Myokines主要是由肌肉透過運動所分泌出來的cytokines，而在生物體中myokines扮演著重要的角色，例如、血管新生、組織再生和修復、維持健康的身體機能，免疫調節和胚胎發育的影響。而在2012年Boströmet al.發現了新myokine並且稱之為FNDC5。 FNDC5也被發現有誘使白色脂肪細胞棕色化並且進一步消耗熱量來達到減肥、減緩糖尿病病症的能力。在目前的研究中，並沒有明確的指出myokine。 FNDC5是否具有調節血管新生以及傷口癒合的功能，而我們透過大腸桿菌系統生產出來的FNDC5來觀測FNDC5對血管新生的功能並發現在內皮細胞中具有促進細胞增生、遷移與管柱形成之功能。此外，在大鼠主動脈環微血管及Tg(fli-1:EGFP)y1斑馬魚幼苗及傷口癒合中也都有明顯的效果。機制研究表示，在血管內皮細胞中，FNDC5具有促進血管內皮生長因子（VEGF）和血管內皮生長因子受體（VEGFR2）以及下游的p-Erk/Erk與pp38/p38。因此，本研究主要探討FNDC5誘導新血管內皮生長，並期許未來能對糖尿病患者的傷口癒合或是心臟病與中風的治療用藥進行發展。

Myokinesis a critical cytokines in organism major secretes from muscle cells after doing exercise. Myokines involved in angiogenesis, tissue regenerate and repair, preserved normal functions of body, affected immune modulation and develop of embryos. Boströmet al. found a new myokine and called FNDC5 in 2012. FNDC5 was founded possessing a ability that can induce White Adipose Tissue becoming Brown Adipose Tissue to lose weight and slow down the symptom of diabetes through increase burning calories. In the resent reports, there is no direct describe that whether FNDC5 can regulates angiogenesis and wound healing. Recombinant FNDC5 (Fibronectin type III domain-containing protein 5) was expressed and purified from E. coli with a molecular weight of 25 kDa. By using cultured endothelial cells, it was found that recombinant FNDC5 stimulated the proliferation, migration and tube formation of endothelial cells. Moreover, FNDC5 promotes vessels sprouting in aortic rings assay and in transgenic Tg(fli-1:EGFP)y1zebrafish. Finally, FNDC5 application enhanced wound healing in diabetic rats. Mechanism studies showed FNDC5 induced the expression of vascular the endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2/FLK-1) pathway and activated the downstream Erk and p38 signaling in endothelial cells. Therefore, this study unveils the novel pro-angiogenic the function and mechanism of FNDC5 in endothelial cells.

The authorization of oral member for research dissertation ………….i
Acknowledgement.……………………………………………………… .ii
Table of contents………………………………………………………….iii
Abstract in Chinese………...…………………………………………….iv
Abstract in English………………………………………………………..v
Table of contents………………………………………………………….vi
Introduction……………………………………………………………….1
Specific Aims………………………………………………………………6
Materials and Methods…………………………………………………...7
Results…………………………………………………………………….18
Purification of the recombinant FNDC5 protein………………….18
FNDC5 expression in endothelial, liver and muscle cells…………18
The stability of FNDC5 proteins at different temperatures………19
FNDC5 promoted multiple angiogenic processes in endothelial cell…………………………………………………………………….19
FNDC5 effected angiogenesis ex vivo and in vivo…………………20
Identification existed of FNDC5 by membrane, cytoplasm, and nucleus fractions……………………………………………………..21
FNDC5 induces VEGF protein level expression in endothelial cells……………………………………………………………………21
FNDC5 elevated the VEGFR2 expression and VEGFR2 phosphorylation protein level in endothelial cells………………….21
FNSDC5 regulated the VEGF/VEGFR2 through the Erk/p38 signal pathway……………………………………………………………….22
FNDC5 not effect NOS signal pathway in endothelial cells……….23
FNDC5 induced VEGF expression by NFκB in endothelial cells at transcriptional level………………………………………………….23
FNDC5 induces VEGF expression by HIF-1α in endothelial cells at transcriptional level………………………………………………….24
Discussion……………………………………………………………..25
Figures and legends…………………………………………………..29
References……………………………………………………………..52

1. Ajisaka, R. (2006) [Health maintenance and exercise training in the elderly]. Nihon Ronen Igakkai zasshi. Japanese journal of geriatrics 43, 585-587
2. Seifi, T., Ghaedi, K., Tanhaei, S., Karamali, F., Kiani-Esfahani, A., Peymani, M., Baharvand, H., and Nasr-Esfahani, M. H. (2014) Identification, Cloning, and Functional Analysis of the TATA-Less Mouse FNDC5 Promoter During Neural Differentiation. Cellular and molecular neurobiology 34, 715-725
3. (!!! INVALID CITATION !!!).
4. Wilmore, J. H. (2003) Aerobic exercise and endurance: improving fitness for health benefits. The Physician and sportsmedicine 31, 45-51
5. Nelson, M. E., Rejeski, W. J., Blair, S. N., Duncan, P. W., Judge, J. O., King, A. C., Macera, C. A., and Castaneda-Sceppa, C. (2007) Physical activity and public health in older adults: recommendation from the American College of Sports Medicine and the American Heart Association. Medicine and science in sports and exercise 39, 1435-1445
6. Chipkin, S. R., Klugh, S. A., and Chasan-Taber, L. (2001) Exercise and diabetes. Cardiology clinics 19, 489-505
7. Hu, F. B., Manson, J. E., Stampfer, M. J., Colditz, G., Liu, S., Solomon, C. G., and Willett, W. C. (2001) Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. The New England journal of medicine 345, 790-797
8. Brandt, C., and Pedersