促红细胞生成素对心肌细胞肥大影响的实验研究
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摘要
促红细胞生成素(Erythropoietin,Epo)是一种低氧诱导的谱系特异性的内分泌激素,可调节红细胞的形成,临床上广泛应用于各种原因引起的贫血,如终末期肾病、获得性免疫缺陷综合征以及需要化疗的非造血系统恶性肿瘤。后来研究发现Epo受体在心血管系统有着广泛的分布,基础研究揭示Epo可保护缺血再灌注心肌和限制心肌梗死的范围。有关Epo对心肌细胞肥大影响的报导在国内外非常有限。本研究以Epo作为干预因素,离体培养新生大鼠心肌细胞作为研究对象,以明确Epo对心肌肥大的影响并初步探讨其肥厚相关的信号通路。
第一部分新生大鼠心肌离体培养和Epo刺激后形态学观察
目的:1)取得稳定的新生大鼠心肌细胞离体培养模型,cTnI免疫组化染色观察Epo刺激不同时间对于心肌细胞大小的影响;2)观察Epo刺激引起的细胞超微结构改变。
方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为2%FBS低血清培液同步化24h。实验分为:空白对照组、AngⅡ组和Epo组。AngⅡ终浓度为1×10~(-6)M;Epo终浓度为10U/ml。于24h、48h和72h固定各组细胞,cTnI免疫组化染色,光镜下测量心肌细胞面积。电镜观察心肌细胞超微结构所用的低血清培养液含0.1%FBS,实验分组同前。于24h、48h和72h固定各组细胞,电镜下观察各组心肌细胞超微结构。
结果:1)干预24h,对照组心肌细胞面积2135.6±524.8μm~2,AngⅡ干预组3202.3±661.2μm~2,Epo组2586.2±165.3μm~2,AngⅡ组相比对照组和Epo组,心肌细胞明显增大,P＜0.05。干预48h,对照组心肌细胞面积1844.5±323.3μm~2,AngⅡ干预组3321.0±428.8μm~2,Epo组3318.9±321.3μm~2,AngⅡ组和Epo组相比对照组,心肌细胞明显增大,P＜0.05;AngⅡ组与Epo组相比,差别无统计学意义。干预72h,对照组心肌细胞面积2213.90±149.5μm~2,AngⅡ干预组3222.20±225.3μm~2,Epo组3317.20±196.1μm~2,AngⅡ组和Epo组相比对照组,心肌细胞明显增大,P＜0.05;AngⅡ组与Epo组相比,差别无统计学意义。Epo干预48h和干预72h相比24h组,细胞面积明显增大,P＜0.05。2)电镜观察,培养72h,对照组心肌细胞肌丝溶解,细胞器大多破坏;AngⅡ组和Epo组细胞核不规则,核膜凹陷,核仁较清晰,染色质浅;线粒体数量增多,肿胀;内质网以及高尔基器等与合成分泌有关的细胞器发达,72小时观察到细胞内出现亚细胞结构的空泡化改变。
结论:Epo刺激可使心肌细胞增大,使心肌细胞出现肥厚的亚细胞结构改变。
第二部分Epo对心肌细胞蛋白激酶活性及肥大相关基因的影响
目的:考察Epo刺激后细胞外信号调节激酶ERK的激活和心肌细胞肥大相关基因ANP、BNP、c-fos mRNA的表达量的变化。
方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为0.1%FBS低血清培液同步化24h,加入Epo干预。1)Epo终浓度为10U/ml,分别在0 min(对照组)、10min、15min、30min、60min和120min终止干预,提取心肌细胞总蛋白,Western blot检测心肌细胞磷酸化ERK1/2(p-ERK1/2)含量;2)Epo终浓度为0 U/ml(对照组)、5 U/ml、10 U/ml、100 U/ml,干预30min,提取心肌细胞总蛋白,Western blot检测心肌细胞p-ERK1/2含量;3)实验分两组,对照组和Epo组,Epo10U/ml干预30min,提取心肌细胞总RNA,RT—PCR法测定两组c-fos mRNA表达量;同样Epo和对照组分组干预2h,RT—PCR法测定ANP mRNA和BNP mRNA表达量。
结果:1)Epo10U/ml干预心肌细胞后p-ERK2表达随时间出现先升高后下降的变化,30min时达到高峰,与干预前和干预后120min相比,P＜0.05;p-ERK1在Epo干预后也观察到先升高后降低的趋势。2)Epo不同浓度干预心肌细胞,随Epo干预浓度增大,p-ERK2水平升高,10 U/ml及100 U/ml浓度干预组与对照组相比,差别有统计学意义(P＜0.05)。p-ERK1在Epo处理后也观察到随浓度升高而升高的趋势。3)Epo10U/ml干预心肌细胞,RT-PCR结果显示,30min c-fosmRNA表达量与对照组相比差异不明显,2h ANP mRNA和BNP mRNA表达量和对照组相比,明显升高,P＜0.05。
结论:1)Epo刺激使心肌细胞p-ERK1/2呈时间相关性和浓度依赖性升高;2)要观察p-ERK1/2变化,可取Epo10U/ml刺激30min做为最佳观察条件;2)Epo刺激促进了心肌肥大相关基因表达。
第三部分Epo引起心肌细胞肥大反应信号通路初探
目的:1)观察Epo刺激对心肌AGT mRNA表达量的影响;2)初步探讨心肌中Epo-ERK信号通路和RAS系统之间是否存在相互关系。
方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为0.1%FBS低血清培养液同步化24h,开始分组干预。
1)Epo10U/ml干预细胞,分别在0h(对照组)、1h、2h、6h、12h、24h提取心肌细胞总RNA,RT-PCR法测定并比较干预不同时间后AGT mRNA表达量。
2)实验分为6组:a)空白对照组;b)AngⅡ组:AngⅡ1×10~(-6)M 8min;c)ARB+AngⅡ组:替米沙坦1×10~(-6)M阻断30min后加入AngⅡ1×10~(-6)M 8min;d)Epo组:Epo 10U/ml 30min;e)ACEI+Epo组:依那普利1×10~(-6)M阻断2h后加入Epo 10U/ml 30min;f)ARB+Epo组:替米沙坦1×10~(-6)M阻断30min后加入Epo 10U/ml 30min。干预结束提取心肌细胞总蛋白,Western blot测定各组心肌细胞p-ERK1/2含量。
3)实验分3组:a)空白对照组;b)Epo组:Epo 10U/ml 6h;c)PD98059+Epo组:PD98059浓度50×10~(-6)M阻断30min后加入Epo 10U/ml 6h。终止干预,提取心肌细胞总RNA,比较三组的AGT mRNA表达量。
结果:1)Epo10U/ml干预心肌细胞24h,使AGT mRNA表达量自2h起升高(P＜0.05),随干预时间延长表现为先上升后下降趋势,6h时AGT mRNA表达量水平最高。2)Epo干预心肌p-ERK1/2的升高可被依那普利和替米沙坦所逆转。3)p-ERK1/2的阻断,并不能逆转Epo对于AGT mRNA表达的促进作用。
结论:Epo可上调心肌细胞中AGT基因的转录,Epo激活ERK信号通路与RAS系统存在相互关系。
Erythropoietin(Epo),a hypoxia-induced,lineage-specific hormone,is essential for normal erythropoiesis.Recombinant human Erythropoietin(rh-Epo) is widely used in the treatment of various kinds of anemia,such as end-stage renal disease,Human Immunodeficiency Virus infection,and in patients with non-myeloid malignancies on chemotherapy.Recently it is found out that Epo receptor is widely existed in cardiovascular system.Animal studies demonstrated that Epo can potentially protect myocardium against ischemia-reperfusion-induced injury in and restrict infarcted size. However,little is known about the role of Epo in the development of cardiac hypertrophy.In this study,we examined the in vitro effects of Epo on hypertrophic responses of cultured cardiomyocytes of neonatal rats.
PART ONE:Epo-induced morphological changes in cultured cardiomyocytes of neonatal rats
Objective:
To observe the morphological changes of cultured cardiomyocytes after addition of Epo for various times.
Methods:
Primary cardiac myocytes were isolated from neonatal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum(FBS) for the first 24 hours,and then in 2%FBS-containing medium for synchronization in the next 24 hours.Three groups were included in these experiments:control group;AngⅡtreated group and Epo treated group.The final concentration of AngⅡwas 1×10~(-6)M; and the final concentration of Epo was 10U/ml.Culture cells were fixed at 24h,48h and 72h,respectively,and immunohistochemical stained with cTnI.The sizes of the cardiomyocytes were measured with light microscopy.Low serum culture medium containing 0.1%FBS was used for myocardium ultrastructure study with transmission electron microscope.Three groups were included as describe before. Cells were fixed at 24h,48h and 72h,and myocardium ultrastructure was observed by a transmission electron microscope.
