【摘要】 目的：血小板源性生长因子(plateletderived growth factor， PDGF)可引起增殖性玻璃体视网膜病变。酪氨酸激酶的激活对生长因子介导的细胞增殖起着重要作用。本研究评价特异性的PDGFα受体酪氨酸激酶阻断剂AG1296和β受体酪氨酸激酶阻断剂AG1295对兔PVR的治疗作用。方法：兔结膜成纤维细胞（rabbit conjunctival fibroblasts，RCF）培养，用MTT法检测PDGFAA 和BB 以及AG1295和AG1296对 兔RCF增殖状况的影响。眼视网膜电图检查和HE染色分析药物的毒性。 建立PVR动物模型，玻璃体腔内分别给予AG1295和AG1296，用牵引性视网膜脱离（tractional retinal detachment， TRD）的发生率评价药物的体内疗效。结果：体外10μmol/L的AG1295和AG1296均可显著抑制由PDGFAA 和BB 诱导的成纤维细胞的增生，体内100μmol/L AG1295和AG1296均减缓了兔TRD的发生，但AG1295的作用仅持续至14d。相同浓度的AG1296和AG1295相比，作用更持久。在两个治疗组中，均未发现明显的视网膜毒性。结论：特异性的PDGFα受体酪氨酸激酶抑制剂AG1296可显著抑制兔TRD的发生，其作用明显强于PDGFβ受体酪氨酸激酶抑制剂AG1295，提示PDGF对PVR的促进作用主要由 α受体介导，这一通路的阻断可能成为治疗PVR的一种方法。
【关键词】 血小板源性生长因子；受体酪氨酸激酶；AG1295； AG1296；增殖性玻璃体视网膜病变
AbstractAIM: Receptor tyrosine kinase (RTK) activation is critical for growth factormediated cell proliferation. The present study was designed to determine the effect of the tyrphostin AG1295 and AG1296, a selective blocker of PDGF β and α RTK, on proliferative vitreoretinopathy (PVR)development.METHODS: Rabbit conjunctival fibroblasts (RCF) cells were cultured. The effects of AG1295, AG1296, PDGFAA and PDGFBB on RCF proliferation are evaluated by MTT assay. Homologous rabbit conjunctival fibroblasts were injected intravitreally to make animal PVR model, followed by injection of 100μmol/L of AG1295 or AG1296 respectively. The presence of tractional retinal detachment (TRD) was assessed to evaluate the effect of AG1295 and AG1296 in vivo. Electroretinography and histologic studies were performed after intravitreal injection of AG1295 into untreated eyes to evaluate toxicity.RESULTS: Both AG1295 and AG1296 (10μmol/L) significantly inhibited rabbit conjunctival fibroblast cell growth stimulated by PDGFAA or BB in vitro. Development of TRD was significantly reduced (P<0.05) with 100 μmol/L of AG1295 or AG1296 in vivo, but the effect of AG1295 only present till day 14. Inhibitive effect of AG1296 is longer than that of AG1295. No significant histologic or retinal functional damage was found in both drugtreated groups.CONCLUSION: PDGF α and β receptor specific inhibitor AG1296 and AG1295 attenuated PVR without significant side effects in rabbits, and AG1296 was better than AG1295. The much longer and stronger therapeutic effect from PDGF α receptor inhibitor indicated that PDGF α receptor is more important in the development of PVR, and inhibition of this pathway could be a useful treatment alternative to prevent PVR.
KEYWORDS: plateletderived growth factor；receptor tyrosine kinase; AG1295; AG1296;proliferative vitreore tinopathy
INTRODUCTION
Proliferative vitreoretinopathy (PVR) is a serious complication of rhegmatogenous retinal detachment and severe ocular trauma and is a leading cause of surgical failure. PDGF, a potent chemoattractant and mitogen for both fibroblasts and RPE cells [1,2], not only promotes dedifferentiation of RPE cells [3] and also enhances contraction of RPE cells and fibroblasts in collagen gel [4,5]. PDGF has five isoforms, PDGFAA, BB, AB, CC, and DD[6], which interact differentially with structurally related receptors designated α and β receptors; each receptor has an extracellular component with five immunoglobulinlike domains and an intracellular component with a tyrosine kinase domain containing a characteristic insert sequence [7]. Tyrosine kinase is activated following binding of the ligand to its receptor and plays a crucial role in signal transduction pathways that regulate a number of cell functions such as proliferation and differentiation, both during normal physiology and in a variety of pathologic disorders [8]. We had ever tried using a specific inhibitor of PDGF β receptor tyrosine kinase, AG1295, to treat rabbit PVR and found this drug is effective to inhibit PVR in the early stage. Ikuno et al found that fibroblasts expressing PDGF α receptors have a stronger intrinsic ability to induce PVR in animal models than those expressing β receptors [9]. Another study showed that TGFβ1dependent contraction of fibroblasts is mediated by the PDGF α receptor[10]. Although the detailed mechanism is not clear at present, they indicated that PDGF receptor might be involved in both proliferation and contraction stages of PVR and plays a more important role than that of PDGFβ receptor.
