促红细胞生成素在小鼠视网膜血管发育与新生血管增殖中表达的研究
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摘要
研究目的:已知促红细胞生成素(EPO)具有血管生成素活性,但其对眼血管生成的作用还不清楚。临床研究表明外源性给予重组人促红细胞生成素(rHuEPO)可能促进早产儿视网膜病变(ROP)的发生和发展。因此,本研究通过建立氧诱导的视网膜病变鼠模型,观察内源性EPO在视网膜血管发育和新生血管形成中的蛋白动态表达情况,并与缺氧诱导因子-1α(HIF-1α)表达规律相比较,以期初步研究EPO在ROP病变发展中的作用。
研究方法:健康生后7天(P7)C57BL/6J幼鼠32只,随机分为模型组、对照组和正常发育组。其中10只建立高氧诱导模型作为模型组;对照组10只和正常发育组12只于自然环境饲养;前两组于生后12、17天各处死5只;正常发育组于生后9、12、15、18天各处死3只。采用视网膜铺片和ADP(二磷酸腺苷酶)染色、病理切片常规HE染色及免疫组织化学检测方法,分别观察视网膜血管的改变、视网膜新生血管内皮细胞核数目及EPO和HIF-1α蛋白的表达情况。Olympus显微摄像系统采集图片,图像分析软件进行蛋白的累积光密度(IOD)值测定。对IOD值进行分析和比较,实验数据以均数±标准差((?)±s)表示,用SPSS12.0软件包处理。正常发育组不同时限的光密度值采用方差分析:模型组和对照组间采用成组设计的t检验;同组两种蛋白表达的相关性,采用Pearson相关性分析。
研究结果:
1、视网膜新生血管分析
ADP染色视网膜铺片显示:正常发育组和对照组随着鼠龄的增加,视网膜完全血管化;模型组P12时视网膜血管的中心区和中周部出现无灌注;P17时在中周部灌注区与无灌注区的连接处出现新生血管丛。HE染色也显示模型组P12时视网膜周边部血管淤滞,神经纤维层存在血管闭锁;P17时毛细血管于神经纤维层和内核层明显增殖,并突破内界膜进入玻璃体内,在视网膜前形成新生血管。
2、正常发育组不同时限视网膜EPO蛋白表达
EPO的阳性反应产物呈棕黄色点状或颗粒状。在视网膜神经节细胞层及神经纤维层可见EPO阳性颗粒。正常组EPO随视网膜血管发育呈动态表达,P9时其IOD值为8.52±0.92,P12有明显升高,达到20.85±0.72,以后三个时段之间没有明显差异,持续在较高水平。
3、高氧诱导模型视网膜EPO蛋白表达
对照组P12、P17视网膜神经节细胞层及神经纤维层可见EPO蛋白表达;模型组P12视网膜各层及内界膜附近棕黄色颗粒明显减少,P17可见EPO蛋白表达显著增加,位于视网膜神经节细胞层、内核层及突破内界膜的新生血管,与正常组相比差异有统计学意义(P＜0.05)。
4、高氧诱导模型视网膜HIF-1α蛋白表达
HIF-1α的阳性反应产物呈棕色。对照组P12、P17及模型组P12视网膜各层及内界膜附近未见HIF-1α阳性颗粒。模型组P17可见HIF-1α蛋白表达在神经节细胞层和突破内界膜的新生血管,与正常组相比差异有统计学意义(P＜0.05)。
5、EPO和HIF-1α蛋白表达相关性
对模型组P17幼鼠视网膜EPO和HIF-1α两种蛋白的IOD值进行相关性分析。模型组P17的EPO和HIF-1α蛋白的IOD值呈正相关(P＜0.05)。
结论
1.视网膜EPO蛋白表达与视网膜血管发育有一定的时空关系,我们推测EPO可能参与正常视网膜血管发育过程。
2.EPO在模型组不同时间位点的动态表达变化与ROP的两期病理变化相一致,我们推测EPO可能参与ROP视网膜新生血管增殖过程
3.在视网膜新生血管增殖中EPO与HIF-1α蛋白表达呈正相关,推测HIF-1α可能参与缺氧状态下视网膜新生血管增殖中EPO的调节过程。
4.EPO在视网膜新生血管增殖中的作用,有可能为ROP的预防和治疗找到全新的干预靶点,尤其在rHuEPO已广泛用于临床治疗早产儿贫血时,更应该警惕EPO对早产儿发生ROP的影响。
Objective:
It had known that erythropoietin(EPO)has a new function:angiogenesis.But the mechanism about ocular vascularization is not yet clear.Clinical research had shown that administration of recombinant human erythropoietin(rHuEPO)may improve the generation and development of retinopathy of prematurity(ROP).This study established the model of oxygen-induced retinopathy and observed the expression of EPO on retinal vascularization and neovascularization,and then compared with hypoxia-inducible factor-1α(HIF-1α),in order to identify the function of EPO on ROP.