Results:
1) After 24 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2135.6±524.8μm~2 for control group,3202.3±661.2μm~2 for AngⅡtreated group,and 2586.2±165.3μm~2 for Epo group.Comparing to the control group and the Epo treated group,the mean size of the ceils was significantly increased for AngⅡtreated group(P＜0.05).After 48 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 1844.5±323.3μm~2 for control group,3321.0±428.8μm~2 for AngⅡtreated group,and 3318.9±321.3μm~2 for Epo treated group.Comparing to the control group,the mean surface area of the cells is significantly increased in AngⅡtreated group and in Epo treated group(P＜0.05).The difference between AngⅡtreated group and Epo treated group was not significant.After 72 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2213.90±149.5μm~2 for control group,3222.20 ±225.3μm~2 for AngⅡtreated group,and 3317.20±196.1μm~2 for Epo treated group.Compared to control group,the mean surface area of the cells was significantly increased in AngⅡtreated group and in Epo treated group(P＜0.05);however,no significant difference was seen between AngⅡtreated group and Epo treated group. Epo intervention for 48h and 72h significantly increased the mean surface area of the cells comparing to the 24h treated group(P＜0.05).
2) Transmission electron microscopy observation showed that in the control group myofilament dissolved gradually,and most of organelle was damaged when exposure to 72hs low-FBS culture medium.Cardiac myocytes in AngⅡtreated group and the Epo treated group had irregular nuclei,distinct nucleolus,faint chromatin,recessed nuclear membrane,lots of swollen mitochondria and prospersous secretion and synthesis related organelles such as endoplasmic reticulum and Golgi body,showed vacuolization occurred within sub-cellular structures when exposed to AngⅡand Epo for 72h.
Conclusion:
Addition of Epo to culture medium can induce enlargement of cardiomyocytes and ultrastructural changes in cardiomyocytes.
PART TWO:Epo-induced upregulation of ERK activation and hypertrophy-related gene expression in cultured cardiomyocytes
Objective:
To investigate whether Epo upregulates ERK activation and some specific gene expression,such as ANP,BNP and c-fos,in cultured cardiomyocytes.
Methods:
Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum(FBS) for the first 24 hours,then in 0.1%FBS-containing medium for synchronization for the next 24 hours, before Epo intervention.1)Epo treated cardiomyocyte with final concentration of Epo 10U/ml,Epo intervention was ended at 0min(control group),10min,15min,30min, 60min and 120min respectively.Total cellular protein was extracted and Western blot detection was used to quantify the myocardial p-ERK1/2;2) Epo treated cardiomyocyte for 30min,with the final concentration 0(control group),5 U /ml,10 U/ml and 100 U/ml respectively.Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 with different Epo concentration treatment;3) Cardiomyocytes were divided into two groups,control group and the Epo treated group(Epo10U/ml treated for 30min).The total RNA of the cardiomyocytes were extracted and RT-PCR was introduced to semi-quantify c-fos mRNA expression.Similarly,ANP and BNP mRNA was extracted from the cardiomyocytes with Epo treatment or blank control for 2hs and gene expression level was measured by RT-PCR.
Results:
1) p-ERK2 expression in cardiac myocytes first increased and then decline in 120min after Epo 10U/ml intervention,and the expression level was at peak when harvested from Epo 10U/ml treatment for 30min,comparing to the control group and the 120min-treated group(P＜0.05).The similar changes of p-ERK1 expression were also seen but the differences were not significant.2) Increasing concentration of Epo intervention resulted in increased p-ERK2 expression levels in cardiac myocytes, p-ERK2 expression was significantly higher in the group of Epo 10 U/ml and 100 U/ ml than that of the control group(P＜0.05).The p-ERK1 expression was also increased with the increasd Epo concentration,yet the differences were not significant. 3) With Epo10U/ml intervention in cardiac myocytes,c-fos mRNA expression was not increased significantly,yet ANP and BNP mRNA expression were significantly higher than that of control group(P＜0.05).
Conclusion:
1) Epo induces ERK1/2 activation in a time- and concentration-dependent manner;
2) Treatment with Epo at 10U/ml for 30min can significantly increase ERK1/2 phosphorylation;
3) Epo can stimulate the expression of ANP and BNP genes,and prone to increase c-fos gene expression.
PART THREE:Involvement of angiotensinⅡin Epo-induced hypertrophic responses of cardiomyocytes
Objective:
1) To examine the impact of Epo on angiotensinogen(AGT) mRNA expression in cardiomyocytes;
2) To examine whether RAS is involved in Epo-induced ERK activation in cardiomyocytes.
Methods:
Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%FBS for the first 24h,and then in 0.1% FBS-containing medium for synchronization for the next 24h.
1) Epo treatment with final concentration of 10U/ml was terminated at 0min(control group),1h,2h,6h,12h,24h respectively.Total cellular RNA were extracted and RT-PCR was introduced to quantify the AGT mRNA expression.
2) 6 groups were included in this study:
a) Control group;
b) AngⅡgroup:AngⅡ1×10~(-6)M for 8min;
c) ARB+AngⅡgroup:Telmisartan 1×10~(-6)M blockage for 30min then AngⅡ1×10~(-6)M for 8min;
d) Epo Group:Epo 10U/ml for 30min;
e) ACEI+Epo group:Enalapril 1×10~(-6)M blockage for 2h then Epo 10U/ml for 30min;
f) ARB+Epo groups:Telmisartan 1×10~(-6)M blockage for 30min then Epo 10U/ml for 30min.
Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 from the groups described above.
3) The cardiomyocytes were divided into 3 groups:
a) Control group;
b) Epo Group:Epo 10U/ml for 6h;
c) PD98059+Epo group:PD98059 50×10~(-6)M blockage for 30min then Epo 10U/ml for 6h.
Total cellular RNA was extracted and RT-PCR was introduced to quantify the AGT mRNA expression.
Results:
1) Cardiomyocytes AGT mRNA expression increased after 2 hours(2h,6h,12h,24h Vs control,P＜0.05) treatment of Epo 10U/ml with the highest expression level at 6hrs treatment.After that,its expression was inclined to decrease.
2) The increase of p-ERK1/2 caused by Epo treatment could be reversed by Enalapril or Telmisartan treatment.
3) p-ERK1/2 pathway blockage with PD98059 could not stop the increase of AGT mRNA expression on the treatment of Epo.
Conclusion:
1) Epo upregulates AGT gene expression in cultured cardiomyocytes.
2) RAS may be involved in Epo-induced ERK activation.
