Browse > Article
http://dx.doi.org/10.14348/molcells.2014.0003

Thymosin Beta4 Regulates Cardiac Valve Formation Via Endothelial-Mesenchymal Transformation in Zebrafish Embryos  

Shin, Sun-Hye (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University)
Lee, Sangkyu (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University)
Bae, Jong-Sup (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University)
Jee, Jun-Goo (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University)
Cha, Hee-Jae (Department of Parasitology and Genetics, Kosin University College of Medicine)
Lee, You Mie (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University)
Publication Information
Molecules and Cells / v.37, no.4, 2014 , pp. 330-336 More about this Journal
Abstract
Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelial-mesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor ${\beta}$ ($TGF{\beta}$) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-${\beta}$-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT.
Keywords
endothelial-mesenchymal transformation; heart valve formation; thymosin beta4; zebrafish;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Armstrong, E.J., and Bischoff, J. (2004). Heart valve development: endothelial cell signaling and differentiation. Circ. Res. 95, 459-470.   DOI   ScienceOn
2 Bakkers, J. (2011). Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc. Res. 91, 279-288.   DOI   ScienceOn
3 Bakkers, J., Verhoeven, M.C., and Abdelilah-Seyfried, S. (2009). Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis. Dev. Biol. 330, 213-220.   DOI   ScienceOn
4 Beis, D., Bartman, T., Jin, S.W., Scott, I.C., D'mico, L.A., Ober, E.A., Verkade, H., Frantsve, J., Field, H.A., Wehman, A., et al. (2005). Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development. Development 132, 4193-4204.   DOI   ScienceOn
5 Bock-Marquette, I., Saxena, A., White, M.D., DiMaio, J.M., and Srivastava, D. (2004). Thymosin ${\beta}4$ activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466-472.   DOI   ScienceOn
6 Derynck, R., and Zhang, Y.E. (2003). Smad-dependent and Smadindependent pathways in TGF-beta family signalling. Nature 425, 577-584.   DOI   ScienceOn
7 Dube, K.N., Bollini, S., Smart, N., and Riley, P.R. (2012). Thymosin beta4 protein therapy for cardiac repair. Curr. Pharm. Des. 18, 799-806.   DOI
8 Goldstein, A.L., Hannappel, E., Sosne, G., and Kleinman, H.K. (2012). Thymosin ${\beta}4$: a multi-functional regenerative peptide. Basic properties and clinical applications. Exp. Opin. Biol. Ther. 12, 37-51.   DOI   ScienceOn
9 Gomez-Marquez, J., Franco del Amo, F., Carpintero, P., and Anadon, R. (1996). High levels of mouse thymosin ${\beta}4$ mRNA in differentiating P19 embryonic cells and during evelopment of cardiovascular tissues. Biochim. Biophys. Acta 1306, 187-193.   DOI   ScienceOn
10 Ji, Y.I., Lee, B.Y., Kang, Y.J., Jo, J.O., Lee, S.H., Kim, H.Y., Kim, Y.O., Lee, C., Koh, S.B., Kim, A., et al. (2013). Expression patterns of Thymosin ${\beta}4$ and cancer stem cell marker CD133 in ovarian cancers. Pathol. Oncol. Res. 19, 237-245.   DOI   ScienceOn
11 Huang, C.J., Tu, C.T., Hsiao, C.D., Hsieh, F.J., and Tsai, H.J. (2003). Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn. 228, 30-40.   DOI   ScienceOn
12 Jang, G.H., Park, I.S., Yang, J.H., Bischoff, J., and Lee, Y.M. (2010). Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells. FEBS Lett. 584, 141-146.   DOI   ScienceOn
13 Jin, S.W., Beis, D., Mitchell, T., Chen, J.N., and Stainier, D.Y.R. (2005). Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199-5209.   DOI   ScienceOn
14 Lee, S.I., Kim, D.S., Lee, H.J., Cha, H.J., and Kim, E.C. (2013). The role of thymosin beta 4 on odontogenic differentiation in human dental pulp cells. PLoS One 8, e61960.   DOI
15 Johnson, E.N., Lee, Y.M., Sander, T.L., Rabkin, E., Schoen, F.J., Kaushal, S., and Bischoff, J. (2003). NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells. J. Biol. Chem. 278, 1686-1692.   DOI   ScienceOn
16 Lee, Y.M., Cope, J.J., Ackermann, G.E., Goishi, K., Armstrong, E.J., Paw, B.H., and Bischoff, J. (2006). Vascular endothelial growth factor receptor signaling is required for cardiac valve formation in zebrafish. Dev. Dyn. 235, 29-37.   DOI   ScienceOn
17 Malinda, K.M., Sidhu, G.S., Mani, H., Banaudha, K., Maheshwari, R.K., Goldstein, A.L., and Kleinman, H.K. (1999). Thymosin ${\beta}4$ accelerates wound healing. J. Invest. Dermatol. 113, 364-368.   DOI   ScienceOn
18 Li, Q., Jones, P., Lafferty, R., Safer, D., and Levy, R. (2002). Thymosin beta4 regulation, expression and function in aortic valve interstitial cells. J. Heart Valve Dis. 11, 726-735.
