心衰的机制与干细胞治疗基础研究
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摘要
课题Ⅰ:SCN5A点突变导致扩心病发生的机制研究
背景:心脏钠通道基因(cardiac sodium channel,SCN5A)突变可导致各类心律失常,现有观点认为SCN5A突变与扩心病有关,但其机理还不清楚并且争议很大。我们曾在一个进行性心脏传导障碍伴扩心病的遗传家系中,发现了一个新的SCN5A基因A1180V突变。本课题即试图在细胞及分子水平上观察该突变位点是否影响钙平衡从而造成心肌损伤。
方法:以PacⅠ线性化重组腺病毒质粒pAdGFP-SCN5A(MT/WT),经线性化后转染到HEK 293细胞中包装成病毒,随后提取DNA进行PCR鉴定和测序鉴定。重组腺病毒Ad-GFP-SCN5A(MT/WT)经HEK293传代、扩增、浓缩与纯化,并测定病毒滴度(TCID_(50)和PFU)。将这些病毒按如下分组接种新生大鼠心肌细胞:对野生型SCN5A和突变型SCN5A分别设置缺氧组、缺氧并加xamoterol处理组、xamoterol组和空白组。接种病毒48小时后,以缺氧和xamoterol处理2小时后,随后进行如下实验:(1)提取RNA,经realtime PCR,观察钠和钙相关基因(L-Ca~(2+)、CaMKⅡ、NaCX、PLB、CaM、SERCA、RyR2)的表达情况;(2)加载钙荧光指示剂X—rhod—1,以激光共聚焦显微镜测定心肌细胞内钙信号的变化情况;(3)用Annexin-V-PE联合碘化丙锭(PI)流式细胞仪检测心肌细胞凋亡率。对数据采用SPSS10.0统计软件进行ANOVA方差分析,p＜0.05为差异显著,p＜0.01为差异极显著。
结果:经PacI酶切线性化的腺病毒质粒pAd-GFP-SCN5A(MT/WT)用脂质体介导转染HEK293细胞后,产生的重组腺病毒对HEK 293细胞有致病作用。用目的基因片段SCN5A引物进行PCR扩增,得到一条约5006p大小片段,测序后经序列分析表明SCN5A未发生新的突变,表明带有目的基因的重组腺病毒Ad-GFP-SCN5A(MT/WT)构建成功。重组腺病毒Ad-GFP-SCN5A(MT/WT)经HEK293传代、扩增、浓缩与纯化,获得滴度分别为PFU 7×10~(8.4)的Ad-GFP-SCN5A(MT)和PFU 7×10~(7.8)的Ad-GFP-SCN5A(WT)。分别接种HEK293和新生鼠心肌细胞,GFP阳性率在90%以上,说明病毒能够感染大部分细胞。从与钠和钙相关基因表达的情况来看,CaM和SERCA其表达量各组之间差异不显著;PLB在SCN5A野生型空白组表达显著高于其它各组(p＜0.05),其它基因其表达规律性不强。SCN5A MT空白组和SCN5A WT Xamoterol处理组之间钙瞬变差异显著(p＜0.05),各组间钙容量差异不显著,钙瞬变和钙容量均在数量上表现出SCN5A MT各组高于SCN5A WT各组的趋势。SCN5A MT缺氧并Xamoterol处理组和SCN5A MT空白组钙升高时间显著高于除这两组之外的其它各组(p＜0.05),除空白组间WT和MT间钙衰减时间差异显著外,其它对应组间差异不显著(p＜0.05),钙升高和钙衰减均在数量上表现出SCN5A MT各组低于SCN5A WT各组的趋势。各SCN5A MT组其细胞凋亡均显著高于相应的SCN5A WT组(p＜0.05)。
结论:钙信号的变化和细胞凋亡的结果一致表现出SCN5A A1180突变可增强心肌细胞内钙信号。这说明SCN5A A1180V突变导致的非失活内向钠电流增加引起了细胞内钙信号加强。这将支持SCN5A A1180V突变可导致心肌细胞内钙稳态失衡从而引起扩心病的假设。
课题Ⅱ:BMP2诱导人MSCs向心肌样细胞分化的可能性及其机制
背景骨形态发生蛋白2(BMP2)可诱导间充质干细胞(MSCs)成骨和脂肪细胞,尚未有经BMP2诱导MSCs向心肌样细胞方向分化的报道。RUNX2和PPARγ分别是向骨和脂肪细胞分化下游通路上两个关键转录因子。BMP2诱导胚胎干细胞ESC和P19CL6可分化成心肌样细胞,MSCs经5-aza诱导也可分化成心肌样细胞,在BMP2通路上,成骨和成脂肪细胞分化是可以切换的,因此,有理由设想也可向心肌细胞方向切换。本课题试图通过下调转录因子RUNX2和PPARγ的表达来观察BMP2诱导下的MSCs可否向心肌样细胞方向分化。
方法第一章:抽取人髂骨骨髓,经Ficoll密度梯度离心分离获得单个核细胞,分别经不同的培养基传代培养。以流式细胞仪鉴定其表面抗原,同时,对这些细胞经向脂肪细胞分化的化学诱导剂诱导。通过上述方法,获得生长性能良好的间充质干细胞株。第二章:以pEGFP-N1构建PPARγ和RUNX2基因的真核表达质粒,将这些基因通过脂质体转染或电转的方式转染到MSCs和HEK293细胞,选择转染率高的方法和高转染的细胞。将经化学合成的PPARγ和RUNX2siRNA与相应的质粒用lipo2000转染到HEK293细胞中,筛选下调基因表达最低的PPARγ和RUNX2 siRNA片段及相应片段的最佳浓度。第三章:以Lipofectamine RNAi MAX将BLOCK-iT~(TM) Alexa Fluor~(?) Red Fluorescent Oligo(Invitrogen USA)转染到MSCs,以评价RNA传送到MSCs的效率。按如下分组诱导MSCs的分化:(1)5-aza、BMP2、PPARγ和RUNX2的siRNA,(2)BMP2、PPARγ和RUNX2的siRNA,(3)5-aza、BMP2,(4)BMP2,(5)5-aza,(6)Control(Blank)。培养基中5-aza的终浓度为0.01uM,BMP2终浓度为100ng/ml,PPARγ和RUNX2的siRNA所加量分别为10pmol/孔(六孔板)。MSCs生长到70%的汇合度时,以5-aza处理细胞24小时,然后换液培养24小时即转染PPARγ和RUNX2的siRNA,6天后再转染一次,BMP2在70%的汇合度时即开始添加,每2天换液一次,12天后收细胞,提取RNA和蛋白,经RT—PCR、realtime PCR和western blotting,检测PPARγ、RUNX2、NKX2.5、GATA4、α-MHC和cTnT基因的表达情况。对数据采用SPSS10.0统计软件进行ANOVA方差分析,p＜0.05为差异显著,p＜0.01为差异极显著。
结果第一章:对抽取的骨髓分离纯化,传代培养,15天即铺满培养瓶底,梭形细胞比例增加,细胞得到纯化,但传至P3,细胞由梭形变为平坦、宽大,细胞立体感逐渐消失,细胞的形状趋向多样,说明细胞很快衰老。换用添加有补充物的MSCs专用培养基培养,MSCs则生长状态良好。样本经流式细胞仪鉴定表面抗原,MSCs高表达CD29(97.69%)和CD166(97.54%),但不表达CD34和CD45。同时,以化学诱导剂(MDI)成功的诱导这些细胞分化成为脂肪细胞。经诱导和鉴定表面抗原,说明所获得的细胞是高纯度的MSCs。第二章:经PCR鉴定和测序鉴定,成功的构建了以pEGFP-N1为载体的PPARγ和RUNX2基因真核表达质粒。将这些质粒与PPARγ和RUNX2 siRNA共转到HEK293,其中PPARγsiRNA的P2片段(5'AGAAUAAUAAGGUGGAGAUGCAGGC3')在mRNA水平和蛋白水平上均显著下调PPARγ的表达(p＜0.05),而RUNX2 siRNA的3片段(R1:5'UUUAAUAGCGUGCUGCCAUUCG AGG3',R2:5'AACAGAUUCAUCCAUUCUGCCACUA3',R3:5'UCAAGCUUCUGUCUGUGCCUUCUGG3')均显著下调RUNX2的表达(p＜0.05),同时,筛选得P2片段的最佳下调浓度为10pmol/孔(六孔板)(p＜0.05),R2片段两个最佳下调浓度为10pmol╱孔和25pmol/孔(p＜0.05)。第三章:将Fluor Red RNA转染到MSCs,发现60%以上的RNA传送到了MSCs,说明有较高的传送效率经BMP2诱导,发现第4组(BMP2)PPARγ的表达显著高于其它各组,RUNX2的表达也在第4组最高,但无统计学上的差异。转录因子Nkx2.5和GATA4和心肌特异性基因TnT和MHC的表达在第1组(5-aza、siRNA和BMP2)一致性的显著升高(p＜0.05),并且显著高于第5组(5-aza),而第5组这些基因的表达也显著高于除第1组之外的其它各组(p＜0.05)。说明在诱导过程中,PPARγ和RUNX2被相应siRNA抑制,单因素5-aza和BMP2均不占主导作用,BMP2与5-aza产生了协同效应,促进了MSCs向心肌样细胞分化。
结论抑制转录因子RUNX2和PPARγ的表达使得BMP2诱导MSCs向心肌样细胞分化是可行的,其机制可能在于BMP2下游信号通路间的串联和切换。
课题Ⅲ:CTSS基因启动子区-25G/A多态对中国人群冠心病的预测
背景:动脉粥样硬化是一种以动脉管壁细胞外基质结构广泛重构为特征的炎症性疾病。最近许多研究显示诸如组织蛋白酶S(CTSS)等的溶酶体半胱氨酸蛋白酶参与动脉粥样硬化形成过程。在CTSS基因的启动子区域核苷酸-25处存在G/A基因多态现象。基于CTSS在冠状动脉粥样硬化中发挥重要作用,而-25G/A多态位于CTSS基因启动子区域,因此,我们推测CTSS -25G/A多态性可能与冠心病之间存在密切联系。本项研究的目的即为观察中国人群中,CTSS-25G/A多态分布及其多态性与冠心病(CAD)危险性之间的关联。
方法:CTSS基因-25G/A多态性由多聚酶链式反应PCR及限制性内切酶降解方法获得。经冠脉造影检查明确的共659名冠心病患者(冠脉狭窄≥50%)及352名非冠心病患者(对照组)纳入本项研究。我们同时收集患者的吸烟史、高血压史及糖尿病史,并测量他们的血脂水平。
结果:所有研究对象中的等位基因G和A的频率分别为0.630及0.370。在病例组及对照组之间未发现其CTSS -25G/A多态性频率(G:0.626 vs.0.633,p＞0.05;A:0.374 vs.0.367,p＞0.05)及基因型分布(G:83.4 vs.85.3,p＞0.05;A:58.0vs.58.5,p＞0.05)存在显著性差异。此外,即使在使用逻辑回归对其他危险因素校正后,CTSS基因型与冠脉狭窄严重程度之间亦未发现存在关联(p＞0.05)。
结论:在中国人群中,基因型AA较基因型GG和GA更为少见。对照组及冠心病组之间的等位基因频率及基因型分布均无显著性差异。CTSS -25G/A多态性与冠状动脉狭窄的发生率及严重程度不相关。
Ⅰ.Novel SCN5A Mutant-Induced Intracellular Calcium Shift in Dilated Cardiomyopathy Pathogenesis
Background Mutations of SCN5A,a cardiac sodium channel gene, have been implicated in the pathogenesis of dilated cardiomyopathy(DCM).Our previous study indicated a role of a novel mutant,SCN5A A1180V,in the onset of DCM in a Chinese family.However,the underlying mechanisms remain unknown. The present study was to investigate the biological significance of SCN5A A1180V in intracellular calcium shift and consequent myocardial damage.
Materials and Methods Recombinant adenoviral vector pAdGFP-SCN5A(MT/WT) linearized by PacⅠenzyme was amplified and confirmed by PCR and DNA sequencing after transfection into HEK 293 by lipofectamine 2000.Amplification was verified by titering analysis.Cardiomyocytes of neonatal rats transfected with Ad-GFP-SCN5A(MT/WT) were then treated with hypoxia,xamoterol,hypoxia+xamoterol respectively.Total RNA were extracted after 2h treatment.Both sodium and calcium-associated genes(L-Ca~(2+),CaMKⅡ,NaCX,PLB,CaM, SERCA and RyR2) were detcted by realtime-PCR.Changes of intracellular calcium loaded with X-rhod-1 were measured by laser confocal microscopy(LCM).Cell apoptosis was analyzed by flow cytometry with Annexin V/PI labeling.Multiple group comparison was done by a one-way analysis of variance(ANOVA) by use of SPSS10.0.Values of P＜0.05 were considered statistically significant.
Results The recombinanta denovirus Ad-SCN5A(MT/WT) with GFP report gene was successfully constructed in 293cell.PCR test proved SCN5A(MT/WT) contained the insertion of SCN5A(MT/WT). The titre of purified recombinant adenovirus of Ad-GFP-SCN5A-MT,and Ad-GFP-SCN5A-WT was 7×10~(8.4) pfu/ml and 7×10~(7.8)/mi,respectvely.GFP posivitve HEK 293 and cardiomyocytes reached 90%or above.Realtime-PCR indicated significant elevation of PLB expression in SCN5A WT control group compared with other groups(p＜0.05).However,no other significant changes were detected in genes expression in SCN5A MT/WT groups.LCM indicated notable calcium transient difference between SCN5A MT control group and SCN5A WT xamoterol group(p＜0.05).SCN5A MT tended to be higher than its SCN5A WT counterparts in both calcium transient and content.However,no significant differences were observed. Time of intracellular calcium increase in SCN5A MT hypoxia+xamoterol and SCN5A MT control were markedly higher than any other group.However,no significant difference were observed in calcium decay time between its SCN5A WT counterparts except in the two control groups of SCN5A MT and SCN5A WT.Apoptosis were significantly higher in SCN5A MT than that in SCN5A WT counterparts(p＜0.05).
Conclusion SCN5A A1180V mutation could induce intracellular calcium overload and consequent cardiac apoptosis via Na+ current increase by Na+ channel inactivation,which may contribute to DCM pathogenesis.
Ⅱ.Differentiation of Bone Marrow derived Human Mesenchymal Stem Cells into Cardiomyocyte-like Cell Induced by BMP2
Background:Pluripotent MSCs can differentiate into several distinct cell types induced by Bone morphogenetic protein 2(BMP2),including osteoblasts and adipocytes.Two key transcription factors,RUNX2 and peroxisome proliferators-activated receptorγ(PPARγ),drive MSCs to differentiate into either osteoblasts or adipocytes, respectively.Also,MSCs can differentiate into cardiomyocytes induced by 5-azacytidine,and ESC or P19CL6 can differentiate into cardiomyocytes induced by BMP2.BMP2 signal pathway appears to play a crucial role in this decision of cellular fate by dose effect,cross talk or molecular switch. Therefore,its reasonable to postulate that it also exists possibility to lead MSCs differentiating into cardiomyocytes induced by BMP2.