In the present study, we compared the effect of AG1295 and AG1296, specific inhibitor of PDGF β and α receptor RTK respectively, on the progression of rabbit PVR, to try to shed more light on the role of PDGF β and α receptor .
MATERIALS AND METHODS
Animal Preparation All animal experiments were conducted according to the tenets of the Association of Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Experimental manipulations were performed on left eyes only. Seventythree pigmented rabbits of either sex weighing 1.52.0kg were included in the study. Before each procedure, the rabbits were anesthetized with intramuscular injections of ketamine hydrochloride (35mg/kg) and xylazine hydrochloride (5mg/kg). The pupils of experimented eyes were dilated with 5g/L tropicamide and 5g/L phenylephrine hydrochloride, and the cornea was anesthetized with 4g/L oxybuprocaine hydrocholoride eyedrops.
After performing a 0.2mL anterior chamber paracentesis, 0.4mL of pure perfluoropropane (C3F8) gas was injected into the vitreous cavity using a 30gauge needle to induce vitreous liquefaction. Cryopexy and fluidgas exchange were not performed to avoid associated complications [11].
Cytotoxicity Studies To confirm the safety of the drugs, the retina was examined after injection of AG1295 or AG1296 (Calbiochiem, Germany). The AG1295 and AG1296 were prepared at a concentration of 100mol/L in dimethyl sulfoxide (DMSO) as a working solution. Then 74μg of AG1295 or 68μg of AG1296 which was diluted into 3μL of DMSO respectively, was dissolved in 0.1mL of balanced saline solution (BSS plus; Santen, Osaka, Japan) and injected into the rabbit vitreous, resulting in a final concentration of approximately 100μmol/L in vivo. The drug was repeatedly injected every week for 28 days into the left eyes, and the fellow control eyes received the same volume of DMSO in 0.1mL BSS. So for both eyes, the intravetreal concentration of DMSO is 1g/L. Ten rabbits were included in AG1295 and AG1296 trial respectively. The eyes were examined by indirect ophthalmoscopy and fundus videography (Topcon, Tokyo, Japan), and scotopic electroretinograms (ERG) were recorded just before AG1295 or AG1296 was injected and at 3, 7, 14, and 28 days after the first injection of drug. The details of each examination are described below.
ERG Studies The eyes were fully dilated and the rabbits the anesthetized and darkadapted for 1 hour. A contact lens electrode (Kyoto Contact Lens, Kyoto, Japan) was placed on the anesthetized cornea (oxybuprocaine hydrochloride 4g/L).
Topical methylcellulose 10g/L was used as the conducting medium. The reference and ground electrodes were made of stainlesssteel surgical needles, which were inserted into the neckback and one ear respectively. The ERG then was recorded (Neuropack 2; Nihon Koden, Tokyo, Japan). The light stimulator was 20cm above the cornea. The darkadapted responses were evoked by a conventional fullfield flash unit that produced flashes with a maximum intensity of 5.76 cd·s/m2. Six waves taken every 60 seconds were averaged for both treated and untreated eyes. To overcome individual and daily variances, the Bwave ratios (the amplitude of the wave in the treated eye divided by its amplitude in the control eye) were calculated [12].
Histologic Studies The animals were sacrificed and the eyes were enucleated on 14, 21, and 28 days. The eyes were cut circumferentially at the limbus to make posterior cups and then fixed in 40g/L paraformaldehyde and 1g/L glutaraldehyde in 0.1mol/L phosphatebuffered saline (PBS) at 4℃ overnight. The specimens were rinsed with distilled water for 30 minutes, dehydrated in a graded ethanol and xylene series, and embedded in paraffin. Sections 2 to 4μm thick were stained with hematoxylin and eosin (HE).
