作者：江伟 邱春亿
作者单位：美国德州大学城德州农工大学健康科学中心医学院眼科药理研究所
【摘要】 目的：研究胼酞嗪对兔脉络膜血流和激光诱导的鼠脉络膜新生血管及人脐静脉内皮细胞管状结构形成的影响。
方法：雌性新西兰白兔左眼眼内压升高至40mmHg后，滴入10g/L胼酞嗪滴眼液，采用彩色微球技术测量眼血流变化。用Nd:YAG激光诱导雄性 Brown Norway大鼠至Bruch膜破裂，而后分别予生理盐水或5，10和20g/L胼酞嗪滴眼液点眼，3次/d，连续4wk。采用眼底荧光血管造影和脉络膜平片测量新生血管的面积。此外亦用不同浓度的胼酞嗪作用于培养之人脐静脉内皮细胞并探讨对其管状结构形成的影响。
结果：10g/L胼酞嗪滴眼液滴入眼内压为40mmHg的兔眼30,60min后，脉络膜血流明显增加。眼底荧光血管造影和脉络膜平片测量均显示，经过4wk药物治疗，5，10，20g/L胼酞嗪滴眼液均明显抑制了鼠眼CNV的形成。3～30mg/L胼酞嗪对在基质凝胶中培养了48h的人脐静脉内皮细胞的管状结构的形成有抑制作用。
结论：胼酞嗪可以抑制体内脉络膜新生血管及体外培养的人脐静脉内皮细胞管状结构的形成，并增强缺血后的兔脉络膜血流。胼酞嗪有望成为治疗年龄相关性黄斑变性的药物。
【关键词】 胼酞嗪 脉络膜 新生血管 人脐静脉内皮细胞 年龄相关性黄斑变性
INTRODUCTION
Angiostatin is a proteolytic fragment of plasminogen (kringle 1 4). It was identified as a potent angiogenic inhibitor which blocks neovascularization of diabetic retinopathy and suppresses tumor growth and metastases[1]. The mechanism responsible for the antiangiogenic activity of angiostatin is currently uncertain. However, angiostatin has been found to inhibit the VEGF(vascular endothelial growth factor) and bFGF(basic fibroblast growth factor)induced activation of the p42/p44 MAP kinase[2]. Recent study has suggested that decreased angiostatin levels in the vitreous may play a role in the development of proliferative diabetic retinopathy[3]. Moreover, recombinant angiostatin has been shown to block retinal neovascularization in a rat model of oxygeninduced retinopathy of prematurity (ROP)[4].
In this study, we have determined the effect of angiostatin on vascular permeability in oxygeninduced ROP and researched into its possible mechanism.
MATERIALS AND METHODS
Animals Timepregnant Brown Norway rats were purchased from Harlan (Indianapolis, IN). Care, use, and treatment of all animals in this study were in strict agreement with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
ROP Model and Intravitreal Injection of Angiostatin ROP was induced as described by Smith method with some modifications[4]. Briefly, rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O2) for 5 days (P712) and then returned to normoxia (room air) to induce retinopathy.
Angiostatin was purchased from Angiogenesis Research Industries, Inc (Chicago, IL) and reconstituted in sterile PBS and diluted to desired concentrations. Angiostatin solution was injected into the vitreous of the right eye (3μL each eye) of the anesthetized rats at P14 through the pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. After the injection, the animals were kept in normoxia until they were analyzed.
Measurement of Vascular Permeability Vascular permeability was quantified by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method following a documented protocol[5]with minor modifications. The rats were anesthetized and Evans blue (30mg/kg) was injected through the femoral vein. The rats were kept on a warm pad for 2 hours to ensure the complete circulation of the Evans bluealbumin complex. Then the rats were perfused via the left ventricle with prewarmed 4g/L paraformaldehyde in citrate buffer (pH=4.2) for 2 minutes under the physiological pressure to clear the dye from the vessel. Immediately after perfusion, the eyes were enucleated and the retina and iris were carefully dissected under an operating microscope. Evans blue dye was extracted by incubating each sample in 150μL of formamide (Sigma) for 18 hours at 70℃. The extract was centrifuged at 70000r/min (Rotor type: TLA1003,TL;Beckman) for 20 minutes at 4℃. Absorbance was measured using 100μL of the supernatant at 620 nm. The concentration of Evans blue in the extracts was calculated from a standard curve of Evans blue in formamide and normalized by the total protein concentration in the tissue. Results were expressed as micrograms of Evans blue per milligrams of total protein in the tissues.