Methods:Chapter 1.MSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time method.Morphology and growth characteristics were examined by phase contrast microscopy. Cell surface markers CD166,CD29,CD34 and CD45 were tested by flow cytometer.To invest the potential of differentiation, MSCs were induced to adipocytes combined with dexamethasone (DM),1-methy-3-isobutylxanthine(IBMX),insulin(IS) and indomethacin(ID) in vitro.Chapter 2.The genes PPARγand RUNX2 were amplified by PCR and cloned into eukaryotic expression vector pEGFP-N1 with EGFP reported gene encoding enhanced green florescence protein,and then identified by PCR and sequeing.The recombinant plasmids pEGFP-N1-PPARγand pEGFP-N1-RUNX2 were transfected into MSCs and HEK293 by the methods mediated by lipofectin and electroporation to investigate the transfection efficiency.To select the better small interfering RNA siRNA segment down-regulating the expression of PPARγand RUNX2,3 pairs of chemically synthetical PPARγsiRNA(P1,P2,P3) and corresponding plasmid pEGFP-N1-PPARγ,RUNX2 siRNA(R1,R2,R3) and corresponding plasmid pEGFP-N1-RUNX2 were co-transfected into HEK293 by use of liposome,respectively.And negative group,positive group and blank were designed as control.The effect of silence was detected by RT-PCR,Real-time PCR,and Western blotting.Chapter 3.To access the delivery ratio of siRNA to MSCs,Fluor Red RNA was ransfected into MSCs with Lipofectamine RNAi MAX.When growing to 70%confluence,MSCs were induced by BMP2 as the following groups(groupl:5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA,group2:BMP2,PPARγsiRNA combined with RUNX2 siRNA,group3:5-aza combined with BMP2,group4:BMP2,group5:5-aza,group6:blank).The dosage of 5-azacytidine(5-aza) and the siRNA was 0.01μM and 10pmol/well(6 well plates),respectively.MSCs were treated by 5-aza for 24 hours in induced dayl,by siRNA in day 2 and day8 for 4 hours.When induecd 12 days,the mRNA of PPARγ,RUNX2, NKX2.5,GATA4,α-MHC and cTnT in hMSCs were tested by RT-PCR and Realtime PCR.Multiple group comparison was done by a one-way analysis of variance(ANOVA) by use of SPSS10.0. Values of P＜0.05 were considered statistically significant.
Results:Chapter 1.After 24 hours in primary culture,the cells adhered to the plastic surface.After 2-3 days,the cells were shuttle-shape.After 15 days,the cells overspread the culture bottle bottom,and kept the same figure characteristics of primary culture.But when the cells grew up to the third passages,shuttle-shape of them changed to plainness and bounty,cytokinesis decreased,and cytoplasm was loosen and there were vacuoles in cytoplasm,and the surface area add up to 5 to 50 fold larger than the normal MSCs.The ageing cells could restore to normal growth status and morphology developed with the special Mesenchymal Stem Cell Medium with Growth Supplement.hMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 (97.69%),CD166(97.54%),and negative for CD34,CD45.After induced with MDI medium,MSCs differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O.Chapter 2.The expression vectors of recombinant plasmid pEGFP-N1-PPARγand pEGFP-N1-RUNX2 were successfully constructed and it can be steadily expressed in HEK293 cells.It provided a platform for the next experiments. plasmid pEGFP-N1-RUNX2 were co-transfected into HEK293 cells.When co-transfected siRNA and recombinant plasmid pEGFP-N1 into HEK293,the expression of mRNA and protein was checked.It resulted that the P2 segment of PPARγsiRNA (5'AGAAUAAUAAGGUGGAGAUGCAGGC3') significantly down-regulated the expression of PPARγgene in mRNA and protein level(p＜0.05). And all 3 RUNX2 siRNA segments(R1:5'UUUAAUAGCGUGCUGCCAUUCG AGG3',R2:5'AACAGAUUCAUCCAUUCUGCCACUA3',R3:5'UCAAGCUUCUGUC UGUGCCUUCUGG3') significantly down-regulated the expression of PPARG gene in mRNA and protein level(p＜0.05).The best concentration of P2 PPARG siRNA was 10pmol/well(6 well plate) (p＜0.05),and the best concentration of R2 RUNX2 siRNA was 10pmol/well or 25pmol/well(6 well plate)(p＜0.05).Chapter 3.Over 60%Fluor Red RNA was transfected into MSCs with Lipofectamine RNAi MAX.It gave a good indication of transfection efficiency and facilitated assessment and optimization of siRNA delivery into MSCs using cationic lipids. The expression of some selective genes was examined when MSCs induced for 12 days.It led that the expression of PPARγgene in group 4(BMP2)increased significantly than the other groups (p＜0.05).Also,the expression of RUNX2 was higher in group 4(BMP2).It was interesting that the expression of cardiac core transfacors Nkx2.5 and GATA4,cardiac structure protein cTnT andαMHC in group 1(5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA) was significantly higher than the other groups including group 5(5-aza) concurrently(p＜0.05),also,the expression of these 4 genes in group 5(5-aza) was significantly higher than the other groups except group 1 (5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA)(p＜0.05). It suggested that PPARγand RUNX2 gene were inhibited.Both 5-aza and BMP2 didn't play a dominant role during the differentiation.However,5-aza combined BMP2 together with siRNA produced synergy action to promote MSCs to differentiate into cardiomyocyte-like cell.
Conclusion:It was feasible that MSCs differentiate into cardiomyocye-like cell induced with BMP2 by down-regulating the the expression of PPARγand RUNX2 gene synchronously.BMP2 signal pathway may act as a molecular rheostat to fine-tune the balance among distinct lineages by dose effect,cross talk or molecular switch.
Ⅲ.CTSS promoter -25G/A:not a risk factor for CHD in Chinese
Background Atherosclerosis(AS) is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Recent data suggested the participation of lysosomal cysteine proteases such as cathepsin S(CTSS) in atherogenesis.The G/A polymorphism at nucleotide -25 was reported to locate in the promoter of the CTSS gene.Because of the importance of CTSS in atherosclerosis and the special location of G/A polymorphism,we supposed that CTSS -25G/A polymorphism maybe have relationship with coronary heat disease.Therefore,the aim of this study is to observe the association between CTSS -25G/A polymorphism and the risk of CHD in Chinese population.
Methods Polymerase chain reaction(PCR) and restriction digestion method were performed to screen the CTSS gene -25G/A polymorphism.Histories of smoking,hypertension and diabetes were investigated and blood lipids were simultaneously measured in patients with coronary heart disease(CHD,coronary artery narrow≥50%,n= 659) and without CHD(control,n = 352).
Results The frequencies of G and A allele in the total population was 0.630 and 0.370 respectively.No significant difference was found in the frequencies of -25G/A polymorphism in CTSS gene(G:0.626 vs.0.633,p＞0.05;A:0.374 vs.0.367, p＞0.05) and genotype distribution(G:83.4 vs.85.3,p＞0.05; A:58.0 vs.58.5,p＞0.05) between CHD patients and control group.Furthermore,CTSS genotype is not associated with severity of coronary stenosis(p＞0.05),even though other risk factors were adjusted by means of logistic regression.
Conclusion The genotype AA was less common than GG and GA in this Chinese population.There was no significant difference in the allele frequencies as well as genotype distribution among the CHD and the Controls.The CTSS -25G/A polymorphism was not related with the vulnerability and the severity of coronary stenosis.
背景:心脏钠通道基因(cardiac sodium channel,SCN5A)突变可导致各类心律失常,现有观点认为SCN5A突变与扩心病有关,但其机理还不清楚并且争议很大。我们曾在一个进行性心脏传导障碍伴扩心病的遗传家系中,发现了一个新的SCN5A基因A1180V突变。本课题即试图在细胞及分子水平上观察该突变位点是否影响钙平衡从而造成心肌损伤。
方法:以PacⅠ线性化重组腺病毒质粒pAdGFP-SCN5A(MT/WT),经线性化后转染到HEK 293细胞中包装成病毒,随后提取DNA进行PCR鉴定和测序鉴定。重组腺病毒Ad-GFP-SCN5A(MT/WT)经HEK293传代、扩增、浓缩与纯化,并测定病毒滴度(TCID_(50)和PFU)。将这些病毒按如下分组接种新生大鼠心肌细胞:对野生型SCN5A和突变型SCN5A分别设置缺氧组、缺氧并加xamoterol处理组、xamoterol组和空白组。接种病毒48小时后,以缺氧和xamoterol处理2小时后,随后进行如下实验:(1)提取RNA,经realtime PCR,观察钠和钙相关基因(L-Ca~(2+)、CaMKⅡ、NaCX、PLB、CaM、SERCA、RyR2)的表达情况;(2)加载钙荧光指示剂X—rhod—1,以激光共聚焦显微镜测定心肌细胞内钙信号的变化情况;(3)用Annexin-V-PE联合碘化丙锭(PI)流式细胞仪检测心肌细胞凋亡率。对数据采用SPSS10.0统计软件进行ANOVA方差分析,p＜0.05为差异显著,p＜0.01为差异极显著。
结果:经PacI酶切线性化的腺病毒质粒pAd-GFP-SCN5A(MT/WT)用脂质体介导转染HEK293细胞后,产生的重组腺病毒对HEK 293细胞有致病作用。用目的基因片段SCN5A引物进行PCR扩增,得到一条约5006p大小片段,测序后经序列分析表明SCN5A未发生新的突变,表明带有目的基因的重组腺病毒Ad-GFP-SCN5A(MT/WT)构建成功。重组腺病毒Ad-GFP-SCN5A(MT/WT)经HEK293传代、扩增、浓缩与纯化,获得滴度分别为PFU 7×10~(8.4)的Ad-GFP-SCN5A(MT)和PFU 7×10~(7.8)的Ad-GFP-SCN5A(WT)。分别接种HEK293和新生鼠心肌细胞,GFP阳性率在90%以上,说明病毒能够感染大部分细胞。从与钠和钙相关基因表达的情况来看,CaM和SERCA其表达量各组之间差异不显著;PLB在SCN5A野生型空白组表达显著高于其它各组(p＜0.05),其它基因其表达规律性不强。SCN5A MT空白组和SCN5A WT Xamoterol处理组之间钙瞬变差异显著(p＜0.05),各组间钙容量差异不显著,钙瞬变和钙容量均在数量上表现出SCN5A MT各组高于SCN5A WT各组的趋势。SCN5A MT缺氧并Xamoterol处理组和SCN5A MT空白组钙升高时间显著高于除这两组之外的其它各组(p＜0.05),除空白组间WT和MT间钙衰减时间差异显著外,其它对应组间差异不显著(p＜0.05),钙升高和钙衰减均在数量上表现出SCN5A MT各组低于SCN5A WT各组的趋势。各SCN5A MT组其细胞凋亡均显著高于相应的SCN5A WT组(p＜0.05)。
结论:钙信号的变化和细胞凋亡的结果一致表现出SCN5A A1180突变可增强心肌细胞内钙信号。这说明SCN5A A1180V突变导致的非失活内向钠电流增加引起了细胞内钙信号加强。这将支持SCN5A A1180V突变可导致心肌细胞内钙稳态失衡从而引起扩心病的假设。
课题Ⅱ:BMP2诱导人MSCs向心肌样细胞分化的可能性及其机制
背景骨形态发生蛋白2(BMP2)可诱导间充质干细胞(MSCs)成骨和脂肪细胞,尚未有经BMP2诱导MSCs向心肌样细胞方向分化的报道。RUNX2和PPARγ分别是向骨和脂肪细胞分化下游通路上两个关键转录因子。BMP2诱导胚胎干细胞ESC和P19CL6可分化成心肌样细胞,MSCs经5-aza诱导也可分化成心肌样细胞,在BMP2通路上,成骨和成脂肪细胞分化是可以切换的,因此,有理由设想也可向心肌细胞方向切换。本课题试图通过下调转录因子RUNX2和PPARγ的表达来观察BMP2诱导下的MSCs可否向心肌样细胞方向分化。
方法第一章:抽取人髂骨骨髓,经Ficoll密度梯度离心分离获得单个核细胞,分别经不同的培养基传代培养。以流式细胞仪鉴定其表面抗原,同时,对这些细胞经向脂肪细胞分化的化学诱导剂诱导。通过上述方法,获得生长性能良好的间充质干细胞株。第二章:以pEGFP-N1构建PPARγ和RUNX2基因的真核表达质粒,将这些基因通过脂质体转染或电转的方式转染到MSCs和HEK293细胞,选择转染率高的方法和高转染的细胞。将经化学合成的PPARγ和RUNX2siRNA与相应的质粒用lipo2000转染到HEK293细胞中,筛选下调基因表达最低的PPARγ和RUNX2 siRNA片段及相应片段的最佳浓度。第三章:以Lipofectamine RNAi MAX将BLOCK-iT~(TM) Alexa Fluor~(?) Red Fluorescent Oligo(Invitrogen USA)转染到MSCs,以评价RNA传送到MSCs的效率。按如下分组诱导MSCs的分化:(1)5-aza、BMP2、PPARγ和RUNX2的siRNA,(2)BMP2、PPARγ和RUNX2的siRNA,(3)5-aza、BMP2,(4)BMP2,(5)5-aza,(6)Control(Blank)。培养基中5-aza的终浓度为0.01uM,BMP2终浓度为100ng/ml,PPARγ和RUNX2的siRNA所加量分别为10pmol/孔(六孔板)。MSCs生长到70%的汇合度时,以5-aza处理细胞24小时,然后换液培养24小时即转染PPARγ和RUNX2的siRNA,6天后再转染一次,BMP2在70%的汇合度时即开始添加,每2天换液一次,12天后收细胞,提取RNA和蛋白,经RT—PCR、realtime PCR和western blotting,检测PPARγ、RUNX2、NKX2.5、GATA4、α-MHC和cTnT基因的表达情况。对数据采用SPSS10.0统计软件进行ANOVA方差分析,p＜0.05为差异显著,p＜0.01为差异极显著。
结果第一章:对抽取的骨髓分离纯化,传代培养,15天即铺满培养瓶底,梭形细胞比例增加,细胞得到纯化,但传至P3,细胞由梭形变为平坦、宽大,细胞立体感逐渐消失,细胞的形状趋向多样,说明细胞很快衰老。换用添加有补充物的MSCs专用培养基培养,MSCs则生长状态良好。样本经流式细胞仪鉴定表面抗原,MSCs高表达CD29(97.69%)和CD166(97.54%),但不表达CD34和CD45。同时,以化学诱导剂(MDI)成功的诱导这些细胞分化成为脂肪细胞。经诱导和鉴定表面抗原,说明所获得的细胞是高纯度的MSCs。第二章:经PCR鉴定和测序鉴定,成功的构建了以pEGFP-N1为载体的PPARγ和RUNX2基因真核表达质粒。将这些质粒与PPARγ和RUNX2 siRNA共转到HEK293,其中PPARγsiRNA的P2片段(5'AGAAUAAUAAGGUGGAGAUGCAGGC3')在mRNA水平和蛋白水平上均显著下调PPARγ的表达(p＜0.05),而RUNX2 siRNA的3片段(R1:5'UUUAAUAGCGUGCUGCCAUUCG AGG3',R2:5'AACAGAUUCAUCCAUUCUGCCACUA3',R3:5'UCAAGCUUCUGUCUGUGCCUUCUGG3')均显著下调RUNX2的表达(p＜0.05),同时,筛选得P2片段的最佳下调浓度为10pmol/孔(六孔板)(p＜0.05),R2片段两个最佳下调浓度为10pmol╱孔和25pmol/孔(p＜0.05)。第三章:将Fluor Red RNA转染到MSCs,发现60%以上的RNA传送到了MSCs,说明有较高的传送效率经BMP2诱导,发现第4组(BMP2)PPARγ的表达显著高于其它各组,RUNX2的表达也在第4组最高,但无统计学上的差异。转录因子Nkx2.5和GATA4和心肌特异性基因TnT和MHC的表达在第1组(5-aza、siRNA和BMP2)一致性的显著升高(p＜0.05),并且显著高于第5组(5-aza),而第5组这些基因的表达也显著高于除第1组之外的其它各组(p＜0.05)。说明在诱导过程中,PPARγ和RUNX2被相应siRNA抑制,单因素5-aza和BMP2均不占主导作用,BMP2与5-aza产生了协同效应,促进了MSCs向心肌样细胞分化。
结论抑制转录因子RUNX2和PPARγ的表达使得BMP2诱导MSCs向心肌样细胞分化是可行的,其机制可能在于BMP2下游信号通路间的串联和切换。
课题Ⅲ:CTSS基因启动子区-25G/A多态对中国人群冠心病的预测
背景:动脉粥样硬化是一种以动脉管壁细胞外基质结构广泛重构为特征的炎症性疾病。最近许多研究显示诸如组织蛋白酶S(CTSS)等的溶酶体半胱氨酸蛋白酶参与动脉粥样硬化形成过程。在CTSS基因的启动子区域核苷酸-25处存在G/A基因多态现象。基于CTSS在冠状动脉粥样硬化中发挥重要作用,而-25G/A多态位于CTSS基因启动子区域,因此,我们推测CTSS -25G/A多态性可能与冠心病之间存在密切联系。本项研究的目的即为观察中国人群中,CTSS-25G/A多态分布及其多态性与冠心病(CAD)危险性之间的关联。
方法:CTSS基因-25G/A多态性由多聚酶链式反应PCR及限制性内切酶降解方法获得。经冠脉造影检查明确的共659名冠心病患者(冠脉狭窄≥50%)及352名非冠心病患者(对照组)纳入本项研究。我们同时收集患者的吸烟史、高血压史及糖尿病史,并测量他们的血脂水平。
结果:所有研究对象中的等位基因G和A的频率分别为0.630及0.370。在病例组及对照组之间未发现其CTSS -25G/A多态性频率(G:0.626 vs.0.633,p＞0.05;A:0.374 vs.0.367,p＞0.05)及基因型分布(G:83.4 vs.85.3,p＞0.05;A:58.0vs.58.5,p＞0.05)存在显著性差异。此外,即使在使用逻辑回归对其他危险因素校正后,CTSS基因型与冠脉狭窄严重程度之间亦未发现存在关联(p＞0.05)。
结论:在中国人群中,基因型AA较基因型GG和GA更为少见。对照组及冠心病组之间的等位基因频率及基因型分布均无显著性差异。CTSS -25G/A多态性与冠状动脉狭窄的发生率及严重程度不相关。
Ⅰ.Novel SCN5A Mutant-Induced Intracellular Calcium Shift in Dilated Cardiomyopathy Pathogenesis
Background Mutations of SCN5A,a cardiac sodium channel gene, have been implicated in the pathogenesis of dilated cardiomyopathy(DCM).Our previous study indicated a role of a novel mutant,SCN5A A1180V,in the onset of DCM in a Chinese family.However,the underlying mechanisms remain unknown. The present study was to investigate the biological significance of SCN5A A1180V in intracellular calcium shift and consequent myocardial damage.
