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Characterization of SMAD3 Gene Variants for Possible Roles in Ventricular Septal Defects and Other Congenital Heart Diseases

  • Fei-Feng Li ,

    Contributed equally to this work with: Fei-Feng Li, Jing Zhou

    Affiliation Genomics Research Center (one of the State-Province Key Laboratory of Biopharmaceutical Engineering, China), Harbin Medical University, Harbin, China

  • Jing Zhou ,

    Contributed equally to this work with: Fei-Feng Li, Jing Zhou

    Affiliation Intensive Care Unit, The Second Affiliated Hospital of Harbin Medical University, Harbin, China

  • Dan-Dan Zhao,

    Affiliation Genomics Research Center (one of the State-Province Key Laboratory of Biopharmaceutical Engineering, China), Harbin Medical University, Harbin, China

  • Peng Yan,

    Affiliation Department of Colorectal Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin, China

  • Xia Li,

    Affiliation Genomics Research Center (one of the State-Province Key Laboratory of Biopharmaceutical Engineering, China), Harbin Medical University, Harbin, China

  • Ying Han,

    Affiliation Genomics Research Center (one of the State-Province Key Laboratory of Biopharmaceutical Engineering, China), Harbin Medical University, Harbin, China

  • Xian-Shu Li,

    Affiliation Daqing People’s Hospital, Daqing, China

  • Gui-Yu Wang ,

    guiywang@gmail.com (GYW); drkaijiang@163.com (KJY); slliu@ucalgary.ca (SLL)

    Affiliation Department of Colorectal Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin, China

  • Kai-Jiang Yu ,

    guiywang@gmail.com (GYW); drkaijiang@163.com (KJY); slliu@ucalgary.ca (SLL)

    Affiliation Intensive Care Unit, The Second Affiliated Hospital of Harbin Medical University, Harbin, China

  • Shu-Lin Liu

    guiywang@gmail.com (GYW); drkaijiang@163.com (KJY); slliu@ucalgary.ca (SLL)

    Affiliations Genomics Research Center (one of the State-Province Key Laboratory of Biopharmaceutical Engineering, China), Harbin Medical University, Harbin, China, Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Canada

Characterization of SMAD3 Gene Variants for Possible Roles in Ventricular Septal Defects and Other Congenital Heart Diseases

  • Fei-Feng Li, 
  • Jing Zhou, 
  • Dan-Dan Zhao, 
  • Peng Yan, 
  • Xia Li, 
  • Ying Han, 
  • Xian-Shu Li, 
  • Gui-Yu Wang, 
  • Kai-Jiang Yu, 
  • Shu-Lin Liu
PLOS
x

Abstract

Background

Nodal/TGF signaling pathway has an important effect at early stages of differentiation of human embryonic stem cells in directing them to develop into different embryonic lineages. SMAD3 is a key intracellular messenger regulating factor in the Nodal/TGF signaling pathway, playing important roles in embryonic and, particularly, cardiovascular system development. The aim of this work was to find evidence on whether SMAD3 variations might be associated with ventricular septal defects (VSD) or other congenital heart diseases (CHD).

Methods

We sequenced the SMAD3 gene for 372 Chinese Han CHD patients including 176 VSD patients and evaluated SNP rs2289263, which is located before the 5’UTR sequence of the gene. The statistical analyses were conducted using Chi-Square Tests as implemented in SPSS (version 13.0). The Hardy-Weinberg equilibrium test of the population was carried out using the online software OEGE.

Results

Three heterozygous variants in SMAD3 gene, rs2289263, rs35874463 and rs17228212, were identified. Statistical analyses showed that the rs2289263 variant located before the 5’UTR sequence of SMAD3 gene was associated with the risk of VSD (P value=0.013 <0.05).

Conclusions

The SNP rs2289263 in the SMAD3 gene is associated with VSD in Chinese Han populations.

Introduction

Congenital heart diseases (CHD) include a series of congenital anatomic malformations, such as pulmonary stenosis, tetralogy of Fallot, patent ductus arteriosus, mitral valve insufficiency, atrial septal and ventricular septal defects etc. [1], which may be complicated by arrhythmias or heart failure and increase the risks of coronary heart diseases [2]. The clinical characteristics of CHDs are very complex, and the diseases have high morbidity and mortality. The incidence of all types of CHDs is about 7.5 present of newborns [3], many of which require clinical intervention (about 1%) [4]. Of all CHD patients, only about 13% are reported with chromosomal variants [5], so currently surgery is still the main treatment for CHD [6]. Although many genetic defects have been revealed in many familiar and sporadic CHD cases by extensive genetic studies [7, 8], the relationships between genetic abnormalities and CHD etiology remain largely unknown.

Ventricular septal defects (VSDs) account for about 40% of CHDs [1, 4]. The prevalence of VSDs varies in different studies due presumably to differences in diagnostic methods and age of participants [911]. VSDs may also be associated with other structural cardiac defects or syndromes, such as aortic coarctation or interruption, tetralogy of Fallot, univentricular atrioventricular connection and Down syndrome [12, 13]. Recently the prevalence of VSDs is increased in newborns due to changes in diagnosis and screening modalities such the use of fetal echocardiography [14, 15].

The heart is among the first formed organs during the embryogenesis and its formation is strictly controlled by gene regulatory networks consisting of many signaling pathways, transcription factors, epigenetic factors, and miRNAs [16, 17]. A large number of defects in genes coding for these factors have been identified [1, 18]. Recently, we reported that SNP rs2295418 in the Lefty2 gene and genotype frequency of rs360057 in Lefty1 gene are associated with the risk of CHD [1]. LEFTY is a crucial transforming growth negative regulation factor in the Nodal/TGF-βsignaling pathway [19], which inhibits the cellular proliferation and differentiation [20, 21]. Importantly, the Nodal/TGF-βsignaling pathway has an important effect in early stages of differentiation of the human embryonic stem (HES) cells, directing them to develop into different embryonic lineages, and errors in the transformation may occur if the pathway has malfunctions [2224].

The HES cells differentiate to various cell types, which develop to ectoderm, endoderm and mesoderm; generation and differentiation of cardiomyocytes and muscle cells take place in the mesoderm [25]. As a key intracellular regulating factor in the Nodal/TGF-βsignaling pathway, SMAD family member 3 (SMAD3) activates or represses gene transcription, thus having important effects on embryonic development that will influence the formation of the cardiovascular system [26, 27]. At the same time, some authors also suggest that Nodal/TGF-βsignaling pathway plays a key roles in the embryogenesis of the heart, valvular pathogenesis and organization of the aortic wall; when activities of the signaling pathway were disrupted, CHDs ensued in animal studies [28]. For elucidation of the mechanisms, the SMAD3 gene knockdown mice could be used as models [29].

To validate possible associations of Smad3 with VSD or other CHDs, we analyzed the transcribed region and splicing sites of the gene and compared the gene sequences between 372 Chinese Han CHD patients (including 176 VSD patients) and 456 controls. We found that the rs2289263 variant before the 5’UTR of the Smad3 gene was closely associated with the risk of VSD but not with the other CHDs.

Results

Patients

We confirmed the clinical diagnosis of all the recruited patients in Linyi people’s Hospital, the second Affiliated Hospital and the fourth Affiliated Hospital of Harbin Medical University. The CHD patients had no history or manifestations of any other systemic abnormalities. We established that their mothers did not have a history of taking medicines or attracting infections during gestation, as those factors have been shown to be associated with heart malformation in pregnancy [30, 31].

The 372 CHD patients contained 176 with ventricular septal defects (VSD), 14 with tetralogy of Fallot, 12 with pulmonary stenosis, 25 with patent ductus arteriosus, 22 with mitral valve insufficiency, 53 with atrial septal defects, and 70 with other complex congenital heart defects. All the CHD patients (n = 372, male 201, female 171, the min and max age were 0.2 and 74 respectively, and the average age was 15.22 years) and unrelated controls (n = 456, male 257, female 199, the min and max age were 0.25 and 41 respectively, and the average age was 14.66 years) were recruited for this study, and there were no statistical differences of the gender composition or age between the two groups (Table 1).

SMAD3 gene analysis

We sequenced the SMAD3 gene to test the hypothesis that germline common genetic variants in SMAD3 may confer susceptibility to CHD. We first compared the transcribed region and splicing sites of SMAD3 and found the variation rs35874463 was located within the translated region and rs17228212 was located within an intron of the SMAD3 gene, but the genetic heterozygosity of the two SNP were very low (S1 Table). The variation rs2289263 was located before 5’UTR of the gene, but its genetic heterozygosity was very high (Fig 1A).