Methods:
32 7-day-old(P7)healthy C57BL/6J mice were randomly divided into 3 groups:model,control and normal development group.10 mice established oxygen-induced model,10 mice in control group and 12 mice in normal development group were raised at room air;the first two groups mice were put to death on P12,17 and the latter were on P9,12,15,18.The ADPase histochemical technique was applicated for retinal flatmount to assess the changes of retinal blood vessels.The proliferative neovascular response was quantified in paraffin section.Immunocytochemistry was performed to detect EPO and HIF-1αprotein expression.Picture were collected with Olympus micro-image system,integrated optical density(IOD)were determined with image analysis system.Analyzed and compared the IOD,data was indicated with mean±standard deviation.Analyzed data with SPSS 12.0.Adopted ANOVA in the different stages of same group,t-test in model and control groups,Peason test in correlation with the two protein expression.
Result:
1.Analysis of retinal neovascularization
ADPase stain result:The retinal vascularization had completed in control and normal development groups.The middle and midperiphery of retinal blood vessel appeared nonperfusion at P12 and new vessels appeared at junction of perfusion and nonperfusion at P 17 in model group.HE stain displayed that periphery vessels were blocked and vessels atresia in nerve fiber layer(NFL)at P12 in model group;the capillary proliferated in NFL and inner nuclear layer(INL),and new vessels broken through interal limiting membrane(ILM)into vitreous at P17.
2.The expression of EPO in retina in normal development group
EPO positive product was buffy granulous.The positive products appeared in ganglion cell layer(GCL)and NFL.The expression of EPO increased coincident with retinal vascularization in normal group.At P9 the IOD was 8.52±0.92,at P12 it increased to 20.85±0.72,afterward maintained at higher level.
3.The expression of EPO in model group
EPO positive products expressed in GCL and NFL at P12 and P17 in control group;The positive products decreased in retinal layers and the surrounding of the ILM at P12 and the expression of EPO increased dramatically at P17(P＜0.05)in model group,the positive products appeared in retinal GCL,INL,the surrounding of ILM and the new vessels broken through ILM.
4.The expression of HIF-1αin model group
HIF-1αpositive product was brown.HIF-1αwas not appeared in retinal layers and the surrounding of the ILM at P12,P17 in control group and P12 in model group.The expression of HIF-1αincreased dramatically at P17(P＜0.05)in model group,and the positive products appeared in retinal GCL and the new vessels broken through ILM.
5.The correlation of the expression of EPO and HIF-1α
The result shown that the expression of EPO and HIF-1αat P17 in model group correlated positively(P＜0.05).
Conclusion:
1.The expression of EPO in retina correlated temporally and spatially with the development of retinal blood vessels.R is supposed that EPO may participated in the process of normal development of retinal blood vessels.
2.The expression changes of EPO protein at different times were coincident with the pathology of ROP.It is supposed that EPO may participate in retinal new vessel formation of ROP.
3.The expression of EPO and HIF-1αcorrelated positively in the process of retinal new vessel formation.It is supposed that HIF-1αmay participate in the regulation of EPO in retinal neovascularization.
4.The function of EPO in the process of retinal neovascularization might be found a new intervention target to prevent or cure ROP.It shoud pay more attention to the effect of EPO on ROP,when rHuEPO was used to treat anemia of prematurity.