Materials and Methods Recombinant adenoviral vector pAdGFP-SCN5A(MT/WT) linearized by PacⅠenzyme was amplified and confirmed by PCR and DNA sequencing after transfection into HEK 293 by lipofectamine 2000.Amplification was verified by titering analysis.Cardiomyocytes of neonatal rats transfected with Ad-GFP-SCN5A(MT/WT) were then treated with hypoxia,xamoterol,hypoxia+xamoterol respectively.Total RNA were extracted after 2h treatment.Both sodium and calcium-associated genes(L-Ca~(2+),CaMKⅡ,NaCX,PLB,CaM, SERCA and RyR2) were detcted by realtime-PCR.Changes of intracellular calcium loaded with X-rhod-1 were measured by laser confocal microscopy(LCM).Cell apoptosis was analyzed by flow cytometry with Annexin V/PI labeling.Multiple group comparison was done by a one-way analysis of variance(ANOVA) by use of SPSS10.0.Values of P＜0.05 were considered statistically significant.
Results The recombinanta denovirus Ad-SCN5A(MT/WT) with GFP report gene was successfully constructed in 293cell.PCR test proved SCN5A(MT/WT) contained the insertion of SCN5A(MT/WT). The titre of purified recombinant adenovirus of Ad-GFP-SCN5A-MT,and Ad-GFP-SCN5A-WT was 7×10~(8.4) pfu/ml and 7×10~(7.8)/mi,respectvely.GFP posivitve HEK 293 and cardiomyocytes reached 90%or above.Realtime-PCR indicated significant elevation of PLB expression in SCN5A WT control group compared with other groups(p＜0.05).However,no other significant changes were detected in genes expression in SCN5A MT/WT groups.LCM indicated notable calcium transient difference between SCN5A MT control group and SCN5A WT xamoterol group(p＜0.05).SCN5A MT tended to be higher than its SCN5A WT counterparts in both calcium transient and content.However,no significant differences were observed. Time of intracellular calcium increase in SCN5A MT hypoxia+xamoterol and SCN5A MT control were markedly higher than any other group.However,no significant difference were observed in calcium decay time between its SCN5A WT counterparts except in the two control groups of SCN5A MT and SCN5A WT.Apoptosis were significantly higher in SCN5A MT than that in SCN5A WT counterparts(p＜0.05).
Conclusion SCN5A A1180V mutation could induce intracellular calcium overload and consequent cardiac apoptosis via Na+ current increase by Na+ channel inactivation,which may contribute to DCM pathogenesis.
Ⅱ.Differentiation of Bone Marrow derived Human Mesenchymal Stem Cells into Cardiomyocyte-like Cell Induced by BMP2
Background:Pluripotent MSCs can differentiate into several distinct cell types induced by Bone morphogenetic protein 2(BMP2),including osteoblasts and adipocytes.Two key transcription factors,RUNX2 and peroxisome proliferators-activated receptorγ(PPARγ),drive MSCs to differentiate into either osteoblasts or adipocytes, respectively.Also,MSCs can differentiate into cardiomyocytes induced by 5-azacytidine,and ESC or P19CL6 can differentiate into cardiomyocytes induced by BMP2.BMP2 signal pathway appears to play a crucial role in this decision of cellular fate by dose effect,cross talk or molecular switch. Therefore,its reasonable to postulate that it also exists possibility to lead MSCs differentiating into cardiomyocytes induced by BMP2.
Methods:Chapter 1.MSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time method.Morphology and growth characteristics were examined by phase contrast microscopy. Cell surface markers CD166,CD29,CD34 and CD45 were tested by flow cytometer.To invest the potential of differentiation, MSCs were induced to adipocytes combined with dexamethasone (DM),1-methy-3-isobutylxanthine(IBMX),insulin(IS) and indomethacin(ID) in vitro.Chapter 2.The genes PPARγand RUNX2 were amplified by PCR and cloned into eukaryotic expression vector pEGFP-N1 with EGFP reported gene encoding enhanced green florescence protein,and then identified by PCR and sequeing.The recombinant plasmids pEGFP-N1-PPARγand pEGFP-N1-RUNX2 were transfected into MSCs and HEK293 by the methods mediated by lipofectin and electroporation to investigate the transfection efficiency.To select the better small interfering RNA siRNA segment down-regulating the expression of PPARγand RUNX2,3 pairs of chemically synthetical PPARγsiRNA(P1,P2,P3) and corresponding plasmid pEGFP-N1-PPARγ,RUNX2 siRNA(R1,R2,R3) and corresponding plasmid pEGFP-N1-RUNX2 were co-transfected into HEK293 by use of liposome,respectively.And negative group,positive group and blank were designed as control.The effect of silence was detected by RT-PCR,Real-time PCR,and Western blotting.Chapter 3.To access the delivery ratio of siRNA to MSCs,Fluor Red RNA was ransfected into MSCs with Lipofectamine RNAi MAX.When growing to 70%confluence,MSCs were induced by BMP2 as the following groups(groupl:5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA,group2:BMP2,PPARγsiRNA combined with RUNX2 siRNA,group3:5-aza combined with BMP2,group4:BMP2,group5:5-aza,group6:blank).The dosage of 5-azacytidine(5-aza) and the siRNA was 0.01μM and 10pmol/well(6 well plates),respectively.MSCs were treated by 5-aza for 24 hours in induced dayl,by siRNA in day 2 and day8 for 4 hours.When induecd 12 days,the mRNA of PPARγ,RUNX2, NKX2.5,GATA4,α-MHC and cTnT in hMSCs were tested by RT-PCR and Realtime PCR.Multiple group comparison was done by a one-way analysis of variance(ANOVA) by use of SPSS10.0. Values of P＜0.05 were considered statistically significant.
Results:Chapter 1.After 24 hours in primary culture,the cells adhered to the plastic surface.After 2-3 days,the cells were shuttle-shape.After 15 days,the cells overspread the culture bottle bottom,and kept the same figure characteristics of primary culture.But when the cells grew up to the third passages,shuttle-shape of them changed to plainness and bounty,cytokinesis decreased,and cytoplasm was loosen and there were vacuoles in cytoplasm,and the surface area add up to 5 to 50 fold larger than the normal MSCs.The ageing cells could restore to normal growth status and morphology developed with the special Mesenchymal Stem Cell Medium with Growth Supplement.hMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 (97.69%),CD166(97.54%),and negative for CD34,CD45.After induced with MDI medium,MSCs differentiated into adipocytes which displayed a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O.Chapter 2.The expression vectors of recombinant plasmid pEGFP-N1-PPARγand pEGFP-N1-RUNX2 were successfully constructed and it can be steadily expressed in HEK293 cells.It provided a platform for the next experiments. plasmid pEGFP-N1-RUNX2 were co-transfected into HEK293 cells.When co-transfected siRNA and recombinant plasmid pEGFP-N1 into HEK293,the expression of mRNA and protein was checked.It resulted that the P2 segment of PPARγsiRNA (5'AGAAUAAUAAGGUGGAGAUGCAGGC3') significantly down-regulated the expression of PPARγgene in mRNA and protein level(p＜0.05). And all 3 RUNX2 siRNA segments(R1:5'UUUAAUAGCGUGCUGCCAUUCG AGG3',R2:5'AACAGAUUCAUCCAUUCUGCCACUA3',R3:5'UCAAGCUUCUGUC UGUGCCUUCUGG3') significantly down-regulated the expression of PPARG gene in mRNA and protein level(p＜0.05).The best concentration of P2 PPARG siRNA was 10pmol/well(6 well plate) (p＜0.05),and the best concentration of R2 RUNX2 siRNA was 10pmol/well or 25pmol/well(6 well plate)(p＜0.05).Chapter 3.Over 60%Fluor Red RNA was transfected into MSCs with Lipofectamine RNAi MAX.It gave a good indication of transfection efficiency and facilitated assessment and optimization of siRNA delivery into MSCs using cationic lipids. The expression of some selective genes was examined when MSCs induced for 12 days.It led that the expression of PPARγgene in group 4(BMP2)increased significantly than the other groups (p＜0.05).Also,the expression of RUNX2 was higher in group 4(BMP2).It was interesting that the expression of cardiac core transfacors Nkx2.5 and GATA4,cardiac structure protein cTnT andαMHC in group 1(5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA) was significantly higher than the other groups including group 5(5-aza) concurrently(p＜0.05),also,the expression of these 4 genes in group 5(5-aza) was significantly higher than the other groups except group 1 (5-aza,BMP2,PPARγsiRNA combined with RUNX2 siRNA)(p＜0.05). It suggested that PPARγand RUNX2 gene were inhibited.Both 5-aza and BMP2 didn't play a dominant role during the differentiation.However,5-aza combined BMP2 together with siRNA produced synergy action to promote MSCs to differentiate into cardiomyocyte-like cell.
Conclusion:It was feasible that MSCs differentiate into cardiomyocye-like cell induced with BMP2 by down-regulating the the expression of PPARγand RUNX2 gene synchronously.BMP2 signal pathway may act as a molecular rheostat to fine-tune the balance among distinct lineages by dose effect,cross talk or molecular switch.
Ⅲ.CTSS promoter -25G/A:not a risk factor for CHD in Chinese
Background Atherosclerosis(AS) is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Recent data suggested the participation of lysosomal cysteine proteases such as cathepsin S(CTSS) in atherogenesis.The G/A polymorphism at nucleotide -25 was reported to locate in the promoter of the CTSS gene.Because of the importance of CTSS in atherosclerosis and the special location of G/A polymorphism,we supposed that CTSS -25G/A polymorphism maybe have relationship with coronary heat disease.Therefore,the aim of this study is to observe the association between CTSS -25G/A polymorphism and the risk of CHD in Chinese population.
Methods Polymerase chain reaction(PCR) and restriction digestion method were performed to screen the CTSS gene -25G/A polymorphism.Histories of smoking,hypertension and diabetes were investigated and blood lipids were simultaneously measured in patients with coronary heart disease(CHD,coronary artery narrow≥50%,n= 659) and without CHD(control,n = 352).
Results The frequencies of G and A allele in the total population was 0.630 and 0.370 respectively.No significant difference was found in the frequencies of -25G/A polymorphism in CTSS gene(G:0.626 vs.0.633,p＞0.05;A:0.374 vs.0.367, p＞0.05) and genotype distribution(G:83.4 vs.85.3,p＞0.05; A:58.0 vs.58.5,p＞0.05) between CHD patients and control group.Furthermore,CTSS genotype is not associated with severity of coronary stenosis(p＞0.05),even though other risk factors were adjusted by means of logistic regression.
Conclusion The genotype AA was less common than GG and GA in this Chinese population.There was no significant difference in the allele frequencies as well as genotype distribution among the CHD and the Controls.The CTSS -25G/A polymorphism was not related with the vulnerability and the severity of coronary stenosis.
引文
1.Burkett,E.L.& Hershberger,R.E.Clinical and genetic issues in familial dilated cardiomyopathy.J Am Coll Cardiol 45,969-81(2005).
2.Smits,J.P.et al.Genotype-phenotype relationship in Brugada syndrome:electrocardiographic features differentiate SCN5A-related patients from non-SCN5A-related patients.J Am Coll Cardiol 40,350-6(2002).
3.Tan,H.L.et al.A sodium-channel mutation causes isolated cardiac conduction disease.Nature 409,1043-7(2001).
4.McNair,W.P.et al.SCN5A mutation associated with dilated cardiomyopathy,conduction disorder,and arrhythmia.Circulation 110,2163-2167(2004).
5.Nguyen,T.P.,Wang,D.W.,Rhodes,T.H.& George,A.L.Divergent biophysical defects caused by mutant sodium channels in dilated cardiomyopathy with arrhythmia.Circulation Research 102,364-371(2008).
6.Groenewegen,W.A.& Wilde,A.A.M.Letter regarding article by McNair et al,"SCNSA mutation associated with dilated cardiomyopathy,conduction disorder,and arrhythmia".Circulation 112,E9-E9(2005).
7.Olson,T.M.et al.Sodium channel mutations and susceptibility to heart failure and atrial fibrillation.Jama-Journal of the American Medical Association 293,447-454(2005).
8.Ge,J.et al.Identification and characterization of a novel SCN5A mutation associated with progressive familial atrioventricular block and dilated cardiomyopathy.Circulation 112,U206-U207(2005).
9.Waddington,S.N.et al.Adenovirus serotype 5 hexon mediates liver gene transfer.Cell 132,397-409(2008).
10.Kaelin,W.G.et al.Expression Cloning of a Cdna-Encoding a Retinoblastoma-Binding Protein with E2f-Like Properties. Cell 70, 351-364 (1992).
11. Zou, L., Cortez, D. & Elledge, S.J. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes & Development 16, 198-208 (2002).
12. Laitinen-Forsblom, P.J. et al. SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias. Journal of Cardiovascular Electrophysiology 17,480-485 (2006).