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Fig 1. Schematic diagrams and DNA sequence chromatograms.

A: Schematic diagrams of rs2289263, rs35874463 and rs17228212 locations in the SMAD3 gene; B: Three genotypes of DNA sequence chromatograms of rs2289263.

https://doi.org/10.1371/journal.pone.0131542.g001

SNP rs2289263 genotyping statistical analysis

To further test any possible associations between SMAD3 and CHD, we conducted SNP analyses and found that the variant rs2289263 before 5’UTR of SMAD3 gene was associated with the risk of VSD in the Chinese Han population but not with the other CHDs (Tables 2 and 3). At the same time, Hardy-Weinberg equilibrium test for the CHD and controls were conducted and it was in line with the equilibrium.

thumbnail
Table 2. The genotype and allele frequency of SNP rs2289263 in 372 CHD patients, 176 VSD patients and 456 non-CHD controls.

https://doi.org/10.1371/journal.pone.0131542.t002

thumbnail
Table 3. SNP rs2289263 before 5’UTR of SMAD3 gene associated with the risk of ventricular septal defect not congenital heart diseases in Chinese populations.

https://doi.org/10.1371/journal.pone.0131542.t003

Discussion

In this study, we analyzed the transcribed regions and splicing sites of the SMAD3 gene in a large cohort of CHD patients and controls and found that the variant rs2289263 in the SMAD3 gene was associated with the risk of VSD in the Chinese Han population, demonstrating the involvement of the SMAD3 gene in the VSD etiology.

Eighteen or nineteen days after fertilization, the human heart starts to form in the mesoderm, and the formation involves strict temporal, spatial, and sequential expression of genes [1]. Nodal/TGF-βsignaling pathway plays a key role during the mammal gastrulation to produce progenitor cells of the mesendoderm [32]. In this process, the expression level of Nodal/TGF-βsignaling pathway can affect the formation of mesendoderm [33].

The mesendoderm progenitor cells form the primitive streak and mutations in the Nodal gene can affect the formation. In mice, the vascular systems arise from extraembryonic mesoderm and migrate through the primitive streak to the presumptive yolk sac [34]. The Nodal gene expression initiates a series of signal transduction and induces some gene and its own expression in later stages of embryonic development [19, 32]. Animal studies also show that TGF-β can induce cardiac fibroblasts proliferation, myocardial fibrosis and cardiomyocytes hypertrophic growth [35], and loss of responsiveness to TGF-βmay lead to fibrosis progresses in the atrial fibrogenesis [14, 36]. Furthermore, many lines of evidence from animal research suggest that TGF-βsignaling is essential in the cardiogenesis, valvular pathogenesis, and organization of the aortic wall [28, 37], and disrupted TGF-βsignaling activities may lead to congenital heart defects [28].

We analyzed genes of the Nodal/TGF-βsignaling pathway, as they have been demonstrated to play vital roles in mesoderm differentiation and heart formation [32]. In this work, we found that the variant rs2289263 before the 5’UTR of SMAD3 gene was associated with increased risk of VSD in the Chinese Han population, while in a previous study, we demonstrated that rs2295418 (g.C925A) in Lefty2 gene is associated with the risk of CHD [1]. Of great significance, LEFTY and SMAD3 both play central roles in the Nodal/TGF-βsignaling pathway, with LEFTY negatively regulating the Nodal/TGF signaling pathway and SMAD3 defects being associated with cardiovascular diseases [26, 29]. To our knowledge, this is the first report showing the association of SMAD3 gene with VSD or other congenital heart defects. Further work will be needed on the the Nodal/TGF-βsignaling pathway genes such as LEFTY and SMAD3 for their involvement in the pathogenesis of CHD at the molecular level.

Materials and Methods

The study population

From Linyi People’s Hospital, the second Affiliated Hospital and the fourth Affiliated Hospital of Harbin Medical University, Harbin, China, we collected specimens of 372 CHD patients including 176 with VSDs for this study. The 456 controls with no reported cardiac phenotypes were also recruited for this study from the Medical Examination Center of the Second Affiliated Hospital of Harbin Medical University (Table 1). All the CHD subjects and controls received comprehensive physical examination, electrocardiogram and ultrasonic echocardiogram examinations. None of the patients showed any other abnormalities in the heart or other body parts and the control members did not have any defects in the heart. From each participant or their parents on behalf of minors, we obtained a written informed consent, and the Ethics Committee of the Harbin Medical University approved this work, consistent with the 1975 Declaration of Helsinki.