研究方法:健康生后7天(P7)C57BL/6J幼鼠32只,随机分为模型组、对照组和正常发育组。其中10只建立高氧诱导模型作为模型组;对照组10只和正常发育组12只于自然环境饲养;前两组于生后12、17天各处死5只;正常发育组于生后9、12、15、18天各处死3只。采用视网膜铺片和ADP(二磷酸腺苷酶)染色、病理切片常规HE染色及免疫组织化学检测方法,分别观察视网膜血管的改变、视网膜新生血管内皮细胞核数目及EPO和HIF-1α蛋白的表达情况。Olympus显微摄像系统采集图片,图像分析软件进行蛋白的累积光密度(IOD)值测定。对IOD值进行分析和比较,实验数据以均数±标准差((?)±s)表示,用SPSS12.0软件包处理。正常发育组不同时限的光密度值采用方差分析:模型组和对照组间采用成组设计的t检验;同组两种蛋白表达的相关性,采用Pearson相关性分析。
研究结果:
1、视网膜新生血管分析
ADP染色视网膜铺片显示:正常发育组和对照组随着鼠龄的增加,视网膜完全血管化;模型组P12时视网膜血管的中心区和中周部出现无灌注;P17时在中周部灌注区与无灌注区的连接处出现新生血管丛。HE染色也显示模型组P12时视网膜周边部血管淤滞,神经纤维层存在血管闭锁;P17时毛细血管于神经纤维层和内核层明显增殖,并突破内界膜进入玻璃体内,在视网膜前形成新生血管。
2、正常发育组不同时限视网膜EPO蛋白表达
EPO的阳性反应产物呈棕黄色点状或颗粒状。在视网膜神经节细胞层及神经纤维层可见EPO阳性颗粒。正常组EPO随视网膜血管发育呈动态表达,P9时其IOD值为8.52±0.92,P12有明显升高,达到20.85±0.72,以后三个时段之间没有明显差异,持续在较高水平。
3、高氧诱导模型视网膜EPO蛋白表达
对照组P12、P17视网膜神经节细胞层及神经纤维层可见EPO蛋白表达;模型组P12视网膜各层及内界膜附近棕黄色颗粒明显减少,P17可见EPO蛋白表达显著增加,位于视网膜神经节细胞层、内核层及突破内界膜的新生血管,与正常组相比差异有统计学意义(P＜0.05)。
4、高氧诱导模型视网膜HIF-1α蛋白表达
HIF-1α的阳性反应产物呈棕色。对照组P12、P17及模型组P12视网膜各层及内界膜附近未见HIF-1α阳性颗粒。模型组P17可见HIF-1α蛋白表达在神经节细胞层和突破内界膜的新生血管,与正常组相比差异有统计学意义(P＜0.05)。
5、EPO和HIF-1α蛋白表达相关性
对模型组P17幼鼠视网膜EPO和HIF-1α两种蛋白的IOD值进行相关性分析。模型组P17的EPO和HIF-1α蛋白的IOD值呈正相关(P＜0.05)。
结论
1.视网膜EPO蛋白表达与视网膜血管发育有一定的时空关系,我们推测EPO可能参与正常视网膜血管发育过程。
2.EPO在模型组不同时间位点的动态表达变化与ROP的两期病理变化相一致,我们推测EPO可能参与ROP视网膜新生血管增殖过程
3.在视网膜新生血管增殖中EPO与HIF-1α蛋白表达呈正相关,推测HIF-1α可能参与缺氧状态下视网膜新生血管增殖中EPO的调节过程。
4.EPO在视网膜新生血管增殖中的作用,有可能为ROP的预防和治疗找到全新的干预靶点,尤其在rHuEPO已广泛用于临床治疗早产儿贫血时,更应该警惕EPO对早产儿发生ROP的影响。
Objective:
It had known that erythropoietin(EPO)has a new function:angiogenesis.But the mechanism about ocular vascularization is not yet clear.Clinical research had shown that administration of recombinant human erythropoietin(rHuEPO)may improve the generation and development of retinopathy of prematurity(ROP).This study established the model of oxygen-induced retinopathy and observed the expression of EPO on retinal vascularization and neovascularization,and then compared with hypoxia-inducible factor-1α(HIF-1α),in order to identify the function of EPO on ROP.
Methods:
32 7-day-old(P7)healthy C57BL/6J mice were randomly divided into 3 groups:model,control and normal development group.10 mice established oxygen-induced model,10 mice in control group and 12 mice in normal development group were raised at room air;the first two groups mice were put to death on P12,17 and the latter were on P9,12,15,18.The ADPase histochemical technique was applicated for retinal flatmount to assess the changes of retinal blood vessels.The proliferative neovascular response was quantified in paraffin section.Immunocytochemistry was performed to detect EPO and HIF-1αprotein expression.Picture were collected with Olympus micro-image system,integrated optical density(IOD)were determined with image analysis system.Analyzed and compared the IOD,data was indicated with mean±standard deviation.Analyzed data with SPSS 12.0.Adopted ANOVA in the different stages of same group,t-test in model and control groups,Peason test in correlation with the two protein expression.
Result:
1.Analysis of retinal neovascularization
ADPase stain result:The retinal vascularization had completed in control and normal development groups.The middle and midperiphery of retinal blood vessel appeared nonperfusion at P12 and new vessels appeared at junction of perfusion and nonperfusion at P 17 in model group.HE stain displayed that periphery vessels were blocked and vessels atresia in nerve fiber layer(NFL)at P12 in model group;the capillary proliferated in NFL and inner nuclear layer(INL),and new vessels broken through interal limiting membrane(ILM)into vitreous at P17.
2.The expression of EPO in retina in normal development group
EPO positive product was buffy granulous.The positive products appeared in ganglion cell layer(GCL)and NFL.The expression of EPO increased coincident with retinal vascularization in normal group.At P9 the IOD was 8.52±0.92,at P12 it increased to 20.85±0.72,afterward maintained at higher level.
3.The expression of EPO in model group
EPO positive products expressed in GCL and NFL at P12 and P17 in control group;The positive products decreased in retinal layers and the surrounding of the ILM at P12 and the expression of EPO increased dramatically at P17(P＜0.05)in model group,the positive products appeared in retinal GCL,INL,the surrounding of ILM and the new vessels broken through ILM.
4.The expression of HIF-1αin model group
HIF-1αpositive product was brown.HIF-1αwas not appeared in retinal layers and the surrounding of the ILM at P12,P17 in control group and P12 in model group.The expression of HIF-1αincreased dramatically at P17(P＜0.05)in model group,and the positive products appeared in retinal GCL and the new vessels broken through ILM.
5.The correlation of the expression of EPO and HIF-1α
The result shown that the expression of EPO and HIF-1αat P17 in model group correlated positively(P＜0.05).
Conclusion:
1.The expression of EPO in retina correlated temporally and spatially with the development of retinal blood vessels.R is supposed that EPO may participated in the process of normal development of retinal blood vessels.
2.The expression changes of EPO protein at different times were coincident with the pathology of ROP.It is supposed that EPO may participate in retinal new vessel formation of ROP.
3.The expression of EPO and HIF-1αcorrelated positively in the process of retinal new vessel formation.It is supposed that HIF-1αmay participate in the regulation of EPO in retinal neovascularization.
4.The function of EPO in the process of retinal neovascularization might be found a new intervention target to prevent or cure ROP.It shoud pay more attention to the effect of EPO on ROP,when rHuEPO was used to treat anemia of prematurity.
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[42]Yasuda S,Arii S,Mori A.Hexokinase2 and VEGF expression in liver tumors:Correlation with hypoxia-inducible factor 1 alpha and its sificance[J].Hepatol,2004,40(1):117-123.
[43]Talks KL,Turley H,Gatter KC.The expression and distribution of the hypoxia inducible factors HIF-1 alpha an d HIF-2 alpha in normal human tissues,cancers,and tumor associated macrophages[J].Am J Pathol,2000,157:411-421.
[44]Ryan HE,Lo J,Johnson RS.HIF-1 alpha is required for solid tumor formation and embryonic vascularizatlon[J].EMBO,1998,7:3005-3015.
[45]邓爱军,姜德咏.缺氧时视网膜血管内皮细胞HIF-1α及其mRNA的表达[J].眼科研究,2006,24:25-27.
[46]Treins C,Giorgett-Peraldi S,Muntaca J.Regulation of vascular endothelial growth factor expression by advanced glyeation end products[J].J Biol Chem,2001,276:43836-43841.
[47]Bhisitkul RB,Ruan DT,Sherwood J.HIF-1-mediated induction of VEGF in RPE cells:upregulation by constitutively active HIF-1 and inhibition by RNA interference[J].Invest Ophthalmol Vis Sci,2004,45:E-Abstract 452.
