;85:323–327. [CrossRef] [PubMed]
Okabe M Ikawa M Kominami K Nakanishi T Nishimune Y . ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 1997;407:313–319. [CrossRef] [PubMed]
Kataoka K Nishiguchi KM Kaneko H van Rooijen N Kachi S Terasaki H . The roles of vitreal macrophages and circulating leukocytes in retinal neovascularization. Invest Ophthalmol Vis Sci. In press.
Kaneko H Nishiguchi KM Nakamura M Kachi S Terasaki H . Characteristics of bone marrow-derived microglia in the normal and injured retina. Invest Ophthalmol Vis Sci. 2008;49:4162–4168. [CrossRef] [PubMed]
Spilsbury K Garrett KL Shen WY Constable IJ Rakoczy PE . Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium engenders the development of choroidal neovascularization. Am J Pathol. 2000;157:135–144. [CrossRef] [PubMed]
Baffi J Byrnes G Chan CC Csaky KG . Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor. Invest Ophthalmol Vis Sci. 2000;41:3582–3589. [PubMed]
Oshima Y Oshima S Nambu H . Increased expression of VEGF in retinal pigmented epithelial cells is insufficient to cause choroidal neovascularization. J Cell Physiol. 2004;201:393–400. [CrossRef] [PubMed]
Lu B Rutledge BJ Gu L . Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J Exp Med. 1998;187:601–608. [CrossRef] [PubMed]
Ambati J Anand A Fernandez S . An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat Med. 2003;9:1390–1397. [CrossRef] [PubMed]
Luhmann UF Robbie S Munro PM . The drusenlike phenotype in aging Ccl2-knockout mice results from an accelerated accumulation of swollen autofluorescent subretinal macrophages. Invest Ophthalmol Vis Sci. 2009;50:5934–5943. [CrossRef] [PubMed]
Capila I Linhardt RJ . Heparin-protein interactions. Angew Chem Int Ed Engl. 2002;41:391–412. [CrossRef] [PubMed]
Salas A Sans M Soriano A . Heparin attenuates TNF-alpha induced inflammatory response through a CD11b dependent mechanism. Gut. 2000;47:88–96. [CrossRef] [PubMed]
Elner VM Burnstine MA Strieter RM Kunkel SL Elner SG . Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: immunochemical and in-situ hybridization analyses. Exp Eye Res. 1997;65:781–789. [CrossRef] [PubMed]
Campochiaro PA . Retinal and choroidal neovascularization. J Cell Physiol. 2000;184:301–310. [CrossRef] [PubMed]
Figure 1.
 
Reduction of CNV by intravitreal heparin injection. (A, B) Representative images of eyes treated with high-dose heparin (Hep; A) and PBS (B). (CE) Relative CNV size in the eyes treated with heparin compared with contralateral eyes treated with PBS. Eyes were treated with either low-dose (n = 8; C), medium-dose (n = 7; D), or high-dose (n = 5; E) heparin. Medium-dose and high-dose heparin reduced CNV size to a similar degree. (F, G) Relative CNV size in eyes treated with heparinase III (HIII) compared with contralateral eyes treated with PBS. Eyes were treated with HIII or PBS 24 hours before (n = 6; F) or immediately after (n = 6; G) laser photocoagulation. No difference was noted in the CNV size between the two conditions. Scale bar, 100 μm.
Figure 1.
 
Reduction of CNV by intravitreal heparin injection. (A, B) Representative images of eyes treated with high-dose heparin (Hep; A) and PBS (B). (CE) Relative CNV size in the eyes treated with heparin compared with contralateral eyes treated with PBS. Eyes were treated with either low-dose (n = 8; C), medium-dose (n = 7; D), or high-dose (n = 5; E) heparin. Medium-dose and high-dose heparin reduced CNV size to a similar degree. (F, G) Relative CNV size in eyes treated with heparinase III (HIII) compared with contralateral eyes treated with PBS. Eyes were treated with HIII or PBS 24 hours before (n = 6; F) or immediately after (n = 6; G) laser photocoagulation. No difference was noted in the CNV size between the two conditions. Scale bar, 100 μm.
Figure 2.
 
Distribution of FITC-labeled heparin after intravitreal injection. (AC) Distribution of injected heparin-FITC (green) 3 hours after laser treatment and GS staining (red). Injected heparin-FITC (arrowhead) was accumulated at the outer nuclear layer immediately adjacent to the laser injury (A; black arrow) and the GS-positive retinal vessels (B, C; white arrow). (DF) Distribution of injected control FITC. FITC colocalized modestly with GS-positive retinal vessels (white arrow). Note the control FITC was evenly distributed throughout all retinal layers. (A, D) Toluidine blue–stained retinal section was overlain with FITC staining. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; C, choroid; S, sclera. Scale bar, 100 μm.
Figure 2.
 
Distribution of FITC-labeled heparin after intravitreal injection. (AC) Distribution of injected heparin-FITC (green) 3 hours after laser treatment and GS staining (red). Injected heparin-FITC (arrowhead) was accumulated at the outer nuclear layer immediately adjacent to the laser injury (A; black arrow) and the GS-positive retinal vessels (B, C; white arrow). (DF) Distribution of injected control FITC. FITC colocalized modestly with GS-positive retinal vessels (white arrow). Note the control FITC was evenly distributed throughout all retinal layers. (A, D) Toluidine blue–stained retinal section was overlain with FITC staining. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; C, choroid; S, sclera. Scale bar, 100 μm.
Figure 3.
 