第一部分新生大鼠心肌离体培养和Epo刺激后形态学观察
目的:1)取得稳定的新生大鼠心肌细胞离体培养模型,cTnI免疫组化染色观察Epo刺激不同时间对于心肌细胞大小的影响;2)观察Epo刺激引起的细胞超微结构改变。
方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为2%FBS低血清培液同步化24h。实验分为:空白对照组、AngⅡ组和Epo组。AngⅡ终浓度为1×10~(-6)M;Epo终浓度为10U/ml。于24h、48h和72h固定各组细胞,cTnI免疫组化染色,光镜下测量心肌细胞面积。电镜观察心肌细胞超微结构所用的低血清培养液含0.1%FBS,实验分组同前。于24h、48h和72h固定各组细胞,电镜下观察各组心肌细胞超微结构。
结果:1)干预24h,对照组心肌细胞面积2135.6±524.8μm~2,AngⅡ干预组3202.3±661.2μm~2,Epo组2586.2±165.3μm~2,AngⅡ组相比对照组和Epo组,心肌细胞明显增大,P＜0.05。干预48h,对照组心肌细胞面积1844.5±323.3μm~2,AngⅡ干预组3321.0±428.8μm~2,Epo组3318.9±321.3μm~2,AngⅡ组和Epo组相比对照组,心肌细胞明显增大,P＜0.05;AngⅡ组与Epo组相比,差别无统计学意义。干预72h,对照组心肌细胞面积2213.90±149.5μm~2,AngⅡ干预组3222.20±225.3μm~2,Epo组3317.20±196.1μm~2,AngⅡ组和Epo组相比对照组,心肌细胞明显增大,P＜0.05;AngⅡ组与Epo组相比,差别无统计学意义。Epo干预48h和干预72h相比24h组,细胞面积明显增大,P＜0.05。2)电镜观察,培养72h,对照组心肌细胞肌丝溶解,细胞器大多破坏;AngⅡ组和Epo组细胞核不规则,核膜凹陷,核仁较清晰,染色质浅;线粒体数量增多,肿胀;内质网以及高尔基器等与合成分泌有关的细胞器发达,72小时观察到细胞内出现亚细胞结构的空泡化改变。
结论:Epo刺激可使心肌细胞增大,使心肌细胞出现肥厚的亚细胞结构改变。
第二部分Epo对心肌细胞蛋白激酶活性及肥大相关基因的影响
目的:考察Epo刺激后细胞外信号调节激酶ERK的激活和心肌细胞肥大相关基因ANP、BNP、c-fos mRNA的表达量的变化。
方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为0.1%FBS低血清培液同步化24h,加入Epo干预。1)Epo终浓度为10U/ml,分别在0 min(对照组)、10min、15min、30min、60min和120min终止干预,提取心肌细胞总蛋白,Western blot检测心肌细胞磷酸化ERK1/2(p-ERK1/2)含量;2)Epo终浓度为0 U/ml(对照组)、5 U/ml、10 U/ml、100 U/ml,干预30min,提取心肌细胞总蛋白,Western blot检测心肌细胞p-ERK1/2含量;3)实验分两组,对照组和Epo组,Epo10U/ml干预30min,提取心肌细胞总RNA,RT—PCR法测定两组c-fos mRNA表达量;同样Epo和对照组分组干预2h,RT—PCR法测定ANP mRNA和BNP mRNA表达量。
结果:1)Epo10U/ml干预心肌细胞后p-ERK2表达随时间出现先升高后下降的变化,30min时达到高峰,与干预前和干预后120min相比,P＜0.05;p-ERK1在Epo干预后也观察到先升高后降低的趋势。2)Epo不同浓度干预心肌细胞,随Epo干预浓度增大,p-ERK2水平升高,10 U/ml及100 U/ml浓度干预组与对照组相比,差别有统计学意义(P＜0.05)。p-ERK1在Epo处理后也观察到随浓度升高而升高的趋势。3)Epo10U/ml干预心肌细胞,RT-PCR结果显示,30min c-fosmRNA表达量与对照组相比差异不明显,2h ANP mRNA和BNP mRNA表达量和对照组相比,明显升高,P＜0.05。
结论:1)Epo刺激使心肌细胞p-ERK1/2呈时间相关性和浓度依赖性升高;2)要观察p-ERK1/2变化,可取Epo10U/ml刺激30min做为最佳观察条件;2)Epo刺激促进了心肌肥大相关基因表达。
第三部分Epo引起心肌细胞肥大反应信号通路初探
目的:1)观察Epo刺激对心肌AGT mRNA表达量的影响;2)初步探讨心肌中Epo-ERK信号通路和RAS系统之间是否存在相互关系。
方法:取新生大鼠原代心肌细胞经10%FBS的DMEM培养液培养24h,更换为0.1%FBS低血清培养液同步化24h,开始分组干预。
1)Epo10U/ml干预细胞,分别在0h(对照组)、1h、2h、6h、12h、24h提取心肌细胞总RNA,RT-PCR法测定并比较干预不同时间后AGT mRNA表达量。
2)实验分为6组:a)空白对照组;b)AngⅡ组:AngⅡ1×10~(-6)M 8min;c)ARB+AngⅡ组:替米沙坦1×10~(-6)M阻断30min后加入AngⅡ1×10~(-6)M 8min;d)Epo组:Epo 10U/ml 30min;e)ACEI+Epo组:依那普利1×10~(-6)M阻断2h后加入Epo 10U/ml 30min;f)ARB+Epo组:替米沙坦1×10~(-6)M阻断30min后加入Epo 10U/ml 30min。干预结束提取心肌细胞总蛋白,Western blot测定各组心肌细胞p-ERK1/2含量。
3)实验分3组:a)空白对照组;b)Epo组:Epo 10U/ml 6h;c)PD98059+Epo组:PD98059浓度50×10~(-6)M阻断30min后加入Epo 10U/ml 6h。终止干预,提取心肌细胞总RNA,比较三组的AGT mRNA表达量。
结果:1)Epo10U/ml干预心肌细胞24h,使AGT mRNA表达量自2h起升高(P＜0.05),随干预时间延长表现为先上升后下降趋势,6h时AGT mRNA表达量水平最高。2)Epo干预心肌p-ERK1/2的升高可被依那普利和替米沙坦所逆转。3)p-ERK1/2的阻断,并不能逆转Epo对于AGT mRNA表达的促进作用。
结论:Epo可上调心肌细胞中AGT基因的转录,Epo激活ERK信号通路与RAS系统存在相互关系。
Erythropoietin(Epo),a hypoxia-induced,lineage-specific hormone,is essential for normal erythropoiesis.Recombinant human Erythropoietin(rh-Epo) is widely used in the treatment of various kinds of anemia,such as end-stage renal disease,Human Immunodeficiency Virus infection,and in patients with non-myeloid malignancies on chemotherapy.Recently it is found out that Epo receptor is widely existed in cardiovascular system.Animal studies demonstrated that Epo can potentially protect myocardium against ischemia-reperfusion-induced injury in and restrict infarcted size. However,little is known about the role of Epo in the development of cardiac hypertrophy.In this study,we examined the in vitro effects of Epo on hypertrophic responses of cultured cardiomyocytes of neonatal rats.
PART ONE:Epo-induced morphological changes in cultured cardiomyocytes of neonatal rats
Objective:
To observe the morphological changes of cultured cardiomyocytes after addition of Epo for various times.
Methods:
Primary cardiac myocytes were isolated from neonatal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum(FBS) for the first 24 hours,and then in 2%FBS-containing medium for synchronization in the next 24 hours.Three groups were included in these experiments:control group;AngⅡtreated group and Epo treated group.The final concentration of AngⅡwas 1×10~(-6)M; and the final concentration of Epo was 10U/ml.Culture cells were fixed at 24h,48h and 72h,respectively,and immunohistochemical stained with cTnI.The sizes of the cardiomyocytes were measured with light microscopy.Low serum culture medium containing 0.1%FBS was used for myocardium ultrastructure study with transmission electron microscope.Three groups were included as describe before. Cells were fixed at 24h,48h and 72h,and myocardium ultrastructure was observed by a transmission electron microscope.
Results:
1) After 24 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2135.6±524.8μm~2 for control group,3202.3±661.2μm~2 for AngⅡtreated group,and 2586.2±165.3μm~2 for Epo group.Comparing to the control group and the Epo treated group,the mean size of the ceils was significantly increased for AngⅡtreated group(P＜0.05).After 48 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 1844.5±323.3μm~2 for control group,3321.0±428.8μm~2 for AngⅡtreated group,and 3318.9±321.3μm~2 for Epo treated group.Comparing to the control group,the mean surface area of the cells is significantly increased in AngⅡtreated group and in Epo treated group(P＜0.05).The difference between AngⅡtreated group and Epo treated group was not significant.After 72 hours exposure to different stimulating factors,the mean surface area of myocardial cells was 2213.90±149.5μm~2 for control group,3222.20 ±225.3μm~2 for AngⅡtreated group,and 3317.20±196.1μm~2 for Epo treated group.Compared to control group,the mean surface area of the cells was significantly increased in AngⅡtreated group and in Epo treated group(P＜0.05);however,no significant difference was seen between AngⅡtreated group and Epo treated group. Epo intervention for 48h and 72h significantly increased the mean surface area of the cells comparing to the 24h treated group(P＜0.05).
2) Transmission electron microscopy observation showed that in the control group myofilament dissolved gradually,and most of organelle was damaged when exposure to 72hs low-FBS culture medium.Cardiac myocytes in AngⅡtreated group and the Epo treated group had irregular nuclei,distinct nucleolus,faint chromatin,recessed nuclear membrane,lots of swollen mitochondria and prospersous secretion and synthesis related organelles such as endoplasmic reticulum and Golgi body,showed vacuolization occurred within sub-cellular structures when exposed to AngⅡand Epo for 72h.
Conclusion:
Addition of Epo to culture medium can induce enlargement of cardiomyocytes and ultrastructural changes in cardiomyocytes.
PART TWO:Epo-induced upregulation of ERK activation and hypertrophy-related gene expression in cultured cardiomyocytes
Objective:
To investigate whether Epo upregulates ERK activation and some specific gene expression,such as ANP,BNP and c-fos,in cultured cardiomyocytes.
Methods:
Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%fetal bovine serum(FBS) for the first 24 hours,then in 0.1%FBS-containing medium for synchronization for the next 24 hours, before Epo intervention.1)Epo treated cardiomyocyte with final concentration of Epo 10U/ml,Epo intervention was ended at 0min(control group),10min,15min,30min, 60min and 120min respectively.Total cellular protein was extracted and Western blot detection was used to quantify the myocardial p-ERK1/2;2) Epo treated cardiomyocyte for 30min,with the final concentration 0(control group),5 U /ml,10 U/ml and 100 U/ml respectively.Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 with different Epo concentration treatment;3) Cardiomyocytes were divided into two groups,control group and the Epo treated group(Epo10U/ml treated for 30min).The total RNA of the cardiomyocytes were extracted and RT-PCR was introduced to semi-quantify c-fos mRNA expression.Similarly,ANP and BNP mRNA was extracted from the cardiomyocytes with Epo treatment or blank control for 2hs and gene expression level was measured by RT-PCR.