[48]Yu X,Shacka JJ,Eells JB,Erythropoietin receptor singalling is required for normal brain development[J].Development,2002,129:505-516.
[49]Bemaudin M,Bellail A,Marti HH.Neurons and astrocytes express EPOmRNA:Oxygensensing mechanisms that involve the redox-system of the brain[J].Glia,2002,30:271-278.
[50]Junk AK,Mammis A,Savitz SI.Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury[J].Proc Nail Acad Sci USA,2002,99:10659-10664.
[51]Roger M,Deborah M.Epolones induce erythropoietin expression via hypoxia-ind ucible factor-1α activation[J].Blood,2000,96:1558-1565.
[52]Ang SO,Chen H,Hirota K.Disruption of oxygen homeostasis underlies congential Chuvash polycythernia[J].Nat Genet,2002,32:614-621.
[53]Fandrey J.Oxygen-dependent and tissue-specific regulation of erythropoietin gen e expression[J].Am J Physiol Regul Intege Comp Physiol,2004,286:R977-988.
[54]Grimm C,Wenzel A,Groszer M.HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration[J].Nation Med,2002,8:718-724.
1 Buemi M,Cavallaro E,Floccari F,et al.Erythropoietin and the brain:from neurodevelopment to neuroprotection[J].Clin Sci,2002,103:275-282
2 Eid T,Brines M.Recombinant human erythropoietin for neuroprotectio- n:what is the evidence[J]? Clin Breast Cancer,2002,3:109-115
3 Silva M,Grillot D,Benito A,et al.Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2[J].Blood,1996,88:1576-1582
4 Tilbrook PA,Klinken SP.Erythropoietin and erythropoietin receptor[J].Growth Factor,1999,17:25-35
5 Bahimann FH,de Groot K,Spandau JM,et al.Erythropoietin regulates endothelial progenitor cells[J].Blood,2004,103:921-926
6 Ribatti D,Presta M,Vacea A,et al.Human erythropoietin induces a pro-angiogenie phenotype in cultured endothelial cells and stimulates neovascularization in vivo[J].Blood,1999,93:2627-2636
7 Jaquet K,Krause K,Tawakol-Khodai M,et al.Erythropoietin and VEGF exhibit equal angiogenic potential[J].Microvasc Res,2002,64:326-333
8 Ribatti D,Vacca A,Roccaro A,et al.Erythropoietin as an angiogenic factor[J].Eur J Clin Invest,2003,33(10):891-896
9 Heeschen C,Aicher A,Lehmann R,et al.Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization[J].Blood,2003,102:1340-1346
10 Galeano M,Altavilla D,Bitto A,et al.Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds[J].Crit Care Med,2006,34(4):1139-1146
11 Yasuda Y,Masuda S,Chikuma M,et al.Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis[J].J Biol Chem,1998,273:25381-25387
12 Ribatti D,Marzullo A,Nico B,et al.Erytropoietin is an angiogenie factor in gastric carcinoma[J].Histpathology,2003,42:246-250
13 Miller JW,Adamis AP,Aiello LP.Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy[J].Diabetes Metab Rev,1997,13:37-50
14 Lioyd PA.Angiogenic pathways in diabetic retinpathy[J].N Engl J Med,2005, 353:839-841
15 Bocker-Meffert S,Rosenstiel P,Rohl C,et al.Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats[J].Invest Ophthalmol Vis Sci,2002,43:2021-2026
16 Hernandez C,Fonollosa A,Garcia-Ramirez M,et al.Erythropoietin is expressed in the human retina and it is highly elevated in the vitreous fluid of patients with diabetic macular edema[J].Diabetes Care,2006,29:2028-2033
17 Grimm C,Wenzel A,Groszer M,et al.HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration[J].Nation Med,2002,8:718-724
18 Junk AK,Mammis A,Savitz SI,et al.Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury.Proc Nail Acad Sci USA,2002,99:10659-1066
19 Ariamaa O,Nikinmaa M.Oxygen-dependent diseases in the retina:role of hypoxia-inducible factors[J].Exp Eye Res,2006,83(3):473-483
