Neovascularization in the cornea is induced by wounding and a wide variety of pathologic conditions, including alkalinity, burns, and inflammation. In the healthy mammalian eye, blood vessels normally are excluded from the cornea and vitreous compartments, which possess antiangiogenic activity. Failure to exclude vessels from the cornea is associated with loss of visual acuity, opacification, and abnormal healing. 1 The mechanisms of corneal neovascularization have been investigated, and various mediators are involved in the process, such as basic fibroblast growth factor (bFGF), 2 vascular endothelial growth factor, 3 and platelet-derived endothelial cell growth factor. 4 In particular, bFGF has been demonstrated to be a major factor in corneal neovascularization. bFGF is released from the cell surface and extracellular matrix by various stresses, including mechanical cell injury and inflammation, and plays an important role in the migration and proliferation of vascular endothelial cells, resulting in formation of a capillary network.
Recent studies have identified potent antiangiogenic factors, including angiostatin, 5 endostatin, 6 thrombospondin-1, 7,8 pigment epithelium–derived factor (PEDF), 9 chondromodulin-1, 10 and platelet factor-4. 11,12 When used for corneal and conjunctival surface reconstruction, amniotic membrane facilitates epithelialization and reduces inflammation, vascularization, and scarring. 13–19 The antiangiogenic activity of amniotic membrane may be controlled by two independent extracellular matrix– and soluble factor–derived mechanisms. Amniotic membrane contains large amounts of extracellular matrix proteins such as collagen α2(IV), laminin-1 and laminin-5, fibronectin, and collagen type VII, which are involved in suppression of corneal neovascularization. 20 On the other hand, recent studies have shown that amniotic cells (AC), including epithelial and mesenchymal cells, produce various kinds of cytokines, including antiangiogenic factors. 19 In this study, antiangiogenic activity in AC culture supernatants was examined using in vivo and in vitro assay systems for neovascularization. The results suggest the applicability of the culture supernatant for treatment of corneal disorders with neovascularization.
MATERIALS AND METHODS
Preparation of Culture Supernatant
In accordance with a protocol approved by the Fukushima Medical University Committee on Human Research, human placentas were obtained shortly after elective cesarean section. The placentas were cleaned with sterile Earle's balanced saline solution containing 50 μg/mL of penicillin, 50 μg/mL of streptomycin, 100 μg/mL of neomycin, and 2.5 μg/mL of amphotericin B, and amniotic membrane was prepared by a method previously described. 21 The amnion was separated by blunt dissection through the potential spaces between these two tissues. The resultant membrane was cut into a 2 × 2 cm 2 disk and treated with collagenase (Sigma Chemical Co., St. Louis, MO, U.S.A.) by incubating at 37°C in a humidified, 5% CO 2 and 95% air atmosphere for 5 hours. The digestion was stopped by addition of fetal calf serum (FCS) to a final concentration of 10%, and tissue debris was removed with a strainer. The cells were collected by gentle centrifugation, resuspended in culture medium (RPMI 1640 containing 10% FCS), and plated into tissue culture flasks. Cells were maintained in the humidified incubator with medium change three times a week. When cells had grown to confluence, the medium was replaced with serum-free RPMI 1640 containing 0.1% bovine serum albumin (BSA), and the culture continued for 24 hours. The resultant supernatant was collected for use in this study.
Corneal Neovascularization Assay
All animal studies were conducted according to the Statement for the Use of Animals in Ophthalmic and Vision Research. Female New Zealand white rabbits (3–3.5 kg) were anesthetized systemically with an intramuscular injection of pentobarbital, and locally with one drop of 0.4% oxybuprocaine on the eye. For assessment of neovascularization in vivo, we used a corneal micropocket assay. 22 In brief, an aliquot of bFGF (Genzyme, Cambridge, MA, U.S.A.) solution was added to 2 μL of 12% (wt/vol) polymer (Hydron type NCC; Interferon Science, New Brunswick, NJ, U.S.A.) in ethanol. Four-microliter aliquots of the mixture were placed on Parafilm (American National Can, Greenwich, CT, U.S.A.) and allowed to dry to produce pellets, each containing 100 ng of bFGF. A partial incision was made 5 mm from the limbus to dissect the corneal stroma and the pellet was placed in this pocket (referred to as day 0). AC culture supernatant was administered from day 1 as a drop in the right eye every day, three times per day, while serum-free medium containing 0.1% BSA was similarly administered to the left eye as a control. Corneal neovascularization induced by pellets containing bFGF was photographed using a slit lamp (Zeiss 30SL-M, Jena, Germany) and the area with angiogenesis was evaluated using NIH Image software. Statistical analysis was carried out using the Student t test. For histologic examination, eyes were collected from the animals at day 10. The tissue samples were enucleated and fixed in 4% paraformaldehyde and 5% glutaraldehyde. The 0.4-μm sections were stained by hematoxylin and eosin and observed with a light microscope.