13. Frustaci, A. et al. Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome. Circulation 112, 3680-3687 (2005).
14. Wilde, A.A.M., Remme, C.A., Derksen, R., Wever, E.F.D. & Hauer, R.N.W. Brugada syndrome. European Heart Journal 23, 675-676 (2002).
15. Roberts, R. & Brugada, R. Genetic aspects of arrhythmias. American Journal of Medical Genetics 97, 310-318 (2000).
16. Puthalakath, H. et al. ER stress triggers apoptosis by activating BH3-only protein Bim. Cell 129,1337-1349(2007).
17. Clapham, D.E. Calcium Signaling. Cell 80, 259-268 (1995).
18. Catterall, W.A. Excitation Contraction Coupling in Vertebrate Ske letal-Muscle - a Tale of 2 Calcium Channels. Cell 64, 871-874 (1991).
19. Fruen, B.R. et al. Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin. Biochemistry 42, 2740-2747 (2003).
20. Fruen, B.R., Norton, L.E., Bloomquist, R.A., Thomas, D.D. & Balog, E.M. Role of CaM Met residues in mediating productive associations with the cardiac ryanodine receptor (RYR2). Biophysical Journal 84, 64A-64A (2003).
21. Ozawa, T. Ryanodine-sensitive Ca2+ release mechanism in non-excitable cells (Review). International Journal of Molecular Medicine 7, 21-25 (2001).
22. Brandl, C.J., Green, N.M., Korczak, B. & Maclennan, D.H. 2 Ca-2+ Atpase Genes -Homologies and Mechanistic Implications of Deduced Amino-Acid-Sequences. Cell 44, 597-607(1986).
23. Kemi, O.J. et al. Aerobic interval training enhances cardiomyocyte contractility and Ca2+ cycling by phosphorylation of CaMKII and Thr-17 of phospholamban. Journal of Molecular and Cellular Cardiology 43, 354-361 (2007).
24. Sag, C.M., DeSantiago, J., Bers, D.M. & Maier, L.S. Recovery of Ca transients, SR Ca reuptake and shortening during acidosis requires the CaMKII/PLB signaling pathway but is impaired in heart failure. European Heart Journal 25, 179-179 (2004).
25. Vasanji, Z., Dhalla, N.S. & Netticadan, T. Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart. Molecular and Cellular Biochemistry 261, 245-249 (2004).
26. Yang, D.M. et al. Ca2+/calmodulin kinase II-dependent phosphorylation of ryanodine receptors stabilizes Ca2+ signaling in intact cardiac myocytes. Circulation 114, 57-57 (2006).
27. Netticadan, T., Temsah, R.M., Kawabata, K. & Dhalla, N.S. Sarcoplasmic reticulum Ca2+/calmodulin-dependent protein kinase is altered in heart failure. Circulation Research 86, 596-605 (2000).
28. Netticadan, T., Temsah, R., Osada, M. & Dhalla, N.S. Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion. American Journal of Physiology-Cell Physiology 277, C384-C391 (1999).
29. Yamaguchi, N., Xu, L., Pasek, D.A., Evans, K..E. & Meissner, G. Molecular basis of calmodulin binding to cardiac muscle Ca2+ release channel (ryanodine receptor). Journal of Biological Chemistry 278,23480-23486(2003).
30.Fruen,B.R.et al.Regulation of RYR1 and RYR2 by CaM mutants deficient in Ca2+ binding.Biophysical Journal 82,291(2002).
31.Balshaw,D.M.,Xu,L.,Yamaguchi,N.,Pasek,D.A.& Meissner,G.Calmodulin binding and inhibition of cardiac muscle calcium release channel(ryanodine receptor).Journal of Biological Chemistry 276,20144-20153(2001).
32.Ashley,C.C.& Ridgway,E.B.Simultaneous Recording of Membrane Potential Calcium Transient and Tension in Single Muscle Fibres.Nature 219,1168-&(1968).
33.Davidson,C.J.,Baum,K.R.,Spitzer,K.W.& Bridge,J.H.B.Exercise reduces the maximal rate of rise of the calcium transient in rat cardiomyocytes.Faseb Journal 20,A1448-A1448(2006).
34.Deng,C.Y.et al.Effect of valsartan on cardiacmyocytes contraction function and calcium transient in heart failure rats.Acta Pharmacologica Sinica 27,189-189(2006).
35.Janssen,P.M.L.,Zeitz,O.,Schumann,H.,Holtz,J.& Hasenfuss,G.Load-Induced Cardiomyocyte Apoptosis in Cultured Multicellular Myocardial Preparations Is Unaltered in Presence of the beta-Adrenoceptor Antagonist Nebivolol.Pharmacology 83,141-147(2009).
36.Xie,P.et al.Atrogin-1/MAFbx Enhances Simulated Ischemia/Reperfusion-induced Apoptosis in Cardiomyocytes through Degradation of MAPK Phosphatase-1 and Sustained JNK Activation.Journal of Biological Chemistry 284,doi:10.1074/jbc.M806487200(2009).
37.Tan,W.Q.et al.Novel Cardiac Apoptotic Pathway The Dephosphorylation of Apoptosis Repressor With Caspase Recruitment Domain by Calcineurin.Circulation 118,2268-2276(2008).
1.Assmann,G;Schulte,H;Cullen,P.New and classical risk factors--the Munster heart study(PROCAM).Eur J Med Res 1997;6:237-42.
2.Kannel WB.The Framingham Study:its 50-year old legacy and future promise.J Atheroscler Thromb 2000;6:60-6.
3.Funke H,Assmann G.Strategies for the assessment of genetic coronary artery disease risk.Curr Opin Lipidol 1999;10:285-91.
4.Gardemann A,Stricker J,Humme Jet al.Angiotensinogen TI74M and M235T gene polymorphisms are associated with the extent of coronary atherosclerosis.Atherosclerosis 1999;145:309-14
5.Lahoz C,Schaefer EJ,Cupples LA et al.Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study.Atherosclerosis 2001;154:529-37.
6.Wilson PWF,Schaefer EJ,Larson MG,Ordovas JM.Apolipoprotein E alleles and risk of coronary disease.A meta-analysis.Arterioscler Thromb Vasc Biol 1996;16:1250-5.
7.Green FR.Fibrinogen polymorphisms and atherothrombotic disease.Ann NY Acad Sci 2001;936:549-59.
8.Folsom AR,Aleksic N,Ahn C et al.Beta-fibrinogen gene-455G/A polymorphism and coronary heart disease incidence.The Atherosclerosis Risk Commtmities (ARIC) Study.Ann Epidemiol 2001;11:166-70.
9.Beyzade S,Zhang S,Wong YK,Day IN,Eriksson P,Ye S Influences of matrix metalloproteinase-3 gene variation on the extent of coronary atherosclerosis and risk of myocardial infarction.J Am Coil Cardiol.2003 Jun 18;41(12):2130-7.
10.Jormsjo S,Wuttge DM,Sirsjo A,Whatling C,Hamsten A,et al.Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice.Am J Pathol 2002;161:939-45.
11.Sukhova GK,Shi GP,Simon DI,Chapman HA,Libby P.Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
12. Sukhova GK, Zhang Y, Pan JH, Wada Y, Yamamoto T, et al. Deficiency of CTSS reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest 2003; 111: 897-906.
13. Pawlak K, Pawlak D, Mysliwiec M. Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis in haemodialysis patients. Thrombosis Research 2007;1: 107.
14. Shi GP, Webb AC, Foster KE, Knoll JH, Lemere CA, et al. Human CTSS: chromosomal localization, gene structure and tissue distribution. J Biol Chem 1994; 269:11530-11536.
15. Cao HN, Robert A, Hegele. Human CTSS gene (CTSS) promoter -25G/A polymorphism. J Hum Genet 2000; 45:94-95.
16. Tang ZH, Russell PT. Candidate genes and confirmed genetic polymorphisms associated with cardiovascular diseases: a tabular assessment. Journal of Thrombosis and Thrombolysis 2001; 11:49-81.
17. Scheer WD, Boudreau DA, Hixson JE, McGill HC, Newman III WP, et al. ACE insert/delete polymorphism and atherosclerosis. Atherosclerosis 2005; 178(2):241-7.
18. Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in Human atheroma and regulation of their procuction in smooth muscle cell. J Clin Invest 1998; 102(3):576-583.
19. Hsing, Lianne C.; Rudensky AY. The lysosomal cysteine proteases in MHC class II antigen presentation. Immunol Rev 2005; 207(1):229-241.
20. Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
21. Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol. 1997;59:63-88.
22. Li Z, Yasuda Y, Li W, Bogyo M, Katz N, et al. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J Biol Chem. 2004;279:5470-5479.
23. Li YH, Chen CH, Yeh PS, Lin HJ, Chang BI, et al. Functional mutation in the promoter region of thrombomodulin gene in relation to carotid atherosclerosis. Atherosclerosis 2001; 154: 713-9.
24. Liu J, Ma LK, Yang JT, Ren A, Sun ZM, et al. Increased serum CTSS in patients with atherosclerosis and diabetes. Atherosclerosis 2006; 186(2):411-9.
25. Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6- 174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001; 22: 2243-52.
1.Guo, S. & Kemphues, K.J. Par-1, a Gene Required for Establishing Polarity in C-Elegans Embryos, Encodes a Putative Ser/Thr Kinase That Is Asymmetrically Distributed. Cell 81,611-620 (1995).
2.Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 (1998).
3.Lin, R.L. & Avery, L. RNA interference - Policing rogue genes. Nature 402, 128-129(1999).
4.Sharp, P.A. & Zamore, P.D. Molecular biology - RNA interference. Science 287, 2431-+(2000).
5.Hannon, GJ. RNA interference. Nature 418,244-251 (2002).
6.Eccleston, A. & Eggleston, A.K. RNA interference. Nature 431, 337-337 (2004).
7.Mello, C.C. & Conte, D. Revealing the world of RNA interference. Nature 431, 338-342 (2004).
8.Check, E. RNA interference - Hitting the on switch. Nature 448, 855-858 (2007).
9.Sharp, P. A. The Centrality of RNA. Cell 136, 577-580 (2009).
10.Siomi, H. & Siomi, M.C. On the road to reading the RNA-interference code. Nature 457, 396-404 (2009).
11.Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101,25-33(2000).
12.Bernstein, E., Caudy, A.A., Hammond, S.M. & Harmon, GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366 (2001).
13.Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411,494-498 (2001).
14.Hutvagner, G.et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834-838 (2001).
15.Knight, S.W. & Bass, B.L. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 2269-2271 (2001).
16.Motamedi, M.R. et al. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789-802 (2004).
17.Filipowicz, W. RNAi: The nuts and bolts of the RISC machine. Cell 122, 17-20 (2005).
18.Ameres, S.L., Martinez, J. & Schroeder, R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130,101-112 (2007).
19.Bass, B.L. Double-stranded RNA as a template for gene silencing. Cell 101, 235-238 (2000).
20.Brown, D.R., McClowry, T.L., Sidner, R.A., Fife, K.H. & Bryan, J.T. Expression of the human papillomavirus type 11 E5A protein from the El boolean AND E4,E5 transcript. Intervirology 41,47-54 (1998).
21.Samarkina, O.N. et al. Universal and rapid method for purification of GFP-like proteins by the ethanol extraction. Protein Expression and Purification 65, 108-113 (2009).
22.Dross, N. et al. Mapping eGFP oligomer mobility in living cell nuclei. PLoS ONE 4, e5041 (2009).
23.Geusens, B. et al. Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes. Journal of Controlled Release 133,214-220 (2009).
24.Marchini, C. et al. Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes. Applied Physics Letters 94(2009).
25.Pappalardo, J.S. et al. Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes. J Control Release 134,41-6 (2009).
26.Martin, J.B., Young, J.L., Benoit, J.N. & Dean, D.A. Gene transfer to intact mesenteric arteries by electroporation. Journal of Vascular Research 37, 372-380 (2000).
1.Attisano,L.& Wrana,J.L.Signal transduction by the TGF-beta superfamily.Science 296,1646-1647(2002).
2.Choy,L.,Skillington,J.& Derynck,R.Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation.Journal of Cell Biology 149,667-681(2000).
3.Flores,M.V.C.,Lam,E.Y.N.,Crosier,K.E.& Crosier,P.S.Osteogenic transcription factor Runx2 is a maternal determinant of dorsoventral patterning in zebrafish.Nature Cell Biology 10,346-U80(2008).
4.Koyanagi,M.et al.Notch signaling contributes to the expression of cardiac markers in human circulating progenitor cells.Circulation Research 101,1139-1145(2007).
5.Prall,O.W.J.et al.An Nkx2-5/Bmp2/Smad1 negative feedback loop orchestrates cardiac progenitor cell proliferation and deployment,and is a molecular target in congenital heart disease.Differentiation 74,O14(2006).
6.Dell'Era,P.et al.Fibroblast growth factor receptor-1 is essential for in vitro cardiomyocyte development.Circulation Research 93,414-420(2003).
7.Yin,Z.Z.& Frasch,M.Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila.Developmental Genetics 22,187-200(1998).
8.Rivera-Feliciano,J.& Tabin,C.J.Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field.Developmental Biology 295,580-588(2006).
9.Schlange,T.,Andree,B.,Arnold,H.H.& Brand,T.BMP2 is required for early heart development during a distinct time period.Mechanisms of Development 91,259-270(2000).
10.Ying,Q.L.,Nichols,J.,Chambers,I.& Smith,A.BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell 115,281-292(2003).
11.Jin,W.Z.et al.Schnurri-2 controls BMP-dependent adipogenesis via interaction with Smad proteins.Developmental Cell 10,461-471(2006).
12.Wrana,J.L.Regulation of Smad activity.Cell 100,189-192(2000).
13.Ellies,D.L.& Krumlauf,R.Bone formation:The nuclear matrix reloaded.Cell 125,840-842(2006).
14.Pallante,B.A.et al.Bone marrow Oct3/4(+) cells differentiate into cardiac myocytes via age-dependent paraerine mechanisms.Circulation Research 100,E1-E11(2007).
15.Yamagishi,T.,Nakajima,Y.,Miyazono,K.& Nakamura,H.Bone morphogenetie protein-2acts synergistically with transforming growth factor-beta 3 during endothelial-mesenchymal transformation in the developing chick heart.Journal of Cellular Physiology 180,35-45 (1999).
16. Lian, J.B. & Stein, G.S. Runx2/Cbfa1: A multifunctional regulator of bone formation. Current Pharmaceutical Design 9,2677-2685 (2003).
17. Wu, C.J. & Lu, H.K. Smad signal pathway in BMP-2-induced osteogenesis - a mini review. Journal of Dental Sciences 3, 13-21 (2008).
18. Wu, M. et al. Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo. Bone 44, 528-536 (2009).
19. Yoshida, Y. et al. Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell 103, 1085-1097 (2000).
20. Marie, P.J. Transcription factors controlling osteoblastogenesis. Archives of Biochemistry and Biophysics 473, 98-105 (2008).
21. Hoffmann, A. et al. The T-box transcription factor Brachyury mediates cartilage development in mesenchymal stem cell line C3H10T1/2. Journal of Cell Science 115, 769-781 (2002).
22. Lin, G.et al. Defining stem and progenitor cells within adipose tissue. Stem Cells Dev 17, 1053-63 (2008).
23. Szabo, E., Qiu, Y.Y., Baksh, S., Michalak, M. & Opas, M. Calreticulin inhibits commitment to adipocyte differentiation. Journal of Cell Biology 182, 103-116 (2008).
24. Yamada, M., Revelli, J.P., Eichele, G., Barron, M. & Schwartz, R.J. Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: Evidence for BMP2 induction of Tbx2. Developmental Biology 228, 95-105 (2000).