Effect of heparin on monocyte/macrophage infiltration. (A, B) Representative images of GS staining of CNV/laser scar and infiltrating monocytes/macrophages 24 hours after photocoagulation and intraocular injection. The number of GS-positive cells (some represented by filled arrowhead) outside the CNV/laser scar was reduced in the eyes treated with heparin (A) compared with the contralateral eyes treated with PBS (B). (C, D) Quantification of GS-positive cells inside and outside of CNV/laser scar (n = 9). The number of GS-positive cells was reduced outside the CNV/laser scar in the heparin-treated eyes. (E, F) Representative images of CNV/laser scar and GFP-positive cells (open arrowhead) 24 hours after photocoagulation and intraocular injection in mice that underwent BMT. (G, H) Quantities of GFP-positive cells inside and outside the CNV/laser scar. There were no differences between heparin-treated and PBS-treated eyes (n = 9). (A, B, E, F) Weak autofluorescence marked the boundaries of the CNV/laser scar. Scale bar, 100 μm.
Figure 3.
 
Effect of heparin on monocyte/macrophage infiltration. (A, B) Representative images of GS staining of CNV/laser scar and infiltrating monocytes/macrophages 24 hours after photocoagulation and intraocular injection. The number of GS-positive cells (some represented by filled arrowhead) outside the CNV/laser scar was reduced in the eyes treated with heparin (A) compared with the contralateral eyes treated with PBS (B). (C, D) Quantification of GS-positive cells inside and outside of CNV/laser scar (n = 9). The number of GS-positive cells was reduced outside the CNV/laser scar in the heparin-treated eyes. (E, F) Representative images of CNV/laser scar and GFP-positive cells (open arrowhead) 24 hours after photocoagulation and intraocular injection in mice that underwent BMT. (G, H) Quantities of GFP-positive cells inside and outside the CNV/laser scar. There were no differences between heparin-treated and PBS-treated eyes (n = 9). (A, B, E, F) Weak autofluorescence marked the boundaries of the CNV/laser scar. Scale bar, 100 μm.
Figure 4.
 
Decreased ARPE19-derived VEGF and CCL2 in the culture media by soluble heparin. (A, B) Measurements of VEGF and CCL2 secretion by cultured human RPE. Addition of lower concentrations of heparin increased APRE19-derived VEGF in the culture media compared with conditions without heparin (A). Further increasing the heparin concentration caused relative decreases in VEGF levels at higher doses. Meanwhile, exogenous heparin decreased the CCL2 level in the media in a dose-dependent manner (B). Supplementation of the media with TNF-α minimally elevated VEGF levels while increasing CCL2 concentrations to a much greater degree. No clear inhibitory effects on TNF-α–mediated release of VEGF and CCL2 by ARPE19 cells were noted. The experiment was conducted in triplicate and repeated.
Figure 4.
 
Decreased ARPE19-derived VEGF and CCL2 in the culture media by soluble heparin. (A, B) Measurements of VEGF and CCL2 secretion by cultured human RPE. Addition of lower concentrations of heparin increased APRE19-derived VEGF in the culture media compared with conditions without heparin (A). Further increasing the heparin concentration caused relative decreases in VEGF levels at higher doses. Meanwhile, exogenous heparin decreased the CCL2 level in the media in a dose-dependent manner (B). Supplementation of the media with TNF-α minimally elevated VEGF levels while increasing CCL2 concentrations to a much greater degree. No clear inhibitory effects on TNF-α–mediated release of VEGF and CCL2 by ARPE19 cells were noted. The experiment was conducted in triplicate and repeated.
Figure 5.
 
Binding inhibition of VEGF and VEGFR2 or CCL2 and CCR2 by heparin or porcine ocular fluid in vitro. (A, B) Inhibition of interaction between VEGF and surface heparin or VEGFR2 by soluble heparin or porcine ocular fluid. Binding of VEGF and VEGFR1 was unaffected. (C) Molecular size effect of heparin on the VEGF-surface heparin interaction. Function of heparin as an inhibitor of VEGF-surface heparin binding was positively correlated with heparin size. (D, E) Inhibition of interaction between CCL2 and surface heparin or CCR2 by soluble heparin or ocular fluid. (F, G) No clear inhibition of interaction between TNF-α and surface heparin or TNFRs by soluble heparin or ocular fluid. Data are presented as averages of three measurements for each experimental condition. The experiment was repeated. OF, porcine ocular fluid; mw, molecular weight.
Figure 5.
 
Binding inhibition of VEGF and VEGFR2 or CCL2 and CCR2 by heparin or porcine ocular fluid in vitro. (A, B) Inhibition of interaction between VEGF and surface heparin or VEGFR2 by soluble heparin or porcine ocular fluid. Binding of VEGF and VEGFR1 was unaffected. (C) Molecular size effect of heparin on the VEGF-surface heparin interaction. Function of heparin as an inhibitor of VEGF-surface heparin binding was positively correlated with heparin size. (D, E) Inhibition of interaction between CCL2 and surface heparin or CCR2 by soluble heparin or ocular fluid. (F, G) No clear inhibition of interaction between TNF-α and surface heparin or TNFRs by soluble heparin or ocular fluid. Data are presented as averages of three measurements for each experimental condition. The experiment was repeated. OF, porcine ocular fluid; mw, molecular weight.
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