【摘要】 促红细胞生成素（Epo）是促红细胞生成的因子，由胎儿肝脏和成人肾脏产生，是贫血及缺氧时的一种应答反应。视网膜异常血管形成，如早产儿视网膜病变（ROP），增殖性糖尿病性视网膜病变（PDR）时Epo水平增高，提示Epo在病理性眼部血管生成中的作用。Epo参与视网膜血管生成，但其与角膜新生血管是否有关尚未见报道。本研究旨在探讨Epo/EpoR是否在正常和新生血管化角膜表达及角膜内注射Epo是否可诱发角膜新生血管的产生，从而了解其与角膜新生血管的联系。
方法：(1)制备碱烧伤诱导鼠角膜新生血管模型，免疫组织化学的方法检测Epo及EpoR是否在正常角膜及新生血管化角膜表达;(2) Epo的克隆、表达及纯化;(3)角膜基质内分别注射Epo（6μL,1μg）及Epo对照（载体对照）及盐水，第14d观察角膜是否有新生血管产生。
结果：Epo及EpoR在正常角膜及碱诱发的新生血管化角膜的角膜上皮细胞，角膜内皮细胞，基质细胞均有表达，并在新生血管化角膜表达加强，同时也在基质内炎症细胞及新生血管均有表达。角膜基质内注射Epo后第14d，6眼中5眼产生新生血管，对照组6眼均未见新生血管。
结论：本文首次报道了Epo及其受体表达于正常角膜和新生血管化角膜。角膜内注射注射Epo可诱发角膜新生血管。Epo及其受体系统与角膜新生血管化的形成有关。
【关键词】 促红细胞生成素 角膜 新生血管
INTRODUCTION
Pathological revascularization within the normally avascular cornea is a serious event that can interfere with normal vision. The regulation of corneal angiogenesis is a complex process which involves the equilibrium between pro and antiangiogenetic factors. Several angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)α and β have a vital role in corneal neovascularization (NV). However, previous strategies to inhibit VEGF action have generally been able to reduce neovascularization by only 30% to 50%. One explanation might be that there are other important angiogenic factors involved that are not affected by the antiVEGF agents. Erythropoietin (Epo) is a hematopoietic cytokine that regulates the production of red blood cells[1]. Epo is produced primarily in the kidney in response to anemia and hypoxia. Its receptor (EpoR), is localized also in nonhematopoietic tissues, eg. liver, uterus, central nervous system, vascular endothelial cells, myocardium, vascular smooth muscle, and mesangial cells and solid tumors. On endothelial cells, erythropoietin reported could induce cell proliferation and induce angiogenesis [2].
Elevated levels of Epo were found in the vitreous samples of patients with proliferative diabetic retinopathy (PDR) [3,4], or with retinopathy of prematurity (ROP) [5], and high Epo concentrations in the human vitreous are more strongly associated with PDR than is VEGF [24]. These instances of Epo/EpoR linking to retinal neovascularization showed a potential association with pathological revascularization in other ocular tissues such as cornea. However, the role of Epo/EpoR in normal cornea and corneal NV is largely unknown. In this study, we hypothesized that Epo/EpoR may also be expressed in the normal cornea and abnormal expression in cornea contributes to the corneal neovascularization.
MATERIALS AND METHODS
All experiments were conducted in accordance with the Declaration of Helsinki and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Epo Cloning and Expression The full length EPO cDNA was amplified from normal persons 1st strand cDNA with primer HEPOpET32aL (SalI): acgcGTCGACaaatggggg tgcacgaatgtcc/HEPOpET32aR (NotI): ataagaatGCGGCCG
Ctcatctgtcccctgtcctgc. PCR fragment was cloned into pET32a vector. Positive clones were identified by bacterial PCR with T7 promoter and T7 terminator primer. Plasmid was extracted from positive clone using Qiagen miniprep kit. The plasmid was transformed into BL21 E. coli strain after verified by enzyme digestion and sequencing. The recombinant protein was purified from E. coli using NiNTA column (Qiagen) after IsopropylβDthiogalactopyranoside (IPTG) induction. Epo control is vector control, pET 32a.
Model of Corneal Neovascularization by Alkaline Burn The C57BL/6 mice used in the experiment were obtained from Kangs Laboratory. All mice were male at the age of 68 weeks.Under intramuscular general anesthesia and additional topical application of lidocaine. NV was induced by application of 2μL of 0.15mmol/L NaOH to the right central cornea of each mouse for 60 seconds, rinsed extensively with phosphatebuffered saline (PBS) for 2 minutes. The corneal epithelium was subsequently scraped off with a corneal knife in a rotary motion parallel to the limbus.The limbal areas were gently massaged over 360° for 3minutes. The ocular surface was then irrigated with 20mL physiological saline. To prevent infection, eyes were administered with antibiotic ointment.
Immunohistochemistry of Corneal Frozen Sections Mice were killed at 14 days after corneal injury. Enucleated eyes were fixed in 40g/L paraformaldehyde, transferred to 150g/L sucrose for 1 hour and then transferred to 300g/L sucrose overnight at 4℃, washed with PBS, and embedded in optimal cutting temperature (OCT) medium (Sakura Finetek, Torrance, CA). Frozen sections of 6μm were washed with 1g/L Triton X100/PBS and blocked for 1 hour with 100mL/L donkey serum before overnight incubation with rabbit polyclonal CD31 (1∶400; Pharmingen,USA) and Goat Epo/EpoR (1∶20; R&D System,Inc, USA). After washed by PBS for 5 minutes each time and 3 times, the sections were subsequently incubated with Rhodamineconjugated polyoclonal donkey antirabbit antibodies(1∶400; InvitrogenMolecular Probes, Eugene,OR) and FITC conjugated polyoclonal donkey antigoat (1∶200) preceded by a 1hour at room temperature.