25. Monzen, K. et al. Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAKl and cardiac transcription factors Csx/Nkx-2.5 and GATA-4. Molecular and Cellular Biology 19, 7096-7105 (1999).
26. Pal, R. & Khanna, A. Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCell((R))hES1, under low serum concentration supplemented with bone morphogenetic protein-2. Differentiation 75, 112-122 (2007).
27. Lowell, B.B. PPAR gamma: An essential regulator of adipogenesis and modulator of fat cell function. Cell 99,239-242 (1999).
28. Gunther, T. & Schule, R. Fat or bone? A non-canonical decision. Nature Cell Biology 9, 1229-1231(2007).
29. Hong, J.H. et al. TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309, 1074-1078 (2005).
30. Saltiel, A.R. Muscle or fat? Rho bridges the GAP. Cell 113, 144-145 (2003).
31. Wotton, D., Lo, R.S., Lee, S. & Massague, J. A Smad transcriptional corepressor. Cell 97, 29-39(1999).
32. Yuasa, S. et al. Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells. Nature Biotechnology 23, 607-611 (2005).
33. Tomita, Y. et al. Application of mesenchymal stem cell-derived cardiomyocytes as bio-pacemakers: current status and problems to be solved. Medical & Biological Engineering & Computing 45, 209-220 (2007).
34. Hakuno, D. et al. Bone marrow-derived regenerated cardiomyocytes (CMG cells) express functional adrenergic and muscarinic receptors. Circulation 105, 380-386 (2002).
35. Makino, S. et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. Journal of Clinical Investigation 103, 697-705 (1999).
36.Zhang,X.et al.Runx2 overexpression enhances osteoblastic differentiation and mineralization in adipose - derived stem cells in vitro and in vivo.Calcified Tissue International 79,169-178(2006).
37.Tontonoz,P.,Hu,E.& Spiegelman,B.M.Stimulation of adipogenesis in fibroblasts by PPAR-gamma-2,a lipid-activated transcription factor.Cell 79,1147-1156(1994).
38.Plutzky,J.PPARs as therapeutic targets:Reverse cardiology? Science 302,406-407(2003).
39.Lehrke,M.& Lazar,M.A.The many faces of PPAR gamma.Cell 123,993-999(2005).
40.Sukhikh,G.T.,Malaitsev,V.V.,Bogdanova,I.M.& Dubrovina,I.V.Mesenchymal stem cells.Bulletin of Experimental Biology and Medicine 133,103-109(2002).
41.Pittenger,M.E et al.Multilineage potential of adult human mesenchymal stem cells.Science 284,143-147(1999).
42.Payushina,O.V.,Domaratskaya,E.L.& Starostin,V.I.Mesenchymal stem cells:Sources,phenotype,and differentiation potential.Biology Bulletin 33,2-18(2006).
43.Musina,R.A.,Bekchanova,E.S.& Sukhikh,G.T.Comparison of mesenchymal stem cells obtained from different human tissues.Bulletin of Experimental Biology and Medicine 139,504-509(2005).
44.McCarty,R.C.,Gronthos,S.,Zannettino,A.C.,Foster,B.K.& Xian,C.J.Characterisation and Developmental Potential of Ovine Bone Marrow Derived Mesenchymal Stem Cells.Journal of Cellular Physiology 219,324-333(2009).
45.Issemann,I.& Green,S.Activation of a Member of the Steroid-Hormone Receptor Superfamily by Peroxisome Proliferators.Nature 347,645-650(1990).
46.Pashmforoush,M.et al.Nkx2-5 pathways and congenital heart disease:Loss of ventricular myocyte lineage progressive cardiomyopathy and complete heart block.Cell 117,373-386(2004).
47.Morrisey,E.E.et al.GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo.Genes & Development 12,3579-3590(1998).
48.Molkentin,J.D.,Lin,Q.,Duncan,S.A.& Olson,E.N.Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.Genes & Development 11,1061-1072(1997).
49.Liberatore,C.M.,Searcy-Schrick,R.D.,Vincent,E.B.& Yutzey,K.E.Nkx-2.5 gene induction in mice is mediated by a smad consensus regulatory region.Developmental Biology 244,243-256(2002).
50.Moskowitz,I.P.G.et al.A molecular pathway including Id2,TbxS,and Nkx2-5 required for cardiac conduction system development.Cell 129,1365-1376(2007).
51.Shin,C.H.et al.Modulation of cardiac growth and development by HOP,an unusual homeodomain protein.Cell 110,725-735(2002).
52.Ericson,J.et al.Pax6 controls progenitor cell identity and neuronal fate in response to graded shh signaling.Cell 90,169-180(1997).
53.Lyons,I.et al.Myogenic and Morphogenetic Defects in the Heart Tubes of Murine Embryos Lacking the Homeo Box Gene Nkx2-5.Genes & Development 9,1654-1666(1995).
54.Kasahara,H.et al.Loss of function and inhibitory effects of human CSX/NKX2.5homeoprotein mutations associated with congenital heart disease.Journal of Clinical Investigation 106,299-308(2000).
55.Schott,J.J.et al.Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108-111 (1998).
56. Schott, J.J. et al. Mutations in the transcription factor Nkx2.5 cause secundum atrial septal defect and atrioventricular block. Circulation 98, 3965 (1998).
57. Fujikura, J. et al. Differentiation of embryonic stem cells is induced by GATA factors. Genes & Development 16, 784-789 (2002).
58. Crispino, J.D. et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes & Development 15, 839-844 (2001).
59. Hosoya-Ohmura, S. et al. GATA-4 incompletely substitutes for GATA-1 in promoting both primitive and definitive erythropoiesis in vivo. Journal of Biological Chemistry 281, 32820-32830 (2006).
60. LaVoie, H.A., McCoy, GL. & Blake, C.A. Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy. Molecular and Cellular Endocrinology 227, 31-40 (2004).
61. Tong, Q. et al. Function of GATA transcription factors in preadipocyte-adipocyte transition. Science 290, 134-138(2000).
62. Hata, A. et al. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100, 229-240 (2000).
63. Hild, M. et al. The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development 126,2149-2159 (1999).
64. Nakashima, K. et al. The novel zinc finger-containing transcription factor Osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29 (2002).
65. Hoodless, P.A. et al. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85,489-500 (1996).
66. Allan, E.H. et al. Differentiation potential of a mouse bone marrow stromal cell line. Journal of Cellular Biochemistry 90, 158-169 (2003).
67. Kimura, N., Matsuo, R., Shibuya, H., Nakashima, K. & Taga, T. BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. Journal of Biological Chemistry 275, 17647-17652 (2000).
68. Paling, N.R.D., Wheadon, H., Bone, H.K. & Welham, M.J. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. Journal of Biological Chemistry 279, 48063-48070 (2004).
69. Suzawa, M. et al. Cytokines suppress adipogenesis and PPAR-function through the TAK1/TAB1/NIK cascade. Nature Cell Biology 5, 224-230 (2003).
70. Izumi, M. et al. Cross-talk between bone morphogenetic protein 2 and leukemia inhibitory factor through ERK 1/2 and Smad1 in protection against doxorubicin-induced injury of cardiomyocytes. Journal of Molecular and Cellular Cardiology 40, 224-233 (2006).
71. Drissi, M.H. et al. Runx2/Cbfal stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smadl on the collagen X promoter in chondrocytes. Journal of Cellular Biochemistry 90, 1287-1298 (2003).
72. Takizawa, T., Ochiai, W., Nakashima, K. & Taga, T, Enhanced gene activation by Notch and BMP signaling cross-talk. Nucleic Acids Research 31, 5723-5731 (2003).
1.Assmann,G;Schulte,H;Cullen,E New and classical risk factors--the Munster heart study(PROCAM).Eur J Med Res 1997;6:237-42.
2.Kannel WB.The Framingham Study:its 50-year old legacy and future promise.J Atheroscler Thromb 2000;6:60-6.
3.Funke H,Assmann G.Strategies for the assessment of genetic coronary artery disease risk.Curr Opin Lipidol 1999;10:285-91.
4.Gardemann A,Stricker J,Humme J et al.Angiotensinogen TI74M and M235T gene polymorphisms are associated with the extent of coronary atherosclerosis.Atherosclerosis 1999;145:309-14
5.Lahoz C,Schaefer E J,Cupples LA et al.Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study.Atherosclerosis 2001;154:529-37.
6.Wilson PWF,Schaefer EJ,Larson MG,Ordovas JM.Apolipoprotein E alleles and risk of coronary disease. A meta-analysis. Arterioscler Thromb Vasc Biol 1996; 16: 1250-5.
7.Green FR. Fibrinogen polymorphisms and atherothrombotic disease. Ann NY Acad Sci 2001; 936: 549-59.
8.Folsom AR, Aleksic N, Ahn C et al. Beta-fibrinogen gene-455G/A polymorphism and coronary heart disease incidence. The Atherosclerosis Risk Communities (ARIC) Study. Ann Epidemiol 2001; 11: 166-70.
9.Beyzade S, Zhang S, Wong YK, Day IN, Eriksson P, Ye S Influences of matrix metalloproteinase-3 gene variation on the extent of coronary atherosclerosis and risk of myocardial infarction. J Am Coll Cardiol. 2003 Jun 18;41(12):2130-7.
10.Jormsjo S, Wuttge DM, Sirsjo A, Whatling C, Hamsten A, et al. Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Pathol 2002; 161: 939-45.
11.Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
12.Sukhova GK, Zhang Y, Pan JH, Wada Y, Yamamoto T, et al. Deficiency of CTSS reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest 2003; 111: 897-906.
13.Pawlak K, Pawlak D, Mysliwiec M. Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis in haemodialysis patients. Thrombosis Research 2007; 1: 107.
14.Shi GP, Webb AC, Foster KE, Knoll JH, Lemere CA, et al. Human CTSS: chromosomal localization, gene structure and tissue distribution. J Biol Chem 1994; 269:11530-11536.
15.Cao HN, Robert A, Hegele. Human CTSS gene (CTSS) promoter -25G/A polymorphism. J Hum Genet 2000; 45:94-95.
16.Tang ZH, Russell PT. Candidate genes and confirmed genetic polymorphisms associated with cardiovascular diseases: a tabular assessment. Journal of Thrombosis and Thrombolysis 2001; 11:49-81.
17.Scheer WD, Boudreau DA, Hixson JE, McGill HC, Newman III WP, et al. ACE insert/delete polymorphism and atherosclerosis. Atherosclerosis 2005; 178(2):241-7.
18.Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in Human atheroma and regulation of their procuction in smooth muscle cell. J Clin Invest 1998; 102(3):576-583.
19.Hsing, Lianne C.; Rudensky AY. The lysosomal cysteine proteases in MHC class II antigen presentation. Immunol Rev 2005; 207(1):229-241.
20.Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
21. Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol. 1997;59:63-88.
22.Li Z, Yasuda Y, Li W, Bogyo M, Katz N, et al. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J Biol Chem. 2004;279:5470-5479.
23.Li YH, Chen CH, Yeh PS, Lin HJ, Chang BI, et al. Functional mutation in the promoter region of thrombomodulin gene in relation to carotid atherosclerosis. Atherosclerosis 2001; 154: 713-9.
24.Liu J, Ma LK, Yang JT, Ren A, Sun ZM, et al. Increased serum CTSS in patients with atherosclerosis and diabetes. Atherosclerosis 2006; 186(2):411-9.
25.Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6- 174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001; 22: 2243-52.
1.Attisano,L.& Wrana,J.L.Signal transduction by the TGF-beta superfamily.Science 296,1646-1647(2002).
2.Choy,L.,Skillington,J.& Derynck,R.Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation.Journal of Cell Biology 149,667-681(2000).
3.Monzen,K.et al.Smads,TAK1,and their common target ATF-2 play a critical role in cardiomyocyte differentiation.J Cell Biol 153,687-98(2001).
4.Monzen,K.,Nagai,R.& Komuro,I.A role for bone morphogenetic protein signaling in cardiomyocyte differentiation.Trends Cardiovasc Med 12,263-9(2002).
5.Monzen,K.et al.Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.Mol Cell Biol 19,7096-105(1999).
6.Rivera-Feliciano,J.& Tabin,C.J.Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field.Developmental Biology 295,580-588(2006).
7.Schlange,T.,Andree,B.,Arnold,H.H.& Brand,T.BMP2 is required for early heart development during a distinct time period.Mechanisms of Development 91,259-270(2000).
8.Ying,Q.L.,Nichols,J.,Chambers,I.& Smith,A.BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115,281-292 (2003).
9. Harada, K. et al. Crossveinless-2 controls bone morphogenetic protein signaling during early cardiomyocyte differentiation in P19 cells. Journal of Biological Chemistry 283,26705-26713 (2008).
10. Prall, O.W.J. et al. An Nkx2-5/Bmp2/Smadl negative feedback loop orchestrates cardiac progenitor cell proliferation and deployment, and is a molecular target in congenital heart disease. Differentiation 74, 014 (2006).
11. Wrana, J.L. Regulation of Smad activity. Cell 100,189-192 (2000).
12. Ellies, D.L. & Krumlauf, R. Bone formation: The nuclear matrix reloaded. Cell 125, 840-842 (2006).
13. Pallante, B.A. et al. Bone marrow Oct3/4(+) cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms. Circulation Research 100, E1-E11 (2007).
14. Yamagishi, T., Nakajima, Y., Miyazono, K. & Nakamura, H. Bone morphogenetic protein-2 acts synergistically with transforming growth factor-beta 3 during endothelial-mesenchymal transformation in the developing chick heart. Journal of Cellular Physiology 180, 35-45 (1999).
15. Lian, J.B. & Stein, G.S. Runx2/Cbfal: A multifunctional regulator of bone formation. Current Pharmaceutical Design 9,2677-2685 (2003).
16. Wu, C.J. & Lu, H.K. Smad signal pathway in BMP-2-induced osteogenesis - a mini review. Journal of Dental Sciences 3,13-21 (2008).
17. Wu, M. et al. Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo. Bone 44, 528-536 (2009).
18. Lin, G. et al. Defining stem and progenitor cells within adipose tissue. Stem Cells Dev 17, 1053-63 (2008).
19. Szabo, E., Qiu, Y.Y., Baksh, S., Michalak, M. & Opas, M. Calreticulin inhibits commitment to adipocyte differentiation. Journal of Cell Biology 182, 103-116(2008).
20. Yin, Z.Z. & Frasch, M. Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila. Developmental Genetics 22, 187-200 (1998).
21. Yamada, M., Revelli, J.P., Eichele, G., Barron, M. & Schwartz, R.J. Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: Evidence for BMP2 induction of Tbx2. Developmental Biology 228, 95-105 (2000).
22. Kim, Y.Y. et al. Use of Long-tenn Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2. Yonsei Medical Journal 49, 819-827 (2008).
23. Lowell, B.B. PPAR gamma: An essential regulator of adipogenesis and modulator of fat cell function. Cell 99,239-242 (1999).
24. de Caestecker, M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev 15,1-11 (2004).
25. Schneider, M.D., Gaussin, V. & Lyons, K.M. Tempting fate: BMP signals for cardiac morphogenesis. Cytokine Growth Factor Rev 14,1-4 (2003).
26. Sachinidis, A., Kolossov, E., Fleischmann, B.K. & Hescheler, J. Generation of cardiomyocytes from embryonic stem cells experimental studies. Herz 27, 589-97 (2002).
27. Nohe, A. et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 277, 5330-8 (2002).
28. Nohe, A., Keating, E., Knaus, P. & Petersen, N.O. Signal transduction of bone morphogenetic protein receptors. Cell Signal 16,291-9 (2004).
29. Fukuda, S. et al. Potentiation of astrogliogenesis by STAT3-mediated activation of bone morphogenetic protein-Smad signaling in neural stem cells. Molecular and Cellular Biology 27,4931-4937 (2007).