Rabbit RPE Cell Culture Rabbit RPE cell cultures were obtained from pigmented rabbits by the method of Flood et al [13] with slight modification. Briefly, the freshly enucleated eyes were immediately submerged in the RPE medium consisting of Eagle’s minimal essential medium （EMEM）(Gibco, Grand Island, NY), 100mL/L fetal bovine serum (BioSource International California, USA) and antibiotics ( penicillin at 100kU/L, streptomycin at 100mg/L). The globes were opened and cornea, lens and vitreous humor were removed by a circumferential cut just posterior to the ora serrata. The neural retina was carefully washed out by the RPE medium. The eye cups were washed with Hanks balanced salt solution (HBSS), and were digested with 0.12g/L tripsin (Nacalai Tesque, Inc. Kyoto Japan) in 0.05g/L EDTA (Nacalai Tesque, Inc. Kyoto Japan) for 1 hour at 37℃. The tripsinization was stopped by adding excessive RPE medium. The dissociated RPE cells were carefully washed out without disturbing the underlying choroids. The RPE cells were first cultured in 12well plates to near confluence with the RPE medium, and then were passed to 25cm2 flasks. The cells were trypsinized and passaged every week.
Cell Proliferation Assay Proliferation of RPE was measured by MTT [3, (4, 5dimethyl2thiazolyl) 2, 5diphenylte 2Htetrazolium bromide] using a commercially available kit purchased from Nacalai Tesque, Kyoto, Japan. Cells were planted in RPE medium containing 100mL/L FBS at a density of 1×104 cells/well in 96 well plates and allowed to adhere for 18 to 24 hours. Cultures were then washed once with PBS and fed with fresh EMEM with 5mL/L FBS containing dilutions of PDGFAA (50 μg/L, R & D Systems, Minneapolis, USA), BB (50μg/L, R&D Systems, Minneapolis, USA) with or without AG1295 or AG1296 (50μmol/L). The cells were incubated for another 72 hours, and finally the cells were treated with 5g/L of MTT for 4 hours at 37℃. MTT solution was aspirated and the formazan crystals were dissolved in detergent reagent for 10 minutes. Relative cell number was determined based on the optical absorbance of the formazan at 570nm using a control wavelength of 655nm measured in an automatic plate reader (BIORAD Model 450, BIORAD Laboratories 2000 Alfred Nobel Drive Hercules, California). AG1295 and AG1296 stock solutions were made in dimethylsulfoxide (DMSO) and diluted in culture medium before adding to the cells. To exclude the effect of DMSO, the concentration of DMSO was decreased to 1g/L, the same as that of in vivo experiment, and sham treatments were used as control.
Experimental PVR Animal Model PVR was induced in the left eyes of 35 pigmented rabbits as described previously [14]. Briefly, gas vitrectomy was performed by injecting 0.4mL C3F8 into the vitreous cavity at 4mm posterior to the corneal limbus. This was through retina after anesthesia was induced. 10 days later, 0.1mL of RPE medium containing 1×105 of RPE cells was injected into the vitreous cavity together with 0.1mL of platelet rich plasma (PRP) using a 30gauge needle. The 6th passage RPE cells were used in this model.
In the treated group, the experimental eye of each rabbit ( n=18) was injected with 74μg AG1295 or 68μg AG1296 dissolved by 3μL DMSO in 0.1mL BSS immediately after cell injection to achieve a final intraocular concentration of 100μmol/L AG1295 or AG1296. For the control group (n=17), 3μL DMSO in 0.1mL BSS was injected. On 7, 14, and 21 days, the treated rabbits continuously received the same volume of AG1295 or AG1296 injection, while rabbits in the control group received a sham treatment.
Each eye was examined by indirect ophthalmoscopy, and fundus video photos were taken at 3, 7, 14, 21, and 28 days after the RPE injection. The development of PVR was evaluated on videography by masked fashion and the PVR was graded according to the scale of Fastenberg et al[15].
Statistical Analysis Students ttest was used for statistical analysis of RPE proliferation and Fishers exact test were used for statistical analysis of tractional retinal detachment. A P value less than 0.05 was considered to be statistically significant.
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