Migration was examined by a double-chamber method 23 using human umbilical vein endothelial cells (HUVEC; Kurabo, Osaka, Japan). HUVEC were cultured in the growth medium, HuMedia (Kurabo), supplemented with 2% FCS, 10 ng/mL of human epidermal growth factor (EGF), 5 ng/mL of human bFGF, 1 μg/mL of hydrocortisone, 50 μg/mL of gentamicin, 50 ng/mL of amphotericin B, and 10 μl/mL of heparin at 37°C in a humidified, 5% CO 2 and 95% air atmosphere. When the cells had grown to confluence, the medium was replaced with growth factor–free, starvation medium—HuMedia containing 2% FCS—and cultivated for 36 hours. Endothelial cell invasion was assessed using transwell inserts of 8.0-μm pore size (Falcon, Franklin Lakes, NJ, U.S.A.). Three hundred microliters of HUVEC suspension in starvation medium (2 × 10 4 cells) was plated into the upper insert. Seven hundred microliters of medium containing 100 ng/mL of bFGF and various amounts of AC culture supernatant was prepared in a 24-well culture plate. An insert was set in each well of this 24-well plate and the cells were incubated for 4 hours. The cells that had migrated to the distal side of the filter were stained with Giemsa, and the number of migrating cells was counted. The results represent the mean ± standard error of the mean (SE) of three independent experiments.
Cell Growth Assay
Cell growth was determined by an assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma). HUVEC were seeded in 96-well microplates (2 × 10 4 cells/well), and incubated in the growth medium at 37°C overnight. The cells were washed and further incubated in the growth medium in the presence or absence of AC culture supernatant for 4 days, with a medium change on the third day. Two hours before the termination of culture, a 5-μL aliquot of MTT (5 mg/mL) was added to each well. At the end of the incubation, 100 μL of dimethyl sulfoxide was added to each culture to solubilize the formazan complex. The optical density at 590 nm was measured using a 96-well multiscanner (Immuno-Mini; Nihon Intermed Co., Tokyo, Japan). The results represent the mean ± SE of three independent experiments.
Suppression of bFGF-Induced Corneal Neovascularization by AC Culture Supernatant
The AC culture supernatant was examined for antiangiogenic activity using a corneal micropocket assay. When a bFGF-containing pellet was implanted in the corneal stroma, neovascularization from the limbus was observed as early as day 3, and a large number of very fine capillaries was observed between the limbus and the pellet on day 8. The control pellet did not induce any neovascularization up to day 14 (data not shown). Using this system, we assessed the effect of AC culture supernatant on neovascularization; AC culture supernatant and control medium were administered in the right and left eye, respectively. As shown in Fig. 1, the area of bFGF-induced neovascularization for control eyes increased in a time-dependent manner and reached a plateau on day 8 after transplantation. In contrast, that for the supernatant-treated eyes did not show any significant changes. Among seven rabbits examined, five showed a significant difference between the right and left eye, whereas two did not respond to the culture supernatant (Fig. 2). Quantitative image analysis for the seven rabbits at day 10 revealed the areas of neovascularization to be 13.2 ± 3.2 mm 2 and 4.0 ± 1.4 mm 2 for the control and treated eyes, respectively (n = 7, p = 0.021). When five rabbits (#1–#5 in Fig. 2) are selected for the analysis, the values are 16.1 ± 3.7 mm 2 and 2.7 ± 1.5 mm 2 for the control and treated eyes, respectively (n = 5, p = 0.009). Thus, corneal neovascularization was significantly inhibited by treatment with AC culture supernatant.
Each eye was examined for histology. As shown in Fig. 3, a number of typical capillaries as well as inflammatory infiltrates were observed in the area between the transplant and corneal stroma in the control eye. By contrast, very little change was observed in the eye treated with AC culture supernatant.
Inhibition of HUVEC Migration and Growth by AC Culture Supernatant
To understand better the antiangiogenic activity in the AC culture supernatant, we examined its effects on endothelial cell migration and growth using HUVEC. bFGF is a potent angiogenic factor, and our HUVEC migration system actually worked when bFGF (100 ng/mL) was used as a chemoattractant. An aliquot of AC culture supernatant added to the bFGF-containing medium suppressed the chemoattractant activity of bFGF in a dose-dependent manner (Fig. 4). Just 50 μL of the AC culture supernatant was enough to suppress significantly the migration-inducing activity of 100 ng/mL bFGF. The control medium did not suppress this activity at all.