30. Tong, Q. et al. Function of GATA transcription factors in preadipocyte-adipocyte transition. Science 290,134-138 (2000).
31. Hata, A. et al. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100,229-240 (2000).
32. Yoshida, Y. et al. Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell 103,1085-1097 (2000).
33. Hild, M. et al. The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development 126, 2149-2159(1999).
34. Nakashima, K. et al. The novel zinc finger-containing transcription factor Osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29(2002).
35. Hoodless, P.A. et al. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85,489-500 (1996).
36. Zwijsen, A., Verschueren, K. & Huylebroeck, D. New Intracellular components of bone morphogenetic protein/Smad signaling cascades. FEBS Lett 546,133-9(2003).
37. Suzawa, M. et al. Cytokines suppress adipogenesis and PPAR-function through the TAK1/TAB1/NIK cascade. Nature Cell Biology 5, 224-230 (2003).
38. Paling, N.R.D., Wheadon, H., Bone, H.K. & Welham, M.J. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. Journal of Biological Chemistry 279,48063-48070 (2004).
39. Fujita, T. et al. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. Journal of Cell Biology 166, 85-95 (2004).
40. Ge, C.X., Xiao, G.Z., Jiang, D. & Franceschi, R.T. Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. Journal of Cell Biology 176, 709-718(2007).
41. Scheid, M.P. & Woodgett, J.R. Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett 546,108-12 (2003).
42. Ghosh-Choudhury, N., Abboud, S.L., Mahimainathan, L., Chandrasekar, B. & Choudhury, G.G. Phosphatidylinositol 3-kinase regulates bone morphogenetic protein-2 (BMP-2)-induced myocyte enhancer factor 2A-dependent transcription of BMP-2 gene in cardiomyocyte precursor cells. J Biol Chem 278,21998-2005(2003).
43. van der Heyden, M.A. & Defize, L.H. Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc Res 58, 292-302 (2003).
44. Eriksson, M. & Leppa, S. Mitogen-activated protein kinases and activator protein 1 are required for proliferation and cardiomyocyte differentiation of P19 embryonal carcinoma cells. J Biol Chem 277, 15992-6001 (2002).
45. Muller, B., Hartmann, B., Pyrowolakis, G., Affolter, M. & Basler, K. Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. Cell 113, 221-233 (2003).
46. Flores, M.V.C., Lam, E.Y.N., Crosier, K.E. & Crosier, P.S. Osteogenic transcription factor Runx2 is a maternal determinant of dorsoventral patterning in zebrafish. Nature Cell Biology 10, 346-U80 (2008).
47. Cohen, M.M. Bone morphogenetic proteins with some comments on fibrodysplasia ossificans progressiva and NOGGIN. American Journal of Medical Genetics 109, 87-92 (2002).
48. Chang, C.B. & Hemmati-Brivanlou, A. Xenopus GDF6, a new antagonist of noggin and a partner of BMPs. Development 126, 3347-3357 (1999).
49. Wylie, C. et al. Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos. Development 122,2987-2996 (1996).
50. Pashmforoush, M. et al. Nkx2-5 pathways and congenital heart disease: Loss of ventricular myocyte lineage progressive cardiomyopathy and complete heart block. Cell 117, 373-386 (2004).
51. Morrisey, E.E. et al. GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes & Development 12, 3579-3590 (1998).
52. Molkentin, J.D., Lin, Q., Duncan, S.A. & Olson, E.N. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes & Development 11,1061-1072 (1997).
53. Moskowitz, I.P.G.et al. A molecular pathway including Id2, Tbx5, and Nkx2-5 required for cardiac conduction system development. Cell 129, 1365-1376 (2007).
54. Shin, C.H. et al. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell 110, 725-735 (2002).
55. Ericson, J. et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded shh signaling. Cell 90,169-180 (1997).
56. Lyons, I. et al. Myogenic and Morphogenetic Defects in the Heart Tubes of Murine Embryos Lacking the Homeo Box Gene Nkx2-5. Genes & Development 9, 1654-1666(1995).
57. Kasahara, H. et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. Journal of Clinical Investigation 106, 299-308 (2000).
58. Schott, J.J. et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108-111 (1998).
59. Schott, J.J. et al. Mutations in the transcription factor Nkx2.5 cause secundum atrial septal defect and atrioventricular block. Circulation 98, 3965 (1998).
60. Schott, J.J. et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281,108-11 (1998).
61. Crispino, J.D. et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes & Development 15, 839-844 (2001).
62. Bisping, E. et al. Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure. Proc NatlAcadSci USA 103, 14471-6 (2006).
63. Heikinheimo, M., Scandrett, J.M. & Wilson, D.B. Localization of transcription factor GATA-4 to regions of the mouse embryo involved in cardiac development. Dev Biol 164, 361-73 (1994).
64. Drissi, M.H. et al. Runx2/Cbfal stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smadl on the collagen X promoter in chondrocytes. Journal of Cellular Biochemistry 90,1287-1298 (2003).
65. Stock, M. & Otto, F. Control of RUNX2 isoform expression: the role of promoters and enhancers. J Cell Biochem 95, 506-17 (2005).
66. Gersbach, C.A., Byers, B.A., Pavlath, G.K. & Garcia, A.J. Runx2/Cbfal stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Exp Cell Res 300,406-17 (2004).
67. Enomoto, H. et al. Runx2 deficiency in chondrocytes causes adipogenic changes in vitro. J Cell Sci 117, 417-25 (2004).
68. Issemann, I. & Green, S. Activation of a Member of the Steroid-Hormone Receptor Superfamily by Peroxisome Proliferators. Nature 347, 645-650 (1990).
69. Fajas, L. et al. The Organization, Promoter Analysis, and Expression of the Human PPARgamma Gene. J. Biol. Chem. 272, 18779-18789 (1997).
70. Li, X.N. et al. CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation. Journal of Cell Biology 181, 959-972 (2008).
71. Tontonoz, P., Hu, E. & Spiegelman, B.M. Stimulation of adipogenesis in fibroblasts by PPAR-gamma-2, a lipid-activated transcription factor. Cell 79, 1147-1156(1994).
72. Lehrke, M. & Lazar, M.A. The many faces of PPAR gamma. Cell 123, 993-999 (2005).
73. Hu, E., Liang, P. & Spiegelman, B.M. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271, 10697-703 (1996).
74. Rosen, E.D. et al. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell 4, 611-7 (1999).
75. Blanquart, C., Barbier, O., Fruchart, J.C., Staels, B. & Glineur, C. Peroxisome proliferator-activated receptors: regulation of transcriptional activities and roles in inflammation. J Steroid Biochem Mol Biol 85,267-73 (2003).
76. Guerre-Millo, M. Extending the glucose/fatty acid cycle: a glucose/adipose tissue cycle. Biochem Soc Trans 31,1161-4 (2003).
2.Smits,J.P.et al.Genotype-phenotype relationship in Brugada syndrome:electrocardiographic features differentiate SCN5A-related patients from non-SCN5A-related patients.J Am Coll Cardiol 40,350-6(2002).
3.Tan,H.L.et al.A sodium-channel mutation causes isolated cardiac conduction disease.Nature 409,1043-7(2001).
4.McNair,W.P.et al.SCN5A mutation associated with dilated cardiomyopathy,conduction disorder,and arrhythmia.Circulation 110,2163-2167(2004).
5.Nguyen,T.P.,Wang,D.W.,Rhodes,T.H.& George,A.L.Divergent biophysical defects caused by mutant sodium channels in dilated cardiomyopathy with arrhythmia.Circulation Research 102,364-371(2008).
6.Groenewegen,W.A.& Wilde,A.A.M.Letter regarding article by McNair et al,"SCNSA mutation associated with dilated cardiomyopathy,conduction disorder,and arrhythmia".Circulation 112,E9-E9(2005).
7.Olson,T.M.et al.Sodium channel mutations and susceptibility to heart failure and atrial fibrillation.Jama-Journal of the American Medical Association 293,447-454(2005).
8.Ge,J.et al.Identification and characterization of a novel SCN5A mutation associated with progressive familial atrioventricular block and dilated cardiomyopathy.Circulation 112,U206-U207(2005).
9.Waddington,S.N.et al.Adenovirus serotype 5 hexon mediates liver gene transfer.Cell 132,397-409(2008).
10.Kaelin,W.G.et al.Expression Cloning of a Cdna-Encoding a Retinoblastoma-Binding Protein with E2f-Like Properties. Cell 70, 351-364 (1992).
11. Zou, L., Cortez, D. & Elledge, S.J. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes & Development 16, 198-208 (2002).
12. Laitinen-Forsblom, P.J. et al. SCN5A mutation associated with cardiac conduction defect and atrial arrhythmias. Journal of Cardiovascular Electrophysiology 17,480-485 (2006).
13. Frustaci, A. et al. Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome. Circulation 112, 3680-3687 (2005).
14. Wilde, A.A.M., Remme, C.A., Derksen, R., Wever, E.F.D. & Hauer, R.N.W. Brugada syndrome. European Heart Journal 23, 675-676 (2002).
15. Roberts, R. & Brugada, R. Genetic aspects of arrhythmias. American Journal of Medical Genetics 97, 310-318 (2000).
16. Puthalakath, H. et al. ER stress triggers apoptosis by activating BH3-only protein Bim. Cell 129,1337-1349(2007).
17. Clapham, D.E. Calcium Signaling. Cell 80, 259-268 (1995).
18. Catterall, W.A. Excitation Contraction Coupling in Vertebrate Ske letal-Muscle - a Tale of 2 Calcium Channels. Cell 64, 871-874 (1991).
19. Fruen, B.R. et al. Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin. Biochemistry 42, 2740-2747 (2003).
20. Fruen, B.R., Norton, L.E., Bloomquist, R.A., Thomas, D.D. & Balog, E.M. Role of CaM Met residues in mediating productive associations with the cardiac ryanodine receptor (RYR2). Biophysical Journal 84, 64A-64A (2003).
21. Ozawa, T. Ryanodine-sensitive Ca2+ release mechanism in non-excitable cells (Review). International Journal of Molecular Medicine 7, 21-25 (2001).
22. Brandl, C.J., Green, N.M., Korczak, B. & Maclennan, D.H. 2 Ca-2+ Atpase Genes -Homologies and Mechanistic Implications of Deduced Amino-Acid-Sequences. Cell 44, 597-607(1986).
23. Kemi, O.J. et al. Aerobic interval training enhances cardiomyocyte contractility and Ca2+ cycling by phosphorylation of CaMKII and Thr-17 of phospholamban. Journal of Molecular and Cellular Cardiology 43, 354-361 (2007).
24. Sag, C.M., DeSantiago, J., Bers, D.M. & Maier, L.S. Recovery of Ca transients, SR Ca reuptake and shortening during acidosis requires the CaMKII/PLB signaling pathway but is impaired in heart failure. European Heart Journal 25, 179-179 (2004).
25. Vasanji, Z., Dhalla, N.S. & Netticadan, T. Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart. Molecular and Cellular Biochemistry 261, 245-249 (2004).
26. Yang, D.M. et al. Ca2+/calmodulin kinase II-dependent phosphorylation of ryanodine receptors stabilizes Ca2+ signaling in intact cardiac myocytes. Circulation 114, 57-57 (2006).
27. Netticadan, T., Temsah, R.M., Kawabata, K. & Dhalla, N.S. Sarcoplasmic reticulum Ca2+/calmodulin-dependent protein kinase is altered in heart failure. Circulation Research 86, 596-605 (2000).
28. Netticadan, T., Temsah, R., Osada, M. & Dhalla, N.S. Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion. American Journal of Physiology-Cell Physiology 277, C384-C391 (1999).
29. Yamaguchi, N., Xu, L., Pasek, D.A., Evans, K..E. & Meissner, G. Molecular basis of calmodulin binding to cardiac muscle Ca2+ release channel (ryanodine receptor). Journal of Biological Chemistry 278,23480-23486(2003).
30.Fruen,B.R.et al.Regulation of RYR1 and RYR2 by CaM mutants deficient in Ca2+ binding.Biophysical Journal 82,291(2002).
31.Balshaw,D.M.,Xu,L.,Yamaguchi,N.,Pasek,D.A.& Meissner,G.Calmodulin binding and inhibition of cardiac muscle calcium release channel(ryanodine receptor).Journal of Biological Chemistry 276,20144-20153(2001).
32.Ashley,C.C.& Ridgway,E.B.Simultaneous Recording of Membrane Potential Calcium Transient and Tension in Single Muscle Fibres.Nature 219,1168-&(1968).
33.Davidson,C.J.,Baum,K.R.,Spitzer,K.W.& Bridge,J.H.B.Exercise reduces the maximal rate of rise of the calcium transient in rat cardiomyocytes.Faseb Journal 20,A1448-A1448(2006).
34.Deng,C.Y.et al.Effect of valsartan on cardiacmyocytes contraction function and calcium transient in heart failure rats.Acta Pharmacologica Sinica 27,189-189(2006).
35.Janssen,P.M.L.,Zeitz,O.,Schumann,H.,Holtz,J.& Hasenfuss,G.Load-Induced Cardiomyocyte Apoptosis in Cultured Multicellular Myocardial Preparations Is Unaltered in Presence of the beta-Adrenoceptor Antagonist Nebivolol.Pharmacology 83,141-147(2009).
36.Xie,P.et al.Atrogin-1/MAFbx Enhances Simulated Ischemia/Reperfusion-induced Apoptosis in Cardiomyocytes through Degradation of MAPK Phosphatase-1 and Sustained JNK Activation.Journal of Biological Chemistry 284,doi:10.1074/jbc.M806487200(2009).
37.Tan,W.Q.et al.Novel Cardiac Apoptotic Pathway The Dephosphorylation of Apoptosis Repressor With Caspase Recruitment Domain by Calcineurin.Circulation 118,2268-2276(2008).
1.Assmann,G;Schulte,H;Cullen,P.New and classical risk factors--the Munster heart study(PROCAM).Eur J Med Res 1997;6:237-42.
2.Kannel WB.The Framingham Study:its 50-year old legacy and future promise.J Atheroscler Thromb 2000;6:60-6.
3.Funke H,Assmann G.Strategies for the assessment of genetic coronary artery disease risk.Curr Opin Lipidol 1999;10:285-91.
4.Gardemann A,Stricker J,Humme Jet al.Angiotensinogen TI74M and M235T gene polymorphisms are associated with the extent of coronary atherosclerosis.Atherosclerosis 1999;145:309-14
5.Lahoz C,Schaefer EJ,Cupples LA et al.Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study.Atherosclerosis 2001;154:529-37.
6.Wilson PWF,Schaefer EJ,Larson MG,Ordovas JM.Apolipoprotein E alleles and risk of coronary disease.A meta-analysis.Arterioscler Thromb Vasc Biol 1996;16:1250-5.
7.Green FR.Fibrinogen polymorphisms and atherothrombotic disease.Ann NY Acad Sci 2001;936:549-59.
8.Folsom AR,Aleksic N,Ahn C et al.Beta-fibrinogen gene-455G/A polymorphism and coronary heart disease incidence.The Atherosclerosis Risk Commtmities (ARIC) Study.Ann Epidemiol 2001;11:166-70.
9.Beyzade S,Zhang S,Wong YK,Day IN,Eriksson P,Ye S Influences of matrix metalloproteinase-3 gene variation on the extent of coronary atherosclerosis and risk of myocardial infarction.J Am Coil Cardiol.2003 Jun 18;41(12):2130-7.
10.Jormsjo S,Wuttge DM,Sirsjo A,Whatling C,Hamsten A,et al.Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice.Am J Pathol 2002;161:939-45.