DNA analysis

We used standard protocols to extract genomic DNA from the peripheral blood leukocytes of the participants. The human SMAD3 gene consisting of nine exons is located on 15q21-22. Using two stage methods, we determined the SNP genotypes in the SMAD3 gene. First, the nine exons and the splicing sites of the gene were amplified using polymerase chain reaction (PCR) method (Table 4), and the products were sequenced using standard protocols [38]. After that, the genotypes of the SNP were determined using PCR and gene sequencing methods [1].

Rs2289263 SMAD3 SNP genotyping analysis and statistical methods

Using two stage methods, we determined genotypes of the rs2289263, rs35874463, and rs17228212 SNP of the SMAD3 gene (Fig 1B). All the measurements were conducted by two independent researchers (Table 5). And then overall CHD meta-analysis was conducted and stratified analysis was carried out according to the types of CHD and sample sizes.

The continuous variable (measurement data, such as age) statistical analyses were conducted using independent-samples T test and the discrete variable (enumeration data, such as gender composition and genotype frequency) statistical analyses were conducted using Chi-Square Tests to calculate odds ratios and P value as implemented in SPSS (version 19.0). P values less than 0.05 were considered statistically significant. The Hardy-Weinberg equilibrium test of the CHD and control population was conducted with the online software OEGE.

Supporting Information

S1 Table. The genotype and allele frequency of SNP rs35874463 and rs17228212 in 372 CHD patients and 456 non-CHD controls.

https://doi.org/10.1371/journal.pone.0131542.s001

(DOC)

Acknowledgments

The authors thank the patients and family members for their cooperation and participation in this study; the physicians for the specimens collection and clinical examinations.

Author Contributions

Conceived and designed the experiments: FFL SLL. Performed the experiments: JZ DDZ PY XL YH. Analyzed the data: FFL GYW KJY JZ. Contributed reagents/materials/analysis tools: FFL GYW KJY JZ XSL. Wrote the paper: FFL SLL. Specimen collection: JZ KJY XL YH XSL.