Results:
1) p-ERK2 expression in cardiac myocytes first increased and then decline in 120min after Epo 10U/ml intervention,and the expression level was at peak when harvested from Epo 10U/ml treatment for 30min,comparing to the control group and the 120min-treated group(P＜0.05).The similar changes of p-ERK1 expression were also seen but the differences were not significant.2) Increasing concentration of Epo intervention resulted in increased p-ERK2 expression levels in cardiac myocytes, p-ERK2 expression was significantly higher in the group of Epo 10 U/ml and 100 U/ ml than that of the control group(P＜0.05).The p-ERK1 expression was also increased with the increasd Epo concentration,yet the differences were not significant. 3) With Epo10U/ml intervention in cardiac myocytes,c-fos mRNA expression was not increased significantly,yet ANP and BNP mRNA expression were significantly higher than that of control group(P＜0.05).
Conclusion:
1) Epo induces ERK1/2 activation in a time- and concentration-dependent manner;
2) Treatment with Epo at 10U/ml for 30min can significantly increase ERK1/2 phosphorylation;
3) Epo can stimulate the expression of ANP and BNP genes,and prone to increase c-fos gene expression.
PART THREE:Involvement of angiotensinⅡin Epo-induced hypertrophic responses of cardiomyocytes
Objective:
1) To examine the impact of Epo on angiotensinogen(AGT) mRNA expression in cardiomyocytes;
2) To examine whether RAS is involved in Epo-induced ERK activation in cardiomyocytes.
Methods:
Primary cardiac myocytes were isolated from natal Sprague-Dawley rats and cultured in DMEM culture medium containing 10%FBS for the first 24h,and then in 0.1% FBS-containing medium for synchronization for the next 24h.
1) Epo treatment with final concentration of 10U/ml was terminated at 0min(control group),1h,2h,6h,12h,24h respectively.Total cellular RNA were extracted and RT-PCR was introduced to quantify the AGT mRNA expression.
2) 6 groups were included in this study:
a) Control group;
b) AngⅡgroup:AngⅡ1×10~(-6)M for 8min;
c) ARB+AngⅡgroup:Telmisartan 1×10~(-6)M blockage for 30min then AngⅡ1×10~(-6)M for 8min;
d) Epo Group:Epo 10U/ml for 30min;
e) ACEI+Epo group:Enalapril 1×10~(-6)M blockage for 2h then Epo 10U/ml for 30min;
f) ARB+Epo groups:Telmisartan 1×10~(-6)M blockage for 30min then Epo 10U/ml for 30min.
Total cellular protein was extracted and western blot detection was used to quantify the myocardial p-ERK1/2 from the groups described above.
3) The cardiomyocytes were divided into 3 groups:
a) Control group;
b) Epo Group:Epo 10U/ml for 6h;
c) PD98059+Epo group:PD98059 50×10~(-6)M blockage for 30min then Epo 10U/ml for 6h.
Total cellular RNA was extracted and RT-PCR was introduced to quantify the AGT mRNA expression.
Results:
1) Cardiomyocytes AGT mRNA expression increased after 2 hours(2h,6h,12h,24h Vs control,P＜0.05) treatment of Epo 10U/ml with the highest expression level at 6hrs treatment.After that,its expression was inclined to decrease.
2) The increase of p-ERK1/2 caused by Epo treatment could be reversed by Enalapril or Telmisartan treatment.
3) p-ERK1/2 pathway blockage with PD98059 could not stop the increase of AGT mRNA expression on the treatment of Epo.
Conclusion:
1) Epo upregulates AGT gene expression in cultured cardiomyocytes.
2) RAS may be involved in Epo-induced ERK activation.
引文
1. Simko F. Left ventricular hypertrophy regression as a process with variable biological implications [J]. Can J Cardiol, 1996,12(5): 507-513.
2. Koury ST, Bondurant MC, Koury MJ. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization [J]. Blood, 1988,71(2):524—527.
3. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia [J]. J Appl Physiol, 2000, 88(4):1474-1480.
4. Wu H, Lee SH, Gao J, et al. Inactivation of erythropoietin leads to defects in cardiac morphogenesis [J]. Development, 1999,126(16):3597—3605.
5. Calvillo L, Latini R, Kajstura J, et al. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling [J]. Proc Natl Acad Sci USA, 2003,100(8): 4802-4806.
6. Tramontano AF, M uniyappa R, Black AD, et al. Erythropoietin protects cardiac m yocytes from hypoxia-induced apoptosis through an Akt-dependent pathway [J]. Biochem Biophys Res Commun, 2003, 308(4): 990-994.
7. Parsa CJ, Matsumoto A, Kim J, et al. A novel protective effect of erythropoietin in the infarcted heart [J]. J CIin Invest, 2003,112(7): 999—1007.
8. Rui T, Feng Q, Lei M, et al. Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1[J].Cardiovasc Res, 2005, 65(3):719 -27.
9. Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades [J]. Nature, 2001,412(6847):641-7.
10. Jaquet K, Krause K, Tawakol-Khodai M, et al. Erythropoietin and VEGF exhibit equal angiogenic potential [J]. Microvasc Res, 2002, 64(2):326-33.
11. Heeschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization [J]. Blood, 2003, 102(4):1340-6.
12. Caiola K, Cheng JW. Use of erythropoietin in heart failure management [J]. Ann Pharmacother, 2004, 38(12):2145-2149.
13.Foley RN, Parfrey PS, Harnett JD, et al . The impact of anemia on cardiomyopathy, morbidity, and mortality in end-stage renal disease [J]. Am J Kidney Dis, 1996,28(1):53-61.
14. Singh NP, Chandrashekhar L, Nair M , et al. The cardiovascular and hemodynamic effects of erythropoietin in chronic renal failure [J].J Assoc Physicians India, 2000, 48(3): 301-306.
15. Greaves SC, Gamble GD, Collins JF, et al. Determinants of left ventricular hypertrophy and systolic dysfunction in chronic renal failure [J] Am J Kidney Dis, 1994, 24(5): 768-776.
16. Hayashi T, Suzuki A, Shoji T, et al. Cardiovascular efect of normalizing the hematocrit level during erythropoietin therapy in predialysis patients with chronic renal failure[J]. Am J Kidney Dis, 2000, 35(2): 250-256.
17. MeMahon LP, Mason K, Skinner SL, et al. Effects of haemoglobin normalization on quality of life and cardiovascular parameters in end stage renal failure [J]. Nephrol Dial Transplant, 2000, 15(9): 1425-1430.
18. Franz H, Messerli, Reinhandiettohut. Left ventricular hypertrophy: A pressure-independent cardiovascular risk factor [J]. J cardiovasc Pharmocol,1993, 22 (Suppl 1) : S7-S13.
19. Silverberg DS, Wexler D, Iaina A. The role of anemia in the progression of congestive heart failure. Is there a place for erythropoietin and intravenous iron[J].J Nephrol, 2004, 17(6): 749-761.
20. Anagnostou A, Lee ES, Kessimian N, et al. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells [J]. Proc Natl Acad Sci USA,1990, 87(15):5978-5982.
21. Barrett JD, Zhang Z, Zhu JH et al. Erythropoietin upregulates angiotensin receptors in cultured rat vascular smooth muscle cells [J]. J Hypertens, 1998,16(12 pt 1): 1749-1757.
22. Goguzev J, Zhu DL, Herembert T, et al. Effect of erythropoietin on DNA synthesis, proto-on-cogene expression and phospholipase C activity in rat vascular smooth muscle cells[J]. Biochem Biophys Res Commun, 1994,199(2):977-983.
23. Ghezzi, P. and Brines, M. Erythropoietin as an antiapoptotic, tissue-protective cytokine [J]. Cell Death Differ, 2004,11 (Suppl. 1), S37-S44.
24.郭志坤.现代心脏组织学[M].北京:人民卫生出版社,2007:176-178.
25. Lundby C, Hellsten Y, Jensen MB, et al. Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle[J]. J Appl Physiol, 2008 ,104(4):1154-60.
26. Seong SR, Lee JW, Lee YK, et al. Stimulation of cell growth by erythropoietin in RAW264.7 cells: association with AP-1 activation[J]. Arch Pharm Res, 2006,29(3):218-23.
27. Ueyama T, Kawashima S, Sakoda T, et al. Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy [J]. J Mol Cell Cardiol, 2000,32(6):947-60.
28. Li X, Tsai P, Wieder ED, et al. Vascular smooth muscle cells grown on Matrigel. A model of the contractile phenotype with decreased activation of mitogen-activated protein kinase [J]. J Biol Chem, 1994, 269(30):19653 -19658.
29. Shi Y, Rafiee P, Su J, et al. Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels [J]. Basic Res Cardiol, 2004, 99():173-182.
30. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts[J]. J Biol Chem,2004,279 (20):20655- 20662.