11.Sukhova GK,Shi GP,Simon DI,Chapman HA,Libby P.Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
12. Sukhova GK, Zhang Y, Pan JH, Wada Y, Yamamoto T, et al. Deficiency of CTSS reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest 2003; 111: 897-906.
13. Pawlak K, Pawlak D, Mysliwiec M. Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis in haemodialysis patients. Thrombosis Research 2007;1: 107.
14. Shi GP, Webb AC, Foster KE, Knoll JH, Lemere CA, et al. Human CTSS: chromosomal localization, gene structure and tissue distribution. J Biol Chem 1994; 269:11530-11536.
15. Cao HN, Robert A, Hegele. Human CTSS gene (CTSS) promoter -25G/A polymorphism. J Hum Genet 2000; 45:94-95.
16. Tang ZH, Russell PT. Candidate genes and confirmed genetic polymorphisms associated with cardiovascular diseases: a tabular assessment. Journal of Thrombosis and Thrombolysis 2001; 11:49-81.
17. Scheer WD, Boudreau DA, Hixson JE, McGill HC, Newman III WP, et al. ACE insert/delete polymorphism and atherosclerosis. Atherosclerosis 2005; 178(2):241-7.
18. Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in Human atheroma and regulation of their procuction in smooth muscle cell. J Clin Invest 1998; 102(3):576-583.
19. Hsing, Lianne C.; Rudensky AY. The lysosomal cysteine proteases in MHC class II antigen presentation. Immunol Rev 2005; 207(1):229-241.
20. Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
21. Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol. 1997;59:63-88.
22. Li Z, Yasuda Y, Li W, Bogyo M, Katz N, et al. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J Biol Chem. 2004;279:5470-5479.
23. Li YH, Chen CH, Yeh PS, Lin HJ, Chang BI, et al. Functional mutation in the promoter region of thrombomodulin gene in relation to carotid atherosclerosis. Atherosclerosis 2001; 154: 713-9.
24. Liu J, Ma LK, Yang JT, Ren A, Sun ZM, et al. Increased serum CTSS in patients with atherosclerosis and diabetes. Atherosclerosis 2006; 186(2):411-9.
25. Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6- 174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001; 22: 2243-52.
1.Guo, S. & Kemphues, K.J. Par-1, a Gene Required for Establishing Polarity in C-Elegans Embryos, Encodes a Putative Ser/Thr Kinase That Is Asymmetrically Distributed. Cell 81,611-620 (1995).
2.Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 (1998).
3.Lin, R.L. & Avery, L. RNA interference - Policing rogue genes. Nature 402, 128-129(1999).
4.Sharp, P.A. & Zamore, P.D. Molecular biology - RNA interference. Science 287, 2431-+(2000).
5.Hannon, GJ. RNA interference. Nature 418,244-251 (2002).
6.Eccleston, A. & Eggleston, A.K. RNA interference. Nature 431, 337-337 (2004).
7.Mello, C.C. & Conte, D. Revealing the world of RNA interference. Nature 431, 338-342 (2004).
8.Check, E. RNA interference - Hitting the on switch. Nature 448, 855-858 (2007).
9.Sharp, P. A. The Centrality of RNA. Cell 136, 577-580 (2009).
10.Siomi, H. & Siomi, M.C. On the road to reading the RNA-interference code. Nature 457, 396-404 (2009).
11.Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101,25-33(2000).
12.Bernstein, E., Caudy, A.A., Hammond, S.M. & Harmon, GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366 (2001).
13.Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411,494-498 (2001).
14.Hutvagner, G.et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834-838 (2001).
15.Knight, S.W. & Bass, B.L. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 2269-2271 (2001).
16.Motamedi, M.R. et al. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119, 789-802 (2004).
17.Filipowicz, W. RNAi: The nuts and bolts of the RISC machine. Cell 122, 17-20 (2005).
18.Ameres, S.L., Martinez, J. & Schroeder, R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130,101-112 (2007).
19.Bass, B.L. Double-stranded RNA as a template for gene silencing. Cell 101, 235-238 (2000).
20.Brown, D.R., McClowry, T.L., Sidner, R.A., Fife, K.H. & Bryan, J.T. Expression of the human papillomavirus type 11 E5A protein from the El boolean AND E4,E5 transcript. Intervirology 41,47-54 (1998).
21.Samarkina, O.N. et al. Universal and rapid method for purification of GFP-like proteins by the ethanol extraction. Protein Expression and Purification 65, 108-113 (2009).
22.Dross, N. et al. Mapping eGFP oligomer mobility in living cell nuclei. PLoS ONE 4, e5041 (2009).
23.Geusens, B. et al. Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes. Journal of Controlled Release 133,214-220 (2009).
24.Marchini, C. et al. Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes. Applied Physics Letters 94(2009).
25.Pappalardo, J.S. et al. Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes. J Control Release 134,41-6 (2009).
26.Martin, J.B., Young, J.L., Benoit, J.N. & Dean, D.A. Gene transfer to intact mesenteric arteries by electroporation. Journal of Vascular Research 37, 372-380 (2000).
1.Attisano,L.& Wrana,J.L.Signal transduction by the TGF-beta superfamily.Science 296,1646-1647(2002).
2.Choy,L.,Skillington,J.& Derynck,R.Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation.Journal of Cell Biology 149,667-681(2000).
3.Flores,M.V.C.,Lam,E.Y.N.,Crosier,K.E.& Crosier,P.S.Osteogenic transcription factor Runx2 is a maternal determinant of dorsoventral patterning in zebrafish.Nature Cell Biology 10,346-U80(2008).
4.Koyanagi,M.et al.Notch signaling contributes to the expression of cardiac markers in human circulating progenitor cells.Circulation Research 101,1139-1145(2007).
5.Prall,O.W.J.et al.An Nkx2-5/Bmp2/Smad1 negative feedback loop orchestrates cardiac progenitor cell proliferation and deployment,and is a molecular target in congenital heart disease.Differentiation 74,O14(2006).
6.Dell'Era,P.et al.Fibroblast growth factor receptor-1 is essential for in vitro cardiomyocyte development.Circulation Research 93,414-420(2003).
7.Yin,Z.Z.& Frasch,M.Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila.Developmental Genetics 22,187-200(1998).
8.Rivera-Feliciano,J.& Tabin,C.J.Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field.Developmental Biology 295,580-588(2006).
9.Schlange,T.,Andree,B.,Arnold,H.H.& Brand,T.BMP2 is required for early heart development during a distinct time period.Mechanisms of Development 91,259-270(2000).
10.Ying,Q.L.,Nichols,J.,Chambers,I.& Smith,A.BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell 115,281-292(2003).
11.Jin,W.Z.et al.Schnurri-2 controls BMP-dependent adipogenesis via interaction with Smad proteins.Developmental Cell 10,461-471(2006).
12.Wrana,J.L.Regulation of Smad activity.Cell 100,189-192(2000).
13.Ellies,D.L.& Krumlauf,R.Bone formation:The nuclear matrix reloaded.Cell 125,840-842(2006).
14.Pallante,B.A.et al.Bone marrow Oct3/4(+) cells differentiate into cardiac myocytes via age-dependent paraerine mechanisms.Circulation Research 100,E1-E11(2007).
15.Yamagishi,T.,Nakajima,Y.,Miyazono,K.& Nakamura,H.Bone morphogenetie protein-2acts synergistically with transforming growth factor-beta 3 during endothelial-mesenchymal transformation in the developing chick heart.Journal of Cellular Physiology 180,35-45 (1999).
16. Lian, J.B. & Stein, G.S. Runx2/Cbfa1: A multifunctional regulator of bone formation. Current Pharmaceutical Design 9,2677-2685 (2003).
17. Wu, C.J. & Lu, H.K. Smad signal pathway in BMP-2-induced osteogenesis - a mini review. Journal of Dental Sciences 3, 13-21 (2008).
18. Wu, M. et al. Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo. Bone 44, 528-536 (2009).
19. Yoshida, Y. et al. Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell 103, 1085-1097 (2000).
20. Marie, P.J. Transcription factors controlling osteoblastogenesis. Archives of Biochemistry and Biophysics 473, 98-105 (2008).
21. Hoffmann, A. et al. The T-box transcription factor Brachyury mediates cartilage development in mesenchymal stem cell line C3H10T1/2. Journal of Cell Science 115, 769-781 (2002).
22. Lin, G.et al. Defining stem and progenitor cells within adipose tissue. Stem Cells Dev 17, 1053-63 (2008).
23. Szabo, E., Qiu, Y.Y., Baksh, S., Michalak, M. & Opas, M. Calreticulin inhibits commitment to adipocyte differentiation. Journal of Cell Biology 182, 103-116 (2008).
24. Yamada, M., Revelli, J.P., Eichele, G., Barron, M. & Schwartz, R.J. Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: Evidence for BMP2 induction of Tbx2. Developmental Biology 228, 95-105 (2000).
25. Monzen, K. et al. Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAKl and cardiac transcription factors Csx/Nkx-2.5 and GATA-4. Molecular and Cellular Biology 19, 7096-7105 (1999).
26. Pal, R. & Khanna, A. Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCell((R))hES1, under low serum concentration supplemented with bone morphogenetic protein-2. Differentiation 75, 112-122 (2007).
27. Lowell, B.B. PPAR gamma: An essential regulator of adipogenesis and modulator of fat cell function. Cell 99,239-242 (1999).
28. Gunther, T. & Schule, R. Fat or bone? A non-canonical decision. Nature Cell Biology 9, 1229-1231(2007).
29. Hong, J.H. et al. TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309, 1074-1078 (2005).
30. Saltiel, A.R. Muscle or fat? Rho bridges the GAP. Cell 113, 144-145 (2003).
31. Wotton, D., Lo, R.S., Lee, S. & Massague, J. A Smad transcriptional corepressor. Cell 97, 29-39(1999).
32. Yuasa, S. et al. Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells. Nature Biotechnology 23, 607-611 (2005).
33. Tomita, Y. et al. Application of mesenchymal stem cell-derived cardiomyocytes as bio-pacemakers: current status and problems to be solved. Medical & Biological Engineering & Computing 45, 209-220 (2007).
34. Hakuno, D. et al. Bone marrow-derived regenerated cardiomyocytes (CMG cells) express functional adrenergic and muscarinic receptors. Circulation 105, 380-386 (2002).
35. Makino, S. et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. Journal of Clinical Investigation 103, 697-705 (1999).
36.Zhang,X.et al.Runx2 overexpression enhances osteoblastic differentiation and mineralization in adipose - derived stem cells in vitro and in vivo.Calcified Tissue International 79,169-178(2006).
37.Tontonoz,P.,Hu,E.& Spiegelman,B.M.Stimulation of adipogenesis in fibroblasts by PPAR-gamma-2,a lipid-activated transcription factor.Cell 79,1147-1156(1994).
38.Plutzky,J.PPARs as therapeutic targets:Reverse cardiology? Science 302,406-407(2003).
39.Lehrke,M.& Lazar,M.A.The many faces of PPAR gamma.Cell 123,993-999(2005).
40.Sukhikh,G.T.,Malaitsev,V.V.,Bogdanova,I.M.& Dubrovina,I.V.Mesenchymal stem cells.Bulletin of Experimental Biology and Medicine 133,103-109(2002).
41.Pittenger,M.E et al.Multilineage potential of adult human mesenchymal stem cells.Science 284,143-147(1999).
42.Payushina,O.V.,Domaratskaya,E.L.& Starostin,V.I.Mesenchymal stem cells:Sources,phenotype,and differentiation potential.Biology Bulletin 33,2-18(2006).
43.Musina,R.A.,Bekchanova,E.S.& Sukhikh,G.T.Comparison of mesenchymal stem cells obtained from different human tissues.Bulletin of Experimental Biology and Medicine 139,504-509(2005).
44.McCarty,R.C.,Gronthos,S.,Zannettino,A.C.,Foster,B.K.& Xian,C.J.Characterisation and Developmental Potential of Ovine Bone Marrow Derived Mesenchymal Stem Cells.Journal of Cellular Physiology 219,324-333(2009).
45.Issemann,I.& Green,S.Activation of a Member of the Steroid-Hormone Receptor Superfamily by Peroxisome Proliferators.Nature 347,645-650(1990).
46.Pashmforoush,M.et al.Nkx2-5 pathways and congenital heart disease:Loss of ventricular myocyte lineage progressive cardiomyopathy and complete heart block.Cell 117,373-386(2004).
47.Morrisey,E.E.et al.GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo.Genes & Development 12,3579-3590(1998).
48.Molkentin,J.D.,Lin,Q.,Duncan,S.A.& Olson,E.N.Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.Genes & Development 11,1061-1072(1997).
49.Liberatore,C.M.,Searcy-Schrick,R.D.,Vincent,E.B.& Yutzey,K.E.Nkx-2.5 gene induction in mice is mediated by a smad consensus regulatory region.Developmental Biology 244,243-256(2002).
50.Moskowitz,I.P.G.et al.A molecular pathway including Id2,TbxS,and Nkx2-5 required for cardiac conduction system development.Cell 129,1365-1376(2007).
51.Shin,C.H.et al.Modulation of cardiac growth and development by HOP,an unusual homeodomain protein.Cell 110,725-735(2002).
52.Ericson,J.et al.Pax6 controls progenitor cell identity and neuronal fate in response to graded shh signaling.Cell 90,169-180(1997).
53.Lyons,I.et al.Myogenic and Morphogenetic Defects in the Heart Tubes of Murine Embryos Lacking the Homeo Box Gene Nkx2-5.Genes & Development 9,1654-1666(1995).
54.Kasahara,H.et al.Loss of function and inhibitory effects of human CSX/NKX2.5homeoprotein mutations associated with congenital heart disease.Journal of Clinical Investigation 106,299-308(2000).
55.Schott,J.J.et al.Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108-111 (1998).
56. Schott, J.J. et al. Mutations in the transcription factor Nkx2.5 cause secundum atrial septal defect and atrioventricular block. Circulation 98, 3965 (1998).
57. Fujikura, J. et al. Differentiation of embryonic stem cells is induced by GATA factors. Genes & Development 16, 784-789 (2002).
58. Crispino, J.D. et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes & Development 15, 839-844 (2001).
59. Hosoya-Ohmura, S. et al. GATA-4 incompletely substitutes for GATA-1 in promoting both primitive and definitive erythropoiesis in vivo. Journal of Biological Chemistry 281, 32820-32830 (2006).
60. LaVoie, H.A., McCoy, GL. & Blake, C.A. Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy. Molecular and Cellular Endocrinology 227, 31-40 (2004).
61. Tong, Q. et al. Function of GATA transcription factors in preadipocyte-adipocyte transition. Science 290, 134-138(2000).
62. Hata, A. et al. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100, 229-240 (2000).
63. Hild, M. et al. The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development 126,2149-2159 (1999).
64. Nakashima, K. et al. The novel zinc finger-containing transcription factor Osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29 (2002).
65. Hoodless, P.A. et al. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85,489-500 (1996).
66. Allan, E.H. et al. Differentiation potential of a mouse bone marrow stromal cell line. Journal of Cellular Biochemistry 90, 158-169 (2003).
67. Kimura, N., Matsuo, R., Shibuya, H., Nakashima, K. & Taga, T. BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. Journal of Biological Chemistry 275, 17647-17652 (2000).
68. Paling, N.R.D., Wheadon, H., Bone, H.K. & Welham, M.J. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. Journal of Biological Chemistry 279, 48063-48070 (2004).
69. Suzawa, M. et al. Cytokines suppress adipogenesis and PPAR-function through the TAK1/TAB1/NIK cascade. Nature Cell Biology 5, 224-230 (2003).