We next examined the effect of the culture supernatant on bFGF-induced growth using HUVEC. bFGF potentially induced endothelial cell growth, and at 100 ng/mL actually enhanced the growth of HUVEC in our system. An aliquot of AC culture supernatant added to the bFGF-containing medium suppressed the growth-inducing activity of bFGF in a dose-dependent manner (Fig. 5). Just 5 μL was enough to abrogate the growth-inducing activity of 100 ng/mL bFGF, whereas the control medium did not suppress growth at all.
Neovascularization in the cornea induced by wounding and pathologic conditions often causes a loss of visual acuity with opacification and abnormal healing. It has been reported that transplantation of amniotic membrane is effective for the reconstruction of the ocular surface by induction of epithelialization 13 and reduction of inflammation, vascularization, and scarring. 18,19 In this study, we demonstrated that the administration of AC culture supernatant as an eye drop significantly suppressed bFGF-induced angiogenesis in a rabbit corneal model. We further revealed that the antiangiogenic activity of the AC culture supernatant is explained by its inhibitory effect on migration and growth of vascular endothelial cells. These results suggest that AC produces cytokine-like soluble factors that strongly abrogate migration and proliferation of vascular endothelial cells induced by angiogenic factors such as bFGF. Moreover, given these results, the AC culture supernatant may be applicable to treatment of corneal diseases with neovascularization. Administering the AC culture supernatant as eye drops would be effective, easy, and convenient.
The results presented here add to our understanding of the biology of the amniotic membrane. It is well known that amniotic membrane contains large amounts of extracellular matrix, and alkaline-treated, cell-free amniotic membranes possess antiangiogenic activity. 20 Presumably, the extracellular matrix induces cell differentiation and suppresses immunologic responses. On the other hand, our results support the hypothesis that epithelial-like and mesenchymal cells derived from amniotic membrane produce soluble factors that abrogate corneal neovascularization. To date, several antiangiogenic cytokines have been reported, including angiostatin, 5 endostatin, 6 thrombospondin-1, 7,8 PEDF, 9 chondromodulin-1, 10 and platelet factor-4. 11,12 In addition, Hao et al., using reverse transcriptase polymerase chain reaction, showed that human amniotic epithelial and mesenchymal cells express antiangiogenic factors such as interleukin-1 receptor antagonist, tissue inhibitors of metalloproteases, collagen 18, interleukin-10, and thrombospondin-1. 19 Our preliminary study on isolating antiangiogenic factors from the AC culture supernatant showed that the supernatant contained lentil lectin–binding factors, suggesting that the AC produce PEDF at least. Further study is required to identify these AC-derived antiangiogenic factors.
A previous attempt at purification revealed that there were angiogenic and mitogenic activities in normal amniochorionic membrane cultures. 24 It also has been shown that AC contain angiogenic factors such as EGF, keratinocyte growth factor, hepatocyte growth factor, and bFGF. 25 In addition, the clinical application of amniotic membrane in corneal transplantation has had mixed results. 26 Moreover, among seven rabbits examined in the current study, two were unresponsive to treatment with AC supernatant (Fig. 2). On the other hand, very little antiangiogenic activity was observed in one lot among four independent AC culture supernatants derived from different amniotic membranes (data not shown). This single case did not affect the bFGF-dependent migration and growth of HUVEC in our in vitro model. Therefore, it is suggested that amniotic membrane and AC possess angiogenic and antiangiogenic activities–that is, a net balance between positive and negative regulators of neovascularization may be important for expression of the antiangiogenic activities of the AC culture supernatant. Although AC consisted of epithelial and mesenchymal cells, more than 85% of cells exhibited epithelial properties in our preparations. Thus, the ratio between two cell types may not be responsible for the different antiangiogenic activities. Rather, it is possible that amniotic epithelial cells consist of different cell types. Identification of antiangiogenic factors will be crucial to understanding this issue.
In summary, our study suggests that AC culture supernatant contains strong inhibitors of bFGF-induced neovascularization. The effect is explained in part by inhibition of migration and growth of vascular endothelial cells. AC culture supernatant may be applicable to the treatment of corneal diseases with neovascularization.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
Antiangiogenesis; Amniotic cells; Corneal neovascularization; Basic fibroblast growth factor; Vascular endothelial cells; Migration; Cell growth