19 Malinda, K., Goldstein, A., and Kleinman, H. (1997). Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells. FASEB J. 11, 474-481.
20 Markwald, R.R., Fitzharris, T.P., and Manasek, F.J. (1977). Structural development of endocardial cushions. Am. J. Anat. 148, 85-119.   DOI   ScienceOn
21 Milan, D.J., Giokas, A.C., Serluca, F.C., Peterson, R.T., and MacRae, C.A. (2006). Notch1b and neuregulin are required for specification of central cardiac conduction tissue. Development 133, 1125-1132.   DOI   ScienceOn
22 Stainier, D.Y., Beis, D., Jungblut, B., and Bartman, T. (2002). Endocardial cushion formation in zebrafish. Cold Spring Harb. Symp. Quant. Biol. 67, 49-56.
23 Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S.J., Rabkin, E., Schoen, F.J., and Bischoff, J. (2001). Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro. Am. J. Pathol. 159, 1335-1343.   DOI   ScienceOn
24 Smart, N., Risebro, C.A., Melville, A.A., Moses, K., Schwartz, R.J., Chien, K.R., and Riley, P.R. (2006). Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization. Nature 445, 177-182.
25 Smart, N., Bollini, S., Dube, K.N., Vieira, J.M., Zhou, B., Davidson, S., Yellon, D., Riegler, J., Price, A.N., and Lythgoe, M.F. (2011). De novo cardiomyocytes from within the activated adult heart after injury. Nature 474, 640-644.   DOI   ScienceOn
26 Sosne, G., Szliter, E.A., Barrett, R., Kernacki, K.A., Kleinman, H., and Hazlett, L.D. (2002). Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury. Exp. Eye Res. 74, 293-299.   DOI   ScienceOn
27 Sosne, G., Xu, L., Prach, L., Mrock, L.K., Kleinman, H.K., Letterio, J.J., Hazlett, L.D., and Kurpakus-Wheater, M. (2004). Thymosin beta 4 stimulates laminin-5 production independent of TGF-beta. Exp. Cell Res. 293, 175-183.   DOI   ScienceOn
28 Sribenja, S., Wongkham, S., Wongkham, C., Yao, Q., and Chen, C. (2013). Roles and mechanisms of ${\beta}$-thymosins in cell migration and cancer metastasis: an update. Cancer Invest. 31, 103-110.   DOI   ScienceOn
29 Stankunas, K., Ma, G.K., Kuhnert, F.J., Kuo, C.J., and Chang, C.P. (2010). VEGF signaling has distinct spatiotemporal roles during heart valve development. Dev. Biol. 347, 325-336.   DOI   ScienceOn
30 Thatcher, J.E., Welch, T., Eberhart, R.C., Schelly, Z.A., and Michael DiMaio, J. (2012). Thymosin ${\beta}4$ sustained release from poly (lactide-co-glycolide) microspheres: synthesis and implications for treatment of myocardial ischemia. Ann. N Y Acad. Sci. 1270, 112-119.   DOI   ScienceOn
31 Westerfield, M. (1993). The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio), (University of Oregon Press, USA).
32 Yang, J.H., Wylie-Sears, J., and Bischoff, J. (2008). Opposing actions of Notch1 and VEGF in post-natal cardiac valve endothelial cells. Biochem. Biophys. Res. Commun. 374, 512-516.   DOI   ScienceOn
33 Yelon, D., Horne, S.A., and Stainier, D.Y. (1999). Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev. Biol. 214, 23-37.   DOI   ScienceOn
34 Kim, S.H., Shin, J., Park, H.C., Yeo, S.Y., Hong, S.K., Han, S., Rhee, M., Kim, C.H., Chitnis, A.B., and Huh, T.L. (2002). Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish. Development 129, 4443-4455.
35 Bock-Marquette, I., Shrivastava, S., Pipes, G., Thatcher, J.E., Blystone, A., Shelton, J.M., Galindo, C.L., Melegh, B., Srivastava, D., and Olson, E.N. (2009). Thymosin ${\beta}4$ mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo. J. Mol. Cell. Cardiol. 46, 728-738.   DOI   ScienceOn
36 Huff, T., Muller, C.S., Otto, A.M., Netzker, R., and Hannappel, E. (2001). ${\beta}$-Thymosins, small acidic peptides with multiple functions. Int. J. Biochem. Cell Biol. 33, 205-220.   DOI   ScienceOn