70. Izumi, M. et al. Cross-talk between bone morphogenetic protein 2 and leukemia inhibitory factor through ERK 1/2 and Smad1 in protection against doxorubicin-induced injury of cardiomyocytes. Journal of Molecular and Cellular Cardiology 40, 224-233 (2006).
71. Drissi, M.H. et al. Runx2/Cbfal stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smadl on the collagen X promoter in chondrocytes. Journal of Cellular Biochemistry 90, 1287-1298 (2003).
72. Takizawa, T., Ochiai, W., Nakashima, K. & Taga, T, Enhanced gene activation by Notch and BMP signaling cross-talk. Nucleic Acids Research 31, 5723-5731 (2003).
1.Assmann,G;Schulte,H;Cullen,E New and classical risk factors--the Munster heart study(PROCAM).Eur J Med Res 1997;6:237-42.
2.Kannel WB.The Framingham Study:its 50-year old legacy and future promise.J Atheroscler Thromb 2000;6:60-6.
3.Funke H,Assmann G.Strategies for the assessment of genetic coronary artery disease risk.Curr Opin Lipidol 1999;10:285-91.
4.Gardemann A,Stricker J,Humme J et al.Angiotensinogen TI74M and M235T gene polymorphisms are associated with the extent of coronary atherosclerosis.Atherosclerosis 1999;145:309-14
5.Lahoz C,Schaefer E J,Cupples LA et al.Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study.Atherosclerosis 2001;154:529-37.
6.Wilson PWF,Schaefer EJ,Larson MG,Ordovas JM.Apolipoprotein E alleles and risk of coronary disease. A meta-analysis. Arterioscler Thromb Vasc Biol 1996; 16: 1250-5.
7.Green FR. Fibrinogen polymorphisms and atherothrombotic disease. Ann NY Acad Sci 2001; 936: 549-59.
8.Folsom AR, Aleksic N, Ahn C et al. Beta-fibrinogen gene-455G/A polymorphism and coronary heart disease incidence. The Atherosclerosis Risk Communities (ARIC) Study. Ann Epidemiol 2001; 11: 166-70.
9.Beyzade S, Zhang S, Wong YK, Day IN, Eriksson P, Ye S Influences of matrix metalloproteinase-3 gene variation on the extent of coronary atherosclerosis and risk of myocardial infarction. J Am Coll Cardiol. 2003 Jun 18;41(12):2130-7.
10.Jormsjo S, Wuttge DM, Sirsjo A, Whatling C, Hamsten A, et al. Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Pathol 2002; 161: 939-45.
11.Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
12.Sukhova GK, Zhang Y, Pan JH, Wada Y, Yamamoto T, et al. Deficiency of CTSS reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest 2003; 111: 897-906.
13.Pawlak K, Pawlak D, Mysliwiec M. Urokinase-type plasminogen activator and metalloproteinase-2 are independently related to the carotid atherosclerosis in haemodialysis patients. Thrombosis Research 2007; 1: 107.
14.Shi GP, Webb AC, Foster KE, Knoll JH, Lemere CA, et al. Human CTSS: chromosomal localization, gene structure and tissue distribution. J Biol Chem 1994; 269:11530-11536.
15.Cao HN, Robert A, Hegele. Human CTSS gene (CTSS) promoter -25G/A polymorphism. J Hum Genet 2000; 45:94-95.
16.Tang ZH, Russell PT. Candidate genes and confirmed genetic polymorphisms associated with cardiovascular diseases: a tabular assessment. Journal of Thrombosis and Thrombolysis 2001; 11:49-81.
17.Scheer WD, Boudreau DA, Hixson JE, McGill HC, Newman III WP, et al. ACE insert/delete polymorphism and atherosclerosis. Atherosclerosis 2005; 178(2):241-7.
18.Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in Human atheroma and regulation of their procuction in smooth muscle cell. J Clin Invest 1998; 102(3):576-583.
19.Hsing, Lianne C.; Rudensky AY. The lysosomal cysteine proteases in MHC class II antigen presentation. Immunol Rev 2005; 207(1):229-241.
20.Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic CTSS and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998;102: 576-583.
21. Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol. 1997;59:63-88.
22.Li Z, Yasuda Y, Li W, Bogyo M, Katz N, et al. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J Biol Chem. 2004;279:5470-5479.
23.Li YH, Chen CH, Yeh PS, Lin HJ, Chang BI, et al. Functional mutation in the promoter region of thrombomodulin gene in relation to carotid atherosclerosis. Atherosclerosis 2001; 154: 713-9.
24.Liu J, Ma LK, Yang JT, Ren A, Sun ZM, et al. Increased serum CTSS in patients with atherosclerosis and diabetes. Atherosclerosis 2006; 186(2):411-9.
25.Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6- 174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001; 22: 2243-52.
1.Attisano,L.& Wrana,J.L.Signal transduction by the TGF-beta superfamily.Science 296,1646-1647(2002).
2.Choy,L.,Skillington,J.& Derynck,R.Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation.Journal of Cell Biology 149,667-681(2000).
3.Monzen,K.et al.Smads,TAK1,and their common target ATF-2 play a critical role in cardiomyocyte differentiation.J Cell Biol 153,687-98(2001).
4.Monzen,K.,Nagai,R.& Komuro,I.A role for bone morphogenetic protein signaling in cardiomyocyte differentiation.Trends Cardiovasc Med 12,263-9(2002).
5.Monzen,K.et al.Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.Mol Cell Biol 19,7096-105(1999).
6.Rivera-Feliciano,J.& Tabin,C.J.Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field.Developmental Biology 295,580-588(2006).
7.Schlange,T.,Andree,B.,Arnold,H.H.& Brand,T.BMP2 is required for early heart development during a distinct time period.Mechanisms of Development 91,259-270(2000).
8.Ying,Q.L.,Nichols,J.,Chambers,I.& Smith,A.BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115,281-292 (2003).
9. Harada, K. et al. Crossveinless-2 controls bone morphogenetic protein signaling during early cardiomyocyte differentiation in P19 cells. Journal of Biological Chemistry 283,26705-26713 (2008).
10. Prall, O.W.J. et al. An Nkx2-5/Bmp2/Smadl negative feedback loop orchestrates cardiac progenitor cell proliferation and deployment, and is a molecular target in congenital heart disease. Differentiation 74, 014 (2006).
11. Wrana, J.L. Regulation of Smad activity. Cell 100,189-192 (2000).
12. Ellies, D.L. & Krumlauf, R. Bone formation: The nuclear matrix reloaded. Cell 125, 840-842 (2006).
13. Pallante, B.A. et al. Bone marrow Oct3/4(+) cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms. Circulation Research 100, E1-E11 (2007).
14. Yamagishi, T., Nakajima, Y., Miyazono, K. & Nakamura, H. Bone morphogenetic protein-2 acts synergistically with transforming growth factor-beta 3 during endothelial-mesenchymal transformation in the developing chick heart. Journal of Cellular Physiology 180, 35-45 (1999).
15. Lian, J.B. & Stein, G.S. Runx2/Cbfal: A multifunctional regulator of bone formation. Current Pharmaceutical Design 9,2677-2685 (2003).
16. Wu, C.J. & Lu, H.K. Smad signal pathway in BMP-2-induced osteogenesis - a mini review. Journal of Dental Sciences 3,13-21 (2008).
17. Wu, M. et al. Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo. Bone 44, 528-536 (2009).
18. Lin, G. et al. Defining stem and progenitor cells within adipose tissue. Stem Cells Dev 17, 1053-63 (2008).
19. Szabo, E., Qiu, Y.Y., Baksh, S., Michalak, M. & Opas, M. Calreticulin inhibits commitment to adipocyte differentiation. Journal of Cell Biology 182, 103-116(2008).
20. Yin, Z.Z. & Frasch, M. Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila. Developmental Genetics 22, 187-200 (1998).
21. Yamada, M., Revelli, J.P., Eichele, G., Barron, M. & Schwartz, R.J. Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: Evidence for BMP2 induction of Tbx2. Developmental Biology 228, 95-105 (2000).
22. Kim, Y.Y. et al. Use of Long-tenn Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2. Yonsei Medical Journal 49, 819-827 (2008).
23. Lowell, B.B. PPAR gamma: An essential regulator of adipogenesis and modulator of fat cell function. Cell 99,239-242 (1999).
24. de Caestecker, M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev 15,1-11 (2004).
25. Schneider, M.D., Gaussin, V. & Lyons, K.M. Tempting fate: BMP signals for cardiac morphogenesis. Cytokine Growth Factor Rev 14,1-4 (2003).
26. Sachinidis, A., Kolossov, E., Fleischmann, B.K. & Hescheler, J. Generation of cardiomyocytes from embryonic stem cells experimental studies. Herz 27, 589-97 (2002).
27. Nohe, A. et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 277, 5330-8 (2002).
28. Nohe, A., Keating, E., Knaus, P. & Petersen, N.O. Signal transduction of bone morphogenetic protein receptors. Cell Signal 16,291-9 (2004).
29. Fukuda, S. et al. Potentiation of astrogliogenesis by STAT3-mediated activation of bone morphogenetic protein-Smad signaling in neural stem cells. Molecular and Cellular Biology 27,4931-4937 (2007).
30. Tong, Q. et al. Function of GATA transcription factors in preadipocyte-adipocyte transition. Science 290,134-138 (2000).
31. Hata, A. et al. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100,229-240 (2000).
32. Yoshida, Y. et al. Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell 103,1085-1097 (2000).
33. Hild, M. et al. The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo. Development 126, 2149-2159(1999).
34. Nakashima, K. et al. The novel zinc finger-containing transcription factor Osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29(2002).
35. Hoodless, P.A. et al. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85,489-500 (1996).
36. Zwijsen, A., Verschueren, K. & Huylebroeck, D. New Intracellular components of bone morphogenetic protein/Smad signaling cascades. FEBS Lett 546,133-9(2003).
37. Suzawa, M. et al. Cytokines suppress adipogenesis and PPAR-function through the TAK1/TAB1/NIK cascade. Nature Cell Biology 5, 224-230 (2003).
38. Paling, N.R.D., Wheadon, H., Bone, H.K. & Welham, M.J. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. Journal of Biological Chemistry 279,48063-48070 (2004).
39. Fujita, T. et al. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. Journal of Cell Biology 166, 85-95 (2004).
40. Ge, C.X., Xiao, G.Z., Jiang, D. & Franceschi, R.T. Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. Journal of Cell Biology 176, 709-718(2007).
41. Scheid, M.P. & Woodgett, J.R. Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett 546,108-12 (2003).
42. Ghosh-Choudhury, N., Abboud, S.L., Mahimainathan, L., Chandrasekar, B. & Choudhury, G.G. Phosphatidylinositol 3-kinase regulates bone morphogenetic protein-2 (BMP-2)-induced myocyte enhancer factor 2A-dependent transcription of BMP-2 gene in cardiomyocyte precursor cells. J Biol Chem 278,21998-2005(2003).
43. van der Heyden, M.A. & Defize, L.H. Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc Res 58, 292-302 (2003).
44. Eriksson, M. & Leppa, S. Mitogen-activated protein kinases and activator protein 1 are required for proliferation and cardiomyocyte differentiation of P19 embryonal carcinoma cells. J Biol Chem 277, 15992-6001 (2002).
45. Muller, B., Hartmann, B., Pyrowolakis, G., Affolter, M. & Basler, K. Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. Cell 113, 221-233 (2003).
46. Flores, M.V.C., Lam, E.Y.N., Crosier, K.E. & Crosier, P.S. Osteogenic transcription factor Runx2 is a maternal determinant of dorsoventral patterning in zebrafish. Nature Cell Biology 10, 346-U80 (2008).
47. Cohen, M.M. Bone morphogenetic proteins with some comments on fibrodysplasia ossificans progressiva and NOGGIN. American Journal of Medical Genetics 109, 87-92 (2002).
48. Chang, C.B. & Hemmati-Brivanlou, A. Xenopus GDF6, a new antagonist of noggin and a partner of BMPs. Development 126, 3347-3357 (1999).
49. Wylie, C. et al. Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos. Development 122,2987-2996 (1996).
50. Pashmforoush, M. et al. Nkx2-5 pathways and congenital heart disease: Loss of ventricular myocyte lineage progressive cardiomyopathy and complete heart block. Cell 117, 373-386 (2004).
51. Morrisey, E.E. et al. GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes & Development 12, 3579-3590 (1998).
52. Molkentin, J.D., Lin, Q., Duncan, S.A. & Olson, E.N. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes & Development 11,1061-1072 (1997).
53. Moskowitz, I.P.G.et al. A molecular pathway including Id2, Tbx5, and Nkx2-5 required for cardiac conduction system development. Cell 129, 1365-1376 (2007).
54. Shin, C.H. et al. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell 110, 725-735 (2002).
55. Ericson, J. et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded shh signaling. Cell 90,169-180 (1997).
56. Lyons, I. et al. Myogenic and Morphogenetic Defects in the Heart Tubes of Murine Embryos Lacking the Homeo Box Gene Nkx2-5. Genes & Development 9, 1654-1666(1995).
57. Kasahara, H. et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. Journal of Clinical Investigation 106, 299-308 (2000).
58. Schott, J.J. et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108-111 (1998).
59. Schott, J.J. et al. Mutations in the transcription factor Nkx2.5 cause secundum atrial septal defect and atrioventricular block. Circulation 98, 3965 (1998).
60. Schott, J.J. et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281,108-11 (1998).
61. Crispino, J.D. et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes & Development 15, 839-844 (2001).
62. Bisping, E. et al. Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure. Proc NatlAcadSci USA 103, 14471-6 (2006).
63. Heikinheimo, M., Scandrett, J.M. & Wilson, D.B. Localization of transcription factor GATA-4 to regions of the mouse embryo involved in cardiac development. Dev Biol 164, 361-73 (1994).
64. Drissi, M.H. et al. Runx2/Cbfal stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smadl on the collagen X promoter in chondrocytes. Journal of Cellular Biochemistry 90,1287-1298 (2003).
65. Stock, M. & Otto, F. Control of RUNX2 isoform expression: the role of promoters and enhancers. J Cell Biochem 95, 506-17 (2005).
66. Gersbach, C.A., Byers, B.A., Pavlath, G.K. & Garcia, A.J. Runx2/Cbfal stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Exp Cell Res 300,406-17 (2004).
67. Enomoto, H. et al. Runx2 deficiency in chondrocytes causes adipogenic changes in vitro. J Cell Sci 117, 417-25 (2004).
68. Issemann, I. & Green, S. Activation of a Member of the Steroid-Hormone Receptor Superfamily by Peroxisome Proliferators. Nature 347, 645-650 (1990).
69. Fajas, L. et al. The Organization, Promoter Analysis, and Expression of the Human PPARgamma Gene. J. Biol. Chem. 272, 18779-18789 (1997).
70. Li, X.N. et al. CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation. Journal of Cell Biology 181, 959-972 (2008).
71. Tontonoz, P., Hu, E. & Spiegelman, B.M. Stimulation of adipogenesis in fibroblasts by PPAR-gamma-2, a lipid-activated transcription factor. Cell 79, 1147-1156(1994).
72. Lehrke, M. & Lazar, M.A. The many faces of PPAR gamma. Cell 123, 993-999 (2005).
73. Hu, E., Liang, P. & Spiegelman, B.M. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271, 10697-703 (1996).
74. Rosen, E.D. et al. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell 4, 611-7 (1999).
75. Blanquart, C., Barbier, O., Fruchart, J.C., Staels, B. & Glineur, C. Peroxisome proliferator-activated receptors: regulation of transcriptional activities and roles in inflammation. J Steroid Biochem Mol Biol 85,267-73 (2003).
76. Guerre-Millo, M. Extending the glucose/fatty acid cycle: a glucose/adipose tissue cycle. Biochem Soc Trans 31,1161-4 (2003).
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