References

  1. 1. Deng X, Zhou J, Li FF, Yan P, Zhao EY, Hao L, et al. Characterization of Nodal/TGF-Lefty Signaling Pathway Gene Variants for Possible Roles in Congenital Heart Diseases. PLoS One. 2014;9(8):e104535. Epub 2014/08/12. PONE-D-14-16633 [pii]. pmid:25111179; PubMed Central PMCID: PMC4128709.
  2. 2. van der Bom T, Zomer AC, Zwinderman AH, Meijboom FJ, Bouma BJ, Mulder BJ. The changing epidemiology of congenital heart disease. Nat Rev Cardiol. 2011;8(1):50–60. Epub 2010/11/04. nrcardio.2010.166 [pii]. pmid:21045784.
  3. 3. Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Coll Cardiol. 2002;39(12):1890–900. Epub 2002/06/27. S0735109702018867 [pii]. pmid:12084585.
  4. 4. Hoffman JI, Kaplan S, Liberthson RR. Prevalence of congenital heart disease. Am Heart J. 2004;147(3):425–39. Epub 2004/03/05. S0002870303007294 [pii]. pmid:14999190.
  5. 5. Pierpont ME, Basson CT, Benson DW Jr, Gelb BD, Giglia TM, Goldmuntz E, et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation. 2007;115(23):3015–38. Epub 2007/05/24. CIRCULATIONAHA.106.183056 [pii] pmid:17519398.
  6. 6. Verheugt CL, Uiterwaal CS, van der Velde ET, Meijboom FJ, Pieper PG, van Dijk AP, et al. Mortality in adult congenital heart disease. Eur Heart J. 2010;31(10):1220–9. Epub 2010/03/09. ehq032 [pii]. pmid:20207625.
  7. 7. Bruneau BG. The developmental genetics of congenital heart disease. Nature. 2008;451(7181):943–8. Epub 2008/02/22. nature06801 [pii]. pmid:18288184.
  8. 8. Richards AA, Garg V. Genetics of congenital heart disease. Curr Cardiol Rev. 2010;6(2):91–7. Epub 2011/05/03. pmid:21532774; PubMed Central PMCID: PMC2892081.
  9. 9. Gelb BD. Recent advances in understanding the genetics of congenital heart defects. Curr Opin Pediatr. 2013. Epub 2013/09/03. https://doi.org/10.1097/MOP.0b013e3283648826 pmid:23995429; PubMed Central PMCID: PMC4049978.
  10. 10. Zaidi S, Choi M, Wakimoto H, Ma L, Jiang J, Overton JD, et al. De novo mutations in histone-modifying genes in congenital heart disease. Nature. 2013;498(7453):220–3. Epub 2013/05/15. nature12141 [pii]. pmid:23665959; PubMed Central PMCID: PMC3706629.
  11. 11. Fahed AC, Gelb BD, Seidman JG, Seidman CE. Genetics of congenital heart disease: the glass half empty. Circ Res. 2013;112(4):707–20. Epub 2013/02/16. 112/4/707 [pii]. pmid:23410880; PubMed Central PMCID: PMC3827691.
  12. 12. Erol O, Sevket O, Keskin S, Yazicioglu HF, Gul A. Natural history of prenatal isolated muscular ventricular septal defects. J Turk Ger Gynecol Assoc. 2014;15(2):96–9. Epub 2014/07/01. jtgga-15-2-96 [pii]. pmid:24976775; PubMed Central PMCID: PMC4072558.
  13. 13. Sharma M, Khera S, Sondhi V, Devgan A. A study to determine the prevalence of pulmonary arterial hypertension in children with Down syndrome and congenital heart disease. Med J Armed Forces India. 2013;69(3):241–5. Epub 2014/03/07. S0377-1237(12)00262-6 [pii]. pmid:24600117; PubMed Central PMCID: PMC3862969.
  14. 14. Khan R, Sheppard R. Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia. Immunology. 2006;118(1):10–24. Epub 2006/04/25. IMM2336 [pii] pmid:16630019; PubMed Central PMCID: PMC1782267.
  15. 15. Dolk H, Loane M, Garne E. Congenital heart defects in Europe: prevalence and perinatal mortality, 2000 to 2005. Circulation. 2011;123(8):841–9. Epub 2011/02/16. CIRCULATIONAHA.110.958405 [pii]. pmid:21321151.
  16. 16. Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 2005;6(11):826–35. Epub 2005/11/24. nrg1710 [pii] pmid:16304598.
  17. 17. van Weerd JH, Koshiba-Takeuchi K, Kwon C, Takeuchi JK. Epigenetic factors and cardiac development. Cardiovasc Res. 2011;91(2):203–11. Epub 2011/05/25. cvr138 [pii]. pmid:21606181; PubMed Central PMCID: PMC3125076.
  18. 18. Gong W, Gottlieb S, Collins J, Blescia A, Dietz H, Goldmuntz E, et al. Mutation analysis of TBX1 in non-deleted patients with features of DGS/VCFS or isolated cardiovascular defects. J Med Genet. 2001;38(12):E45. Epub 2001/12/19. pmid:11748311; PubMed Central PMCID: PMC1734783.
  19. 19. Tabibzadeh S, Hemmati-Brivanlou A. Lefty at the crossroads of "stemness" and differentiative events. Stem Cells. 2006;24(9):1998–2006. Epub 2006/05/27. 2006–0075 [pii] pmid:16728558.