Western Blot Analysis and Immunohistochemistry of VEGF VEGF Western blot analysis was performed as described previously[6]. Immunohistochemistry was carried out following a documented protocol[7]. Briefly, retinal sections were incubated with 1∶100 dilution of the antiVEGF antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4℃. After extensive wash, the sections were incubated with biotin labeled monoclonal antirabbit antibody for 60 minutes at 37℃, and then developed using the ABC Complex (Vector Laboratories, Burligame, USA), with 3.3’ diaminobenzidine (0.25g/L in 0.05 mmol/L Tris, pH 7.4, containing 0.3mL/L hydrogen peroxide) as a chromogen.
Statistical Analysis Statistical analysis employed the Students t test. The paired t test was used for comparison of the angiostatininjected eye with the PBSinjected contralateral controls from the same animal, while the unpaired test was used for interanimal comparison.
RESULTS
The Oxygeninduced ROP Rats Showed Significant Increases of Vascular Permeability in the Retina and Iris Vascular permeability was measured in the retina and iris of ROP rats at age P16 (4 days after the rats returned to normoxia) and compared with those in the agematched normal rats. ROP rats showed significant increases of vascular permeability in the retina and iris (P<0.01) (Figure 1).
To evaluate the influence of intravitreal injection on vascular permeability, ROP rats and agematched normal rats received an intravitreal injection of 3 μL of sterile PBS into the right eye at age P14. The retinal vascular permeability was measured
Figure 1 Vascular permeability in oxygeninduced ROP rats
Figure 2 Angiostatin dosedependent reduction of vascular permeability in retina of ROP rats 1：normal rats received an injection of PBS；2：ROP rats with a PBS injection;3, 4 and 5：ROP rats with an injection of 1.88, 3.75 and 7.5 μg/eye of angiostatin, respectively. Vascular permeability in the retina was measured using the Evans blue method and normalized by total protein concentrations. Permeability was expressed as μg of Evans blue per mg of protein (mean±SD, n=4) aP<0.05,bP<0.01 vs Group 2at P16, 2 days following the injection. Intravitreal injection of PBS significantly increased permeability in the retina and iris over that in the uninjected contralateral eye (P<0.05),(Figure 1), possibly due to injury responses. In both tissues, ROP rats with PBS injection showed significantly higher vascular permeability than those in the agematched normal rats with the same injection, suggesting that intravitreal injection does not affect the comparison between the ROP rats and their normal controls (Figure 1).
Angiostatin Reduces Vascular Permeability in a Dosedependent Manner in the Retina of ROP Rats To determine the effect of angiostatin on vascular permeability, ROP rats (P14) received an intravitreal injection of 3μL of angiostatin with different concentrations into the right eyes, to reach doses of 1.88, 3.75 and 7.5 μg each eye, and the same volume of PBS into the left eyes for controls. Vascular permeability was measured at P16 using the Evans blue method. In the eyes injected with angiostatin, vascular permeability was reduced in an angiostatin dosedependent manner (Figure 2). At dose of 3.75 and 7.5μg each eye, angiostatin decreased the vascular permeability, respectively (P<0.05,P<0.01, respectively), while the low dose of angiostatin (1.88μg each eye) showed no significant reduction in permeability (Figure 2). No significant reduction of vascular permeability was detected in the iris of ROP rats after the injection of angiostatin at all the doses used.
Figure 3 Time course of the angiostatininduced reduction in vascular permeability The permeability was normalized by the total retinal protein concentrations (mean±SD, n=4)aP<0.05,bP<0.01, vs PBS
Figure 4 Angiostatinmediated downregulation of VEGF expression in the retina of ROP rats Retinal VEGF levels were determined by Western blot analysis using an antiVEGF antibody 1 day after the injection. The same membranes were striped and reblotted with the antiβactin antibody
Time Course of the Angiostatin Induced Reduction of Vascular Permeability in Retina of ROP Rat At P14, the right eye of ROP rats received an intravitreal injection of angiostatin (7.5 μg each eye) and left eye received PBS as the control. Vascular permeability was measured at 1, 2 and 3 days after the injection. Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2fold (P<0.05 , P<0.01, respectively) at P15 and P16, respectively (Figure 3).At P17,3 days after the injection, vascular permeability returned to the level of PBSinjected contralateral control.
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