31.Kiemer AK, Vollmar AM, Bilzer M, et al. Atrial natriuretic peptide reduces expression of TNF-alpha mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1 [J]. J Hepatol, 2000, 33(2):236-246.
32. Porat O, Neumann D, Zamir O, et al. Erythropoietin stimulates atrial natriuretic peptide secretion from adult rat cardiac atrium[J]. J Pharmacol Exp Ther, 1996,276(3): 1162-1168.
33. Nishigaki K, Tomita M, Kagawa K, et al. Marked expression of plasma brain natriuretic peptide is a special feature of hypertrophic obstructive cardiomyopathy [J].J Am Coll Cardiol, 1996, 128(5): 1234-42.
34. Piuhola J, Kerkela R, Keenan JI, et al. Direct cardiac actions of erythropoietin (EPO): effects on cardiac contractility, BNP secretion and ischaemia/reperfusion injury[J]. Clin Sci (Lond), 2008, 114(4):293-304.
35. Briest W, Homagk L, Baba HA, et al. Cardiac remodeling in erythropoietin-transgenic mice [J]. Cell Physiol Biochem, 2004, 14(4-6):277-84.
36. Piuhola J, Szokodi I, Ruskoaho H. Endothelin-1 and angiotensin II contribute to BNP but not c-fos gene expression response to elevated load in isolated mice hearts [J]. Biochim Biophys Acta, 2007, 772(3):338-44.
37. Wolny A, Clozel JP, Rein J, et al. Functional and biochemical analysis of angiotensin II forming pathways in the human heart [J]. Circ Res, 1997, 80 (2) :219-227.
38. Dzau VJ. Circulating versus local renin angiotensin system in cardiovascular homeostasis [J]. Circulation, 1998, 77 (6 Pt 2): 14-13.
39. Tipnis SR, Hooper NM, Hyde R, et al. A human homolog of angiotensin-converting enzyme.cloning and functional expression as a captoprilinsensitive carboxypeptidase [J]. J Biol Chem, 2000, 275(4): 33238-33243.
40. Ferrario CM, B rosnihan KB, Diz DI, et al. A new hormone of the angiotensin system [J]. Hypertension, 1991, 18 [5 suppl]: III126-133.
41. Akimoto T, Kusano E, Fujita N et al. Erythropoietin modulates angiotensin II - or noradrenaline-induced Ca mobilization in cultured rat vascular smooth-muscle cells [J]. Nephrol Dial Transplant, 2001, 16(3):491-499.
42. Carlini RG, Gupta A, Liapis H, et al. Endothelin-1 release by erythropoietin involves calcium signaling in endothelial cells [J]. J Cardiovasc Pharmacol, 1995,26(6):889-892
1. Bonsdorff E, Jalavisto E. A humoral mechanism in anoxic erythrocytosis [J]. Acta Physiologica Scandinavica, 1948, 6:150-170.
2. Ng T, Marx G, Littlewood T, et al. Recombinant erythropoietin in clinical practice [J]. Postgrad Med J, 2003, 79(933):367- 376.
3. Ozaki K, Leonard WJ. Cytokine and cytokine receptor pleiotropy and redundancy [J]. J Biol Chem, 2002,277(33):29355-29358.
4. Dame C, Fahnenstich H, Freitag P, et al. Erythropoietin mRNA in human fetal and neonatal tissue[J]. Blood, 1998, 92(9):3218-3225.
5. Koury ST, Bondurant MC, Koury MJ. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization [J]. Blood, 1988, 71(2):524-527.
6. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia [J]. J Appl Physiol, 2000, 88(4): 1474-1480.
7. Masuda S, Okano M, Yamagishi K, et al. A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes [J]. J Biol Chem, 1994, 269(30): 19488- 19493.
8. Bernaudin M, Bellail A, Marti HH, et al. Neurons and astrocytes express EPO mRNA: oxygen-sensing mechanisms that involve the redox-state of the brain [J].Glia, 2000, 30(3):271-8.
9. Vogt C, Pentz S, Rich IN. A role for the macrophage in normal hemopoiesis: III.In vitro and in vivo erythropoietin gene expression in macrophages detected by in situ hybridization [J]. Exp Hematol, 1989, 17(5):391-397.
10. Conrad KP, Benyo DF, Westerhausen-Larsen A, et al. Expression of erythropoietin by the human placenta [J]. FASEB J, 1996,10(7):760-768.
11. Juul SE, Zhao Y, Dame JB, et al. Origin and fate of erythropoietin in human milk[J]. Pediatr Res, 2000, 48(5):660-667.
12. Bazan JF. Structural design and molecular evolution of a cytokine receptor superfamily[J]. Proc Natl Acad Sci USA, 1990, 87(18):6934-6938.
13. Fraser JK, Tan AS, Lin F-K, et al. Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes [J]. Exp Hematol, 1989,17(1):10-16.
14. Anagnostou A, Liu Z, Steiner M et al. Erythropoietin receptor mRNA expression in human endothelial cells [J]. Proc Natl Acad Sci USA, 1994,91(9):3974-3978.
15. Nagai T, Akizawa T, Kohjiro S et al. rHuEPO enhances the production of plasminogen activator inhibitor-1 in cultured endothelial cells [J]. Kidney Int,1996, 50(1):102-107.
16. Anagnostou A, Lee ES, Kessimian N, et al. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells [J]. Proc Natl Acad Sci USA, 1990,87(15):5978-5982.
17. Ogilvie M, Yu X, Nicolas-Metral V, et al. Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts[J]. J Biol Chem, 2000,275(50):39754-39761.
18. Masuda S, Nagao M, Takahata K, et al. Functional erythropoietin receptor of the cells with neural characteristics. Comparison with receptor properties of erythroid cells [J]. J Biol Chem, 1993,268(15): 11208-11216.
19. Westenfelder C, Biddle DL, Baranowski RL. Human, rat, and mouse kidney cells express functional erythropoietin receptors [J]. Kidney Int, 1999, 55(3):808-820.
20. Arcasoy MO, Amin K, Karayal AF, et al. Functional significance of erythropoietin receptor expression in breast cancer[J]. Lab Invest, 2002,82(7):911-918.
21. Junk AK, Mammis A, Savitz SI, et al. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury [J]. Proc Natl Acad Sci USA, 2002, 99(16):10659-10664.
22. Tramontano AF, Muniyappa R, Black AD, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway [J].Biochem Biophys Res Commun, 2003,308 (4):990- 994.
23. Wright GL, Hanlon P, Amin K, et al. Erythropoietin receptor expression in adult rat cardiomyocytesis associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury[J]. FASEB J, 2004,18 (9):1031- 1033.
24. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts [J]. J Biol Chem,2004, 279 (20): 20655- 20662.
25. Harris KW, Winkelmann JC. Enzyme-linked immunosorbent assay detects a potential soluble form of the erythropoietin receptor in human plasma [J]. Am J Hematol, 1996, 52(1):8-13.
26. Maeda Y, Sakaguchi M, Naiki Y, et al. Possible involvement of soluble erythropoietin receptor in resistance to erythropoietin in patients with renal anemia[J]. Am J Nephrol, 2001,21(5):426.
27. Shimizu R, Komatsu N, Miura Y. Dominant negative effect of a truncated erythropoietin receptor (EPOR-T) on erythropoietin-induced erythroid differentiation: possible involvement of EPOR-T in ineffective erythropoiesis of myeloblastic syndrome [J]. Exp Hematol, 1999, 27(2):229-233.
28. Brines M, Grasso G, Fiordaliso F, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor [J]. Proc Natl Acad Sci U S A, 2004, 101(41):14907-14912.
29. Richmond TD, Chohan M, Barber DL. Turning cells red: signal transduction mediated by erythropoietin [J]. Trends Cell Biol 2005; 15(3):146-55.
30. Witthuhn A, Quelle FW, Silvennoinen O, et al. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin [J]. Cell, 1993, 74(2):227-236.
31. Yamaura G, Turoczi T, Yamamoto F, et al. STAT signaling in ischemic heart: a role of STAT5A in ischemic preconditioning [J]. Am J Physiol Heart Circ Physiol,2003,285 (2):H476-H482.
32. Gobert S, Duprez V, Lacombe C, et al. The signal transduction pathway of erythropoietin involves three forms of mitogen-activated protein (MAP) kinase in UT7 erythroleukemia cells [J]. Eur J Biochem, 1995, 234 (1): 75-83.
33. Rafiee P, Shi Y, Kong X, et al. Activation of protein kinases in chronically hypoxic infant human and rabbit hearts: role in cardioprotection[J]. Circulation,2002, 106(2): 239-245.
34. Shi Y, Rafiee P, Su J, et al. Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels [J]. Basic Res Cardiol, 2004, 99(3): 173-182.
35. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts [J]. J Biol Chem,2004,279 (20): 20655-20662.
36. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury [J]. N Engl J Med,2007, 357(11):1121-1135.
37. Zimmermann S, Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B) [J]. Science, 1999,286 (5445): 1741- 1744.
38. Rodriguez-Viciana P, Warne PH, Khwaja A, et al. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras[J]. Cell,1997, 89(3): 457-467.
39. Figueroa C, Vojtek AB. Akt negatively regulates translation of the ternary complex factor Elk-1[J]. Oncogene, 2003, 22 (36):5554-5561.
40. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway [J]. Cardiovasc Res, 2004, 61(3): 448^60.
41. Wu XC, Johns EJ, Richards NT. Relationship between erythropoietin and nitric oxide in the contraction of rat renal arcuate arteries and human umbilical vein endothelial cells[J]. Clin Sci (Lond), 1999, 97 (4):413-419.
42. Bullard AJ, Govewalla P, Yellon DM, et al. Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo [J]. Basic Res Cardiol,2005, 100(5): 397-403.
43. Burger D, Lei M, Geoghegan-Morphet N, et al. Erythropoietin protects cardiomyocytes from apoptosis via up-regulation of endothelial nitric oxide synthase [J]. Cardiovasc Res, 2006, 72(1): 51-59.
44. Greaves SC, Ganlble GD, Colins JF, et al. Determinants of left ventricular hypertrophy and systolic dysfunction in chronic renal failure[J]. Am J Kidney Dis, 1994, 24(5): 768-776.
45. Wizemann V, Kaufmann J, Kramer W. Effect of erythropoietin on ischemia tolerance in anemic hemodialysis patients with confirmed coronary artery disease[J] Nephron, 1992, 62(2): 161-165.
46. Singh NP, Chandrashekhar L, Nair M, et al . The cardiovascular and hemodynamic effects of erythropoietin in chronic renal failure [J]. J Assoc Physicians India, 2000, 48(3): 301-306.
47. Hayashi T, Suzuki A, Shoji T, et al. Cardiovascular efect of normalizing the hematocrit level during erythropoietin therapy in predialysis patients with chronic renal failure. Am J Kidney Dis, 2000, 35(2): 250-256.
48. Silverherg DS, Wexier D, Blum M, et al. The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe,resistant congestive heart failure improves cardiac and renal funotion and functional cardiac class, and markedly reduces hospitalization[J]J Am Coil Cardiol, 2000, 35(7): 1737-1744.
49. McClellan WM, Flanders WD, Langston RD, et al. Anemia and renal insufficiency are independent risk factors for death among patients with congestive heart failure admitted to community hospitals: A population-based study[J]. J Am Soc Nephrology, 2002, 13(7): 1928-1936.
50. Horwich TB, Fonarow GC, Hamilton MA, et al. Anemia is associated with worse symptoms, greater impairment in functional capacity and a significant increase in mortality in patients with advanced heart failure[J]. J Am Coll Cardiol, 2002,39(11): 1780-1786.
51. Van der Meer P, Voors AA, Lipsic E, et al. Prognostic value of plasma erythropoietin on mortality in patients with chronic heart failure[J]. J Am Coll Cardiol, 2004, 44(1): 63-67.
52. Silverberg DS, Wexier D, Iaina A. The role of anemia in the progression of congestive heart failure. Is there a place for erythropoietin and intravenous iron [J].Nephrol, 2004, 17(6): 749-761.
53. Silverberg DS, Wexier D, Sheps D, et al. The efect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study[J]. J Am Coll Cardiol, 2001, 37(7): 1775-1780.
54. Calvillo L, Latini R, Kajstura J, et al. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling [J]. Proc Natl Acad Sci USA, 2003, 100(8):4802-4806.
55. Tramontano AF, M uniyappa R, Black AD, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway [J]. Biochem Biophys Res Commun. 2003. 308(4): 990-994.
56. Parsa CJ, Matsumoto A, Kim J, et al. A novel protective effect of erythropoietin in the infarcted heart [J]. J Clin Invest, 2003, 112 (7): 999-1007.
57. Cai Z, Manalo DJ, Wei G, et al. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury [J].Circulation, 2003, 108(1): 79-85.
58. Moon C, Krawczyk M, Ahn D, et al. Erythropoietin reduces m yocardial infarction and left ventricular functional decline after coronary artery ligation in rats [J]. Proc Natl Acad Sci U S A, 2003, 100(20): 11612-11617.
59. van der Meer P. et al. Erythropoietin induces neovascularization and improves cardiac function in rats with heart failure after myocardial infarction [J]. Am Coll Cardiol, 2005,46(1): 125-133.
60. Gao E, Boucher M, Chuprun JK, et al. Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart [J]. Am J Physiol Heart Circ Physiol, 2007, 293(1): H60-H68.
61. Moon C, Krawczyk M, Paik D, et al. Cardioprotection by recombinant human erythropoietin following acute experimental myocardial infarction: dose response and therapeutic window [J]. Cardiovasc Drugs Ther, 2005, 19(4): 243-250.
62. Hirata A, Minamino T, Asanuma H, et al. Erythropoietin enhances neovascularization of ischemic myocardium and improves left ventricular dysfunction after myocardial infarction in dogs [J]. J Am Coll Cardiol, 2006,48(1): 176-184.
63. Westenbrink BD, Lipsic E, Van der Meer P, et al. Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization [J]. Eur Heart J, 2007,28(1): 2018-2027.
64. Prunier F, Pfister O, Hadri L, et al. Delayed erythropoietin therapy reduces post-MI cardiac remodeling only at a dose that mobilizes endothelial progenitor cells [J]. Am J Physiol Heart Circ Physiol, 2007, 292(1): H522-H529.
65. Riksen NP, Hausenloy DJ, Yellon DM.[J]. Erythropoietin: ready for prime-time cardioprotection [J]. Trends Pharmacol Sci, 2008, 29(5):258-267.
66. Lipsic E, Van der Meer P, Voors AA, et al. A single bolus of a long-acting erythropoietin analogue darbepoetin alfa in patients with acute myocardial infarction: a randomized feasibility and safety study [J]. Cardiovasc Drugs Ther,2006,20(2): 135-141.
67. Liem A, Van de Woestijne, Bruijns E, et al. Effect of EPO administration on myocardial infarct size in patients with non-STE acute coronary syndromes;results from a pilot study [J]. Int J Cardiol, 2009, 131(2):285-7.
2. Koury ST, Bondurant MC, Koury MJ. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization [J]. Blood, 1988,71(2):524—527.
3. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia [J]. J Appl Physiol, 2000, 88(4):1474-1480.
4. Wu H, Lee SH, Gao J, et al. Inactivation of erythropoietin leads to defects in cardiac morphogenesis [J]. Development, 1999,126(16):3597—3605.
5. Calvillo L, Latini R, Kajstura J, et al. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling [J]. Proc Natl Acad Sci USA, 2003,100(8): 4802-4806.
6. Tramontano AF, M uniyappa R, Black AD, et al. Erythropoietin protects cardiac m yocytes from hypoxia-induced apoptosis through an Akt-dependent pathway [J]. Biochem Biophys Res Commun, 2003, 308(4): 990-994.
7. Parsa CJ, Matsumoto A, Kim J, et al. A novel protective effect of erythropoietin in the infarcted heart [J]. J CIin Invest, 2003,112(7): 999—1007.
8. Rui T, Feng Q, Lei M, et al. Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1[J].Cardiovasc Res, 2005, 65(3):719 -27.
9. Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades [J]. Nature, 2001,412(6847):641-7.
10. Jaquet K, Krause K, Tawakol-Khodai M, et al. Erythropoietin and VEGF exhibit equal angiogenic potential [J]. Microvasc Res, 2002, 64(2):326-33.
11. Heeschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization [J]. Blood, 2003, 102(4):1340-6.
12. Caiola K, Cheng JW. Use of erythropoietin in heart failure management [J]. Ann Pharmacother, 2004, 38(12):2145-2149.
13.Foley RN, Parfrey PS, Harnett JD, et al . The impact of anemia on cardiomyopathy, morbidity, and mortality in end-stage renal disease [J]. Am J Kidney Dis, 1996,28(1):53-61.
14. Singh NP, Chandrashekhar L, Nair M , et al. The cardiovascular and hemodynamic effects of erythropoietin in chronic renal failure [J].J Assoc Physicians India, 2000, 48(3): 301-306.
15. Greaves SC, Gamble GD, Collins JF, et al. Determinants of left ventricular hypertrophy and systolic dysfunction in chronic renal failure [J] Am J Kidney Dis, 1994, 24(5): 768-776.
16. Hayashi T, Suzuki A, Shoji T, et al. Cardiovascular efect of normalizing the hematocrit level during erythropoietin therapy in predialysis patients with chronic renal failure[J]. Am J Kidney Dis, 2000, 35(2): 250-256.