  20. 20. Ikushima H, Miyazono K. TGFbeta signalling: a complex web in cancer progression. Nat Rev Cancer. 2010;10(6):415–24. Epub 2010/05/25. nrc2853 [pii]. pmid:20495575.
  21. 21. Heldin CH, Landstrom M, Moustakas A. Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr Opin Cell Biol. 2009;21(2):166–76. Epub 2009/02/25. S0955-0674(09)00019-2 [pii]. pmid:19237272.
  22. 22. Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, et al. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci U S A. 2008;105(11):4329–34. Epub 2008/03/13. 0800467105 [pii]. pmid:18334633; PubMed Central PMCID: PMC2393795.
  23. 23. Costa FF, Seftor EA, Bischof JM, Kirschmann DA, Strizzi L, Arndt K, et al. Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment. Epigenomics. 2009;1(2):387–98. Epub 2010/05/25. pmid:20495621; PubMed Central PMCID: PMC2872497.
  24. 24. Malchenko S, Galat V, Seftor EA, Vanin EF, Costa FF, Seftor RE, et al. Cancer hallmarks in induced pluripotent cells: new insights. J Cell Physiol. 2010;225(2):390–3. Epub 2010/06/23. pmid:20568225; PubMed Central PMCID: PMC3180883.
  25. 25. Schuldiner M, Benvenisty N. Factors controlling human embryonic stem cell differentiation. Methods Enzymol. 2003;365:446–61. Epub 2003/12/31. pmid:14696364.
  26. 26. van de Laar IM, Oldenburg RA, Pals G, Roos-Hesselink JW, de Graaf BM, Verhagen JM, et al. Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis. Nat Genet. 2011;43(2):121–6. Epub 2011/01/11. ng.744 [pii]. pmid:21217753.
  27. 27. Liying J, Yuchun T, Youcheng W, Yingchen W, Chunyu J, Yanling Y, et al. A SMAD3 gene polymorphism is related with osteoarthritis in a Northeast Chinese population. Rheumatol Int. 2013;33(7):1763–8. Epub 2013/01/08. pmid:23292212.
  28. 28. Arthur HM, Bamforth SD. TGFbeta signaling and congenital heart disease: Insights from mouse studies. Birth Defects Res A Clin Mol Teratol. 2011;91(6):423–34. Epub 2011/05/04. pmid:21538815.
  29. 29. van der Linde D, van de Laar IM, Bertoli-Avella AM, Oldenburg RA, Bekkers JA, Mattace-Raso FU, et al. Aggressive cardiovascular phenotype of aneurysms-osteoarthritis syndrome caused by pathogenic SMAD3 variants. J Am Coll Cardiol. 2012;60(5):397–403. Epub 2012/05/29. S0735-1097(12)01238-7 [pii]. pmid:22633655.
  30. 30. van Driel LM, Smedts HP, Helbing WA, Isaacs A, Lindemans J, Uitterlinden AG, et al. Eight-fold increased risk for congenital heart defects in children carrying the nicotinamide N-methyltransferase polymorphism and exposed to medicines and low nicotinamide. Eur Heart J. 2008;29(11):1424–31. Epub 2008/04/29. ehn170 [pii]. pmid:18441319.
  31. 31. Kebed KY, Bishu K, Al Adham RI, Baddour LM, Connolly HM, Sohail MR, et al. Pregnancy and Postpartum Infective Endocarditis: A Systematic Review. Mayo Clin Proc. 2014;89(8):1143–52. Epub 2014/07/06. S0025-6196(14)00388-7 [pii] pmid:24997091.
  32. 32. Dvash T, Sharon N, Yanuka O, Benvenisty N. Molecular analysis of LEFTY-expressing cells in early human embryoid bodies. Stem Cells. 2007;25(2):465–72. Epub 2006/10/14. 2006–0179 [pii] pmid:17038673.
  33. 33. Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol. 2003;19:589–621. Epub 2003/10/23. pmid:14570583.
  34. 34. Barroso-delJesus A, Lucena-Aguilar G, Sanchez L, Ligero G, Gutierrez-Aranda I, Menendez P. The Nodal inhibitor Lefty is negatively modulated by the microRNA miR-302 in human embryonic stem cells. FASEB J. 2011;25(5):1497–508. Epub 2011/01/27. fj.10-172221 [pii]. pmid:21266536.
  35. 35. Rosenkranz S. TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res. 2004;63(3):423–32. Epub 2004/07/28. S0008636304002020 [pii]. pmid:15276467.
  36. 36. Gramley F, Lorenzen J, Koellensperger E, Kettering K, Weiss C, Munzel T. Atrial fibrosis and atrial fibrillation: the role of the TGF-beta1 signaling pathway. Int J Cardiol. 2010;143(3):405–13. Epub 2009/04/28. S0167-5273(09)00396-9 [pii]. pmid:19394095.
  37. 37. Armstrong EJ, Bischoff J. Heart valve development: endothelial cell signaling and differentiation. Circ Res. 2004;95(5):459–70. Epub 2004/09/04. [pii]. pmid:15345668; PubMed Central PMCID: PMC2810618.
  38. 38. Tan ZX, Li FF, Qu YY, Liu J, Liu GR, Zhou J, et al. Identification of a known mutation in Notch 3 in familiar CADASIL in China. PLoS One. 2012;7(5):e36590. Epub 2012/05/25. PONE-D-11-24676 [pii]. pmid:22623959; PubMed Central PMCID: PMC3356370.