20 Watanabe D,Suzuma K,Matsui S,et al.Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy[J].N Engl J Med,2005,353:782-792
21 Katsura Y,Okano T,Matsuno K,et al.Erythropoietin is highly elevated in vitreous fluid of patients with proliferative diabetic retinopathy[J].Diabete Care,2005,28:2252-2254
22 Assouline Y,Engel HM,Vega-Rich C.The development of retinopathy of prematurity in neonates treated with recombinant human erythropoietin[J].Invest Ophthalmol Vis Sci,2004,45:4040
23 Manzoni P,Maestri A,Gomirato G,et al.Erythropoietin as a retinal angiogenic factor[J].N Engl J Med,2005,353:2190-2191
24 Mark S,Anna E,Eric K,et al.Association between higher cumulative dose of recombinant erythropoietin and risk for retinopathy of prematurity[J].JAAPOS,2006,10:143-149
25 Morita M,Ohneda O,Yamashita T,et al.HLF/HIF-2alpha is a key factor in retinopathy ofprematurity in association with erythropoietin[J].EMBOJ,2003,22:1134-1146
26 Chen J,Liu N,Kachara Z,et al.Early administration of erythropoietin prevents oxygen induced retinal vascular degeneration in retinopathy of prematurity[J].Invest Ophthalmol Vis Sci,2006,47:3219
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[39]van Wijngaarden P,Brereton HM,Coster DJ.Genetic influences on susceptibility to oxygen-induced retinopathy[J].Invest Ophthalmol Vis Sci,2007,48:1761-1767.
[40]Thornton RD,Lane P,Borghaei RE.Interleukin 1 induces hypoxia-inducible factor-1 in human gingival and synoval fibroblasta[J].Biochem J,2000,350:307-312.
[41]Albina JE,Vessella JA.HIF-1 expressing in healing wounds:HIF-1 alpha induction in primary inflammatory cells by TNF- alpha[J].Cell Physiol,2001,281:1971-1977.
[42]Yasuda S,Arii S,Mori A.Hexokinase2 and VEGF expression in liver tumors:Correlation with hypoxia-inducible factor 1 alpha and its sificance[J].Hepatol,2004,40(1):117-123.
[43]Talks KL,Turley H,Gatter KC.The expression and distribution of the hypoxia inducible factors HIF-1 alpha an d HIF-2 alpha in normal human tissues,cancers,and tumor associated macrophages[J].Am J Pathol,2000,157:411-421.
[44]Ryan HE,Lo J,Johnson RS.HIF-1 alpha is required for solid tumor formation and embryonic vascularizatlon[J].EMBO,1998,7:3005-3015.
[45]邓爱军,姜德咏.缺氧时视网膜血管内皮细胞HIF-1α及其mRNA的表达[J].眼科研究,2006,24:25-27.
[46]Treins C,Giorgett-Peraldi S,Muntaca J.Regulation of vascular endothelial growth factor expression by advanced glyeation end products[J].J Biol Chem,2001,276:43836-43841.
[47]Bhisitkul RB,Ruan DT,Sherwood J.HIF-1-mediated induction of VEGF in RPE cells:upregulation by constitutively active HIF-1 and inhibition by RNA interference[J].Invest Ophthalmol Vis Sci,2004,45:E-Abstract 452.
[48]Yu X,Shacka JJ,Eells JB,Erythropoietin receptor singalling is required for normal brain development[J].Development,2002,129:505-516.
[49]Bemaudin M,Bellail A,Marti HH.Neurons and astrocytes express EPOmRNA:Oxygensensing mechanisms that involve the redox-system of the brain[J].Glia,2002,30:271-278.
[50]Junk AK,Mammis A,Savitz SI.Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury[J].Proc Nail Acad Sci USA,2002,99:10659-10664.
[51]Roger M,Deborah M.Epolones induce erythropoietin expression via hypoxia-ind ucible factor-1α activation[J].Blood,2000,96:1558-1565.
[52]Ang SO,Chen H,Hirota K.Disruption of oxygen homeostasis underlies congential Chuvash polycythernia[J].Nat Genet,2002,32:614-621.
[53]Fandrey J.Oxygen-dependent and tissue-specific regulation of erythropoietin gen e expression[J].Am J Physiol Regul Intege Comp Physiol,2004,286:R977-988.
[54]Grimm C,Wenzel A,Groszer M.HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration[J].Nation Med,2002,8:718-724.