17. MeMahon LP, Mason K, Skinner SL, et al. Effects of haemoglobin normalization on quality of life and cardiovascular parameters in end stage renal failure [J]. Nephrol Dial Transplant, 2000, 15(9): 1425-1430.
18. Franz H, Messerli, Reinhandiettohut. Left ventricular hypertrophy: A pressure-independent cardiovascular risk factor [J]. J cardiovasc Pharmocol,1993, 22 (Suppl 1) : S7-S13.
19. Silverberg DS, Wexler D, Iaina A. The role of anemia in the progression of congestive heart failure. Is there a place for erythropoietin and intravenous iron[J].J Nephrol, 2004, 17(6): 749-761.
20. Anagnostou A, Lee ES, Kessimian N, et al. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells [J]. Proc Natl Acad Sci USA,1990, 87(15):5978-5982.
21. Barrett JD, Zhang Z, Zhu JH et al. Erythropoietin upregulates angiotensin receptors in cultured rat vascular smooth muscle cells [J]. J Hypertens, 1998,16(12 pt 1): 1749-1757.
22. Goguzev J, Zhu DL, Herembert T, et al. Effect of erythropoietin on DNA synthesis, proto-on-cogene expression and phospholipase C activity in rat vascular smooth muscle cells[J]. Biochem Biophys Res Commun, 1994,199(2):977-983.
23. Ghezzi, P. and Brines, M. Erythropoietin as an antiapoptotic, tissue-protective cytokine [J]. Cell Death Differ, 2004,11 (Suppl. 1), S37-S44.
24.郭志坤.现代心脏组织学[M].北京:人民卫生出版社,2007:176-178.
25. Lundby C, Hellsten Y, Jensen MB, et al. Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle[J]. J Appl Physiol, 2008 ,104(4):1154-60.
26. Seong SR, Lee JW, Lee YK, et al. Stimulation of cell growth by erythropoietin in RAW264.7 cells: association with AP-1 activation[J]. Arch Pharm Res, 2006,29(3):218-23.
27. Ueyama T, Kawashima S, Sakoda T, et al. Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy [J]. J Mol Cell Cardiol, 2000,32(6):947-60.
28. Li X, Tsai P, Wieder ED, et al. Vascular smooth muscle cells grown on Matrigel. A model of the contractile phenotype with decreased activation of mitogen-activated protein kinase [J]. J Biol Chem, 1994, 269(30):19653 -19658.
29. Shi Y, Rafiee P, Su J, et al. Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels [J]. Basic Res Cardiol, 2004, 99():173-182.
30. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts[J]. J Biol Chem,2004,279 (20):20655- 20662.
31.Kiemer AK, Vollmar AM, Bilzer M, et al. Atrial natriuretic peptide reduces expression of TNF-alpha mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1 [J]. J Hepatol, 2000, 33(2):236-246.
32. Porat O, Neumann D, Zamir O, et al. Erythropoietin stimulates atrial natriuretic peptide secretion from adult rat cardiac atrium[J]. J Pharmacol Exp Ther, 1996,276(3): 1162-1168.
33. Nishigaki K, Tomita M, Kagawa K, et al. Marked expression of plasma brain natriuretic peptide is a special feature of hypertrophic obstructive cardiomyopathy [J].J Am Coll Cardiol, 1996, 128(5): 1234-42.
34. Piuhola J, Kerkela R, Keenan JI, et al. Direct cardiac actions of erythropoietin (EPO): effects on cardiac contractility, BNP secretion and ischaemia/reperfusion injury[J]. Clin Sci (Lond), 2008, 114(4):293-304.
35. Briest W, Homagk L, Baba HA, et al. Cardiac remodeling in erythropoietin-transgenic mice [J]. Cell Physiol Biochem, 2004, 14(4-6):277-84.
36. Piuhola J, Szokodi I, Ruskoaho H. Endothelin-1 and angiotensin II contribute to BNP but not c-fos gene expression response to elevated load in isolated mice hearts [J]. Biochim Biophys Acta, 2007, 772(3):338-44.
37. Wolny A, Clozel JP, Rein J, et al. Functional and biochemical analysis of angiotensin II forming pathways in the human heart [J]. Circ Res, 1997, 80 (2) :219-227.
38. Dzau VJ. Circulating versus local renin angiotensin system in cardiovascular homeostasis [J]. Circulation, 1998, 77 (6 Pt 2): 14-13.
39. Tipnis SR, Hooper NM, Hyde R, et al. A human homolog of angiotensin-converting enzyme.cloning and functional expression as a captoprilinsensitive carboxypeptidase [J]. J Biol Chem, 2000, 275(4): 33238-33243.
40. Ferrario CM, B rosnihan KB, Diz DI, et al. A new hormone of the angiotensin system [J]. Hypertension, 1991, 18 [5 suppl]: III126-133.
41. Akimoto T, Kusano E, Fujita N et al. Erythropoietin modulates angiotensin II - or noradrenaline-induced Ca mobilization in cultured rat vascular smooth-muscle cells [J]. Nephrol Dial Transplant, 2001, 16(3):491-499.
42. Carlini RG, Gupta A, Liapis H, et al. Endothelin-1 release by erythropoietin involves calcium signaling in endothelial cells [J]. J Cardiovasc Pharmacol, 1995,26(6):889-892
1. Bonsdorff E, Jalavisto E. A humoral mechanism in anoxic erythrocytosis [J]. Acta Physiologica Scandinavica, 1948, 6:150-170.
2. Ng T, Marx G, Littlewood T, et al. Recombinant erythropoietin in clinical practice [J]. Postgrad Med J, 2003, 79(933):367- 376.
3. Ozaki K, Leonard WJ. Cytokine and cytokine receptor pleiotropy and redundancy [J]. J Biol Chem, 2002,277(33):29355-29358.
4. Dame C, Fahnenstich H, Freitag P, et al. Erythropoietin mRNA in human fetal and neonatal tissue[J]. Blood, 1998, 92(9):3218-3225.
5. Koury ST, Bondurant MC, Koury MJ. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization [J]. Blood, 1988, 71(2):524-527.
6. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia [J]. J Appl Physiol, 2000, 88(4): 1474-1480.
7. Masuda S, Okano M, Yamagishi K, et al. A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes [J]. J Biol Chem, 1994, 269(30): 19488- 19493.
8. Bernaudin M, Bellail A, Marti HH, et al. Neurons and astrocytes express EPO mRNA: oxygen-sensing mechanisms that involve the redox-state of the brain [J].Glia, 2000, 30(3):271-8.
9. Vogt C, Pentz S, Rich IN. A role for the macrophage in normal hemopoiesis: III.In vitro and in vivo erythropoietin gene expression in macrophages detected by in situ hybridization [J]. Exp Hematol, 1989, 17(5):391-397.
10. Conrad KP, Benyo DF, Westerhausen-Larsen A, et al. Expression of erythropoietin by the human placenta [J]. FASEB J, 1996,10(7):760-768.
11. Juul SE, Zhao Y, Dame JB, et al. Origin and fate of erythropoietin in human milk[J]. Pediatr Res, 2000, 48(5):660-667.
12. Bazan JF. Structural design and molecular evolution of a cytokine receptor superfamily[J]. Proc Natl Acad Sci USA, 1990, 87(18):6934-6938.
13. Fraser JK, Tan AS, Lin F-K, et al. Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes [J]. Exp Hematol, 1989,17(1):10-16.
14. Anagnostou A, Liu Z, Steiner M et al. Erythropoietin receptor mRNA expression in human endothelial cells [J]. Proc Natl Acad Sci USA, 1994,91(9):3974-3978.
15. Nagai T, Akizawa T, Kohjiro S et al. rHuEPO enhances the production of plasminogen activator inhibitor-1 in cultured endothelial cells [J]. Kidney Int,1996, 50(1):102-107.
16. Anagnostou A, Lee ES, Kessimian N, et al. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells [J]. Proc Natl Acad Sci USA, 1990,87(15):5978-5982.
17. Ogilvie M, Yu X, Nicolas-Metral V, et al. Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts[J]. J Biol Chem, 2000,275(50):39754-39761.
18. Masuda S, Nagao M, Takahata K, et al. Functional erythropoietin receptor of the cells with neural characteristics. Comparison with receptor properties of erythroid cells [J]. J Biol Chem, 1993,268(15): 11208-11216.
19. Westenfelder C, Biddle DL, Baranowski RL. Human, rat, and mouse kidney cells express functional erythropoietin receptors [J]. Kidney Int, 1999, 55(3):808-820.
20. Arcasoy MO, Amin K, Karayal AF, et al. Functional significance of erythropoietin receptor expression in breast cancer[J]. Lab Invest, 2002,82(7):911-918.
21. Junk AK, Mammis A, Savitz SI, et al. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury [J]. Proc Natl Acad Sci USA, 2002, 99(16):10659-10664.