Corneal Intrastromal Injection Under direct microscopic observation, a nick in the epithelium and anterior stroma of the mouse cornea was made in the midperiphery with a 0.5in, 30gauge needle (BD Biosciences, Franklin Lakes, NJ). A 0.5in., 33gauge needle with a 30° bevel on a 10μL gastight syringe (Hamilton, Reno, NV) was introduced into the corneal stroma and advanced 1.5mm to the corneal center. 6μL of Epo protein (extracted and purified in E.coli), or Control (6μL of vector control or saline) was forcibly injected into the stroma [6].
RESULTS
Epo/EpoR Expressed in Normal Cornea To test the hypothesis that Epo/EpoR was expressed in the normal cornea, we first analyzed the presence of Epo/EpoR in murine cornea by using immunohistochemistry. As revealed in Figure 1, Epo was expressed in corneal epithelial cells, capillary endothelial cells, and also expressed in keratocytes. EpoR was observed in the same cells.
Epo/EpoR Expressed in NV A preliminary study revealed that corneal neovascularization was maximal in 57BL/6 mice around 14 days after scraping. Therefore, on day 14 we examined corneas for coexpression of the endothelial cell marker CD31 and Epo /EpoR. Results showed that the CD31 marked vessels within the cornea coexpressed the Epo (Figure 2A,B,C) or EpoR (Figure 2D,E,F). Furthermore, Epo/EpoR was expressed more strongly on inflammatory cells, epithelial cells, endothelial cells and keratocytes in NV than those in normal cornea (Figure 2 G,H,I).
NV Induced by Corneal Intrastromal Injection of Epo No vessels were observed in all normal eyes (n=6) at day 14 after vector control or saline intrastromal injection (6μL) in normal corneas (Figure 3A). In contrast, some new vessels were observed in 5 out of 6 eyes at day 14 after Epo injection (6μL, 1.0μg). The picture (Figure 3B) showed the most obvious new vessels in cornea of all cases. The new vessels were growing through the limbal area stretching to the center but not reaching the center of cornea at day 14.
DISCUSSION
Ocular abnormal angiogenesis is the copathological character with a hallmark of abnormal and excessive blood vessel growth in retina, as well as in cornea. The influence of Epo on angiogenesis of retinopathy is beginning to be defined[7]. Some evidence suggests that the role of Epo extends beyond orthogenesis. Epo has been found to promote endothelial cell proliferation and vessel growth [8]. In the eye, Epo levels are elevated in the vitreous of patients with proliferative diabetic retinopathy, and Epo in the proliferative phase in the mouse model of retinopathy can inhibit retinal neovascularization [3,4]. It is important to understand the role of Epo in the development of abnormal new vessels in ocular diseases. The effect of Epo on angiogenesis is likely to be important not only for retinopathy but also for other diseases as well as cornea neovascularization. However, the role of Epo in the ocular neovascularization has not been described.
Our study revealed that the Epo/EpoR was expressed in normal cornea, including epithelial cells, endothelial cells and keratocytes. We also found that the Epo and EpoR were coexpressed on the new vessels in the NV induced by alkali. Furthermore, Epo/EpoR was also observed being expressed on inflammatory cells and may have enhanced expression in epithelial cells, endothelial cells and keratocytes in NV. These evidences suggested that Epo/EpoR existed in normal cornea and on new vessels of vascularized cornea induced by alkali.
However, since previous studies which examined Epo receptor localization in the retina have been contradictory [9,10]. This inconsistency might partially reflect the finding that Epo receptor antibodies are unreliable for immunohistochemistry[11]. To overcome antibody nonspecificity, laser capture microdissection of corneal layers and vessels with quantitative realtime RTPCR to localize mRNA expression of Epo and EpoR should be used in the cornea in the further study.
VEGF is probably the most important cytokine in the pathogenesis of NV. It enhances endothelial proliferation, migration,basement membrane degradation,and permeability [12]. Although Epo and VEGF exhibit similar angiogenic potential [13], the role of Epo in the vessel development has not been well described. Epo production appears to be independent of VEGF [3,14]. Exogenous Epo does not change VEGF or VEGF receptor expression. In animals, an intrastromal pellet loaded with 500 or 750ng VEGF can induce corneal NV [15].
Next, to test whether Epo protein injection can induce cornea neovascularization, we directly injected Epo protein to corneal stroma. In this study, 5 out of 6 mice were observed to have new vessels in cornea after Epo injection. However, we noticed that the new vessels did not cover the whole cornea or stretch to the center of cornea at the day 14 after injection. Epo seemed to induce corneal neovascularization not as strongly as VEGF did. One possible reason is that the Epos halflife is 34 hours in blood, and its effect probably wont be lasting long enough by only one injection without intrastromal pellet. Anyway, more models of Epo intrastromal injection are necessary and the effect of Epo should be studied further.
In summary, the current findings first provide the demonstration of Epo/EpoR expressed in normal cornea. Epo/ EpoR has been associated with neovascularization in the eye, not only in retina, but also in cornea. It suggests that Epo is another important candidate factor for ocular neovascularization. It is likely that future treatments will address this and that we will have a mixture of angiogenesis inhibitors that target several angiogenic factors at the same time in treating ocular neovascularization[16].
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