1 Buemi M,Cavallaro E,Floccari F,et al.Erythropoietin and the brain:from neurodevelopment to neuroprotection[J].Clin Sci,2002,103:275-282
2 Eid T,Brines M.Recombinant human erythropoietin for neuroprotectio- n:what is the evidence[J]? Clin Breast Cancer,2002,3:109-115
3 Silva M,Grillot D,Benito A,et al.Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2[J].Blood,1996,88:1576-1582
4 Tilbrook PA,Klinken SP.Erythropoietin and erythropoietin receptor[J].Growth Factor,1999,17:25-35
5 Bahimann FH,de Groot K,Spandau JM,et al.Erythropoietin regulates endothelial progenitor cells[J].Blood,2004,103:921-926
6 Ribatti D,Presta M,Vacea A,et al.Human erythropoietin induces a pro-angiogenie phenotype in cultured endothelial cells and stimulates neovascularization in vivo[J].Blood,1999,93:2627-2636
7 Jaquet K,Krause K,Tawakol-Khodai M,et al.Erythropoietin and VEGF exhibit equal angiogenic potential[J].Microvasc Res,2002,64:326-333
8 Ribatti D,Vacca A,Roccaro A,et al.Erythropoietin as an angiogenic factor[J].Eur J Clin Invest,2003,33(10):891-896
9 Heeschen C,Aicher A,Lehmann R,et al.Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization[J].Blood,2003,102:1340-1346
10 Galeano M,Altavilla D,Bitto A,et al.Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds[J].Crit Care Med,2006,34(4):1139-1146
11 Yasuda Y,Masuda S,Chikuma M,et al.Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis[J].J Biol Chem,1998,273:25381-25387
12 Ribatti D,Marzullo A,Nico B,et al.Erytropoietin is an angiogenie factor in gastric carcinoma[J].Histpathology,2003,42:246-250
13 Miller JW,Adamis AP,Aiello LP.Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy[J].Diabetes Metab Rev,1997,13:37-50
14 Lioyd PA.Angiogenic pathways in diabetic retinpathy[J].N Engl J Med,2005, 353:839-841
15 Bocker-Meffert S,Rosenstiel P,Rohl C,et al.Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats[J].Invest Ophthalmol Vis Sci,2002,43:2021-2026
16 Hernandez C,Fonollosa A,Garcia-Ramirez M,et al.Erythropoietin is expressed in the human retina and it is highly elevated in the vitreous fluid of patients with diabetic macular edema[J].Diabetes Care,2006,29:2028-2033
17 Grimm C,Wenzel A,Groszer M,et al.HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration[J].Nation Med,2002,8:718-724
18 Junk AK,Mammis A,Savitz SI,et al.Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury.Proc Nail Acad Sci USA,2002,99:10659-1066
19 Ariamaa O,Nikinmaa M.Oxygen-dependent diseases in the retina:role of hypoxia-inducible factors[J].Exp Eye Res,2006,83(3):473-483
20 Watanabe D,Suzuma K,Matsui S,et al.Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy[J].N Engl J Med,2005,353:782-792
21 Katsura Y,Okano T,Matsuno K,et al.Erythropoietin is highly elevated in vitreous fluid of patients with proliferative diabetic retinopathy[J].Diabete Care,2005,28:2252-2254
22 Assouline Y,Engel HM,Vega-Rich C.The development of retinopathy of prematurity in neonates treated with recombinant human erythropoietin[J].Invest Ophthalmol Vis Sci,2004,45:4040
23 Manzoni P,Maestri A,Gomirato G,et al.Erythropoietin as a retinal angiogenic factor[J].N Engl J Med,2005,353:2190-2191
24 Mark S,Anna E,Eric K,et al.Association between higher cumulative dose of recombinant erythropoietin and risk for retinopathy of prematurity[J].JAAPOS,2006,10:143-149
25 Morita M,Ohneda O,Yamashita T,et al.HLF/HIF-2alpha is a key factor in retinopathy ofprematurity in association with erythropoietin[J].EMBOJ,2003,22:1134-1146
26 Chen J,Liu N,Kachara Z,et al.Early administration of erythropoietin prevents oxygen induced retinal vascular degeneration in retinopathy of prematurity[J].Invest Ophthalmol Vis Sci,2006,47:3219