22. Tramontano AF, Muniyappa R, Black AD, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway [J].Biochem Biophys Res Commun, 2003,308 (4):990- 994.
23. Wright GL, Hanlon P, Amin K, et al. Erythropoietin receptor expression in adult rat cardiomyocytesis associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury[J]. FASEB J, 2004,18 (9):1031- 1033.
24. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts [J]. J Biol Chem,2004, 279 (20): 20655- 20662.
25. Harris KW, Winkelmann JC. Enzyme-linked immunosorbent assay detects a potential soluble form of the erythropoietin receptor in human plasma [J]. Am J Hematol, 1996, 52(1):8-13.
26. Maeda Y, Sakaguchi M, Naiki Y, et al. Possible involvement of soluble erythropoietin receptor in resistance to erythropoietin in patients with renal anemia[J]. Am J Nephrol, 2001,21(5):426.
27. Shimizu R, Komatsu N, Miura Y. Dominant negative effect of a truncated erythropoietin receptor (EPOR-T) on erythropoietin-induced erythroid differentiation: possible involvement of EPOR-T in ineffective erythropoiesis of myeloblastic syndrome [J]. Exp Hematol, 1999, 27(2):229-233.
28. Brines M, Grasso G, Fiordaliso F, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor [J]. Proc Natl Acad Sci U S A, 2004, 101(41):14907-14912.
29. Richmond TD, Chohan M, Barber DL. Turning cells red: signal transduction mediated by erythropoietin [J]. Trends Cell Biol 2005; 15(3):146-55.
30. Witthuhn A, Quelle FW, Silvennoinen O, et al. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin [J]. Cell, 1993, 74(2):227-236.
31. Yamaura G, Turoczi T, Yamamoto F, et al. STAT signaling in ischemic heart: a role of STAT5A in ischemic preconditioning [J]. Am J Physiol Heart Circ Physiol,2003,285 (2):H476-H482.
32. Gobert S, Duprez V, Lacombe C, et al. The signal transduction pathway of erythropoietin involves three forms of mitogen-activated protein (MAP) kinase in UT7 erythroleukemia cells [J]. Eur J Biochem, 1995, 234 (1): 75-83.
33. Rafiee P, Shi Y, Kong X, et al. Activation of protein kinases in chronically hypoxic infant human and rabbit hearts: role in cardioprotection[J]. Circulation,2002, 106(2): 239-245.
34. Shi Y, Rafiee P, Su J, et al. Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels [J]. Basic Res Cardiol, 2004, 99(3): 173-182.
35. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts [J]. J Biol Chem,2004,279 (20): 20655-20662.
36. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury [J]. N Engl J Med,2007, 357(11):1121-1135.
37. Zimmermann S, Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B) [J]. Science, 1999,286 (5445): 1741- 1744.
38. Rodriguez-Viciana P, Warne PH, Khwaja A, et al. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras[J]. Cell,1997, 89(3): 457-467.
39. Figueroa C, Vojtek AB. Akt negatively regulates translation of the ternary complex factor Elk-1[J]. Oncogene, 2003, 22 (36):5554-5561.
40. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway [J]. Cardiovasc Res, 2004, 61(3): 448^60.
41. Wu XC, Johns EJ, Richards NT. Relationship between erythropoietin and nitric oxide in the contraction of rat renal arcuate arteries and human umbilical vein endothelial cells[J]. Clin Sci (Lond), 1999, 97 (4):413-419.
42. Bullard AJ, Govewalla P, Yellon DM, et al. Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo [J]. Basic Res Cardiol,2005, 100(5): 397-403.
43. Burger D, Lei M, Geoghegan-Morphet N, et al. Erythropoietin protects cardiomyocytes from apoptosis via up-regulation of endothelial nitric oxide synthase [J]. Cardiovasc Res, 2006, 72(1): 51-59.
44. Greaves SC, Ganlble GD, Colins JF, et al. Determinants of left ventricular hypertrophy and systolic dysfunction in chronic renal failure[J]. Am J Kidney Dis, 1994, 24(5): 768-776.
45. Wizemann V, Kaufmann J, Kramer W. Effect of erythropoietin on ischemia tolerance in anemic hemodialysis patients with confirmed coronary artery disease[J] Nephron, 1992, 62(2): 161-165.
46. Singh NP, Chandrashekhar L, Nair M, et al . The cardiovascular and hemodynamic effects of erythropoietin in chronic renal failure [J]. J Assoc Physicians India, 2000, 48(3): 301-306.
47. Hayashi T, Suzuki A, Shoji T, et al. Cardiovascular efect of normalizing the hematocrit level during erythropoietin therapy in predialysis patients with chronic renal failure. Am J Kidney Dis, 2000, 35(2): 250-256.
48. Silverherg DS, Wexier D, Blum M, et al. The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe,resistant congestive heart failure improves cardiac and renal funotion and functional cardiac class, and markedly reduces hospitalization[J]J Am Coil Cardiol, 2000, 35(7): 1737-1744.
49. McClellan WM, Flanders WD, Langston RD, et al. Anemia and renal insufficiency are independent risk factors for death among patients with congestive heart failure admitted to community hospitals: A population-based study[J]. J Am Soc Nephrology, 2002, 13(7): 1928-1936.
50. Horwich TB, Fonarow GC, Hamilton MA, et al. Anemia is associated with worse symptoms, greater impairment in functional capacity and a significant increase in mortality in patients with advanced heart failure[J]. J Am Coll Cardiol, 2002,39(11): 1780-1786.
51. Van der Meer P, Voors AA, Lipsic E, et al. Prognostic value of plasma erythropoietin on mortality in patients with chronic heart failure[J]. J Am Coll Cardiol, 2004, 44(1): 63-67.
52. Silverberg DS, Wexier D, Iaina A. The role of anemia in the progression of congestive heart failure. Is there a place for erythropoietin and intravenous iron [J].Nephrol, 2004, 17(6): 749-761.
53. Silverberg DS, Wexier D, Sheps D, et al. The efect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study[J]. J Am Coll Cardiol, 2001, 37(7): 1775-1780.
54. Calvillo L, Latini R, Kajstura J, et al. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling [J]. Proc Natl Acad Sci USA, 2003, 100(8):4802-4806.
55. Tramontano AF, M uniyappa R, Black AD, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway [J]. Biochem Biophys Res Commun. 2003. 308(4): 990-994.
56. Parsa CJ, Matsumoto A, Kim J, et al. A novel protective effect of erythropoietin in the infarcted heart [J]. J Clin Invest, 2003, 112 (7): 999-1007.
57. Cai Z, Manalo DJ, Wei G, et al. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury [J].Circulation, 2003, 108(1): 79-85.
58. Moon C, Krawczyk M, Ahn D, et al. Erythropoietin reduces m yocardial infarction and left ventricular functional decline after coronary artery ligation in rats [J]. Proc Natl Acad Sci U S A, 2003, 100(20): 11612-11617.
59. van der Meer P. et al. Erythropoietin induces neovascularization and improves cardiac function in rats with heart failure after myocardial infarction [J]. Am Coll Cardiol, 2005,46(1): 125-133.
60. Gao E, Boucher M, Chuprun JK, et al. Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart [J]. Am J Physiol Heart Circ Physiol, 2007, 293(1): H60-H68.
61. Moon C, Krawczyk M, Paik D, et al. Cardioprotection by recombinant human erythropoietin following acute experimental myocardial infarction: dose response and therapeutic window [J]. Cardiovasc Drugs Ther, 2005, 19(4): 243-250.
62. Hirata A, Minamino T, Asanuma H, et al. Erythropoietin enhances neovascularization of ischemic myocardium and improves left ventricular dysfunction after myocardial infarction in dogs [J]. J Am Coll Cardiol, 2006,48(1): 176-184.
63. Westenbrink BD, Lipsic E, Van der Meer P, et al. Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization [J]. Eur Heart J, 2007,28(1): 2018-2027.
64. Prunier F, Pfister O, Hadri L, et al. Delayed erythropoietin therapy reduces post-MI cardiac remodeling only at a dose that mobilizes endothelial progenitor cells [J]. Am J Physiol Heart Circ Physiol, 2007, 292(1): H522-H529.
65. Riksen NP, Hausenloy DJ, Yellon DM.[J]. Erythropoietin: ready for prime-time cardioprotection [J]. Trends Pharmacol Sci, 2008, 29(5):258-267.
66. Lipsic E, Van der Meer P, Voors AA, et al. A single bolus of a long-acting erythropoietin analogue darbepoetin alfa in patients with acute myocardial infarction: a randomized feasibility and safety study [J]. Cardiovasc Drugs Ther,2006,20(2): 135-141.
67. Liem A, Van de Woestijne, Bruijns E, et al. Effect of EPO administration on myocardial infarct size in patients with non-STE acute coronary syndromes;results from a pilot study [J]. Int J Cardiol, 2009, 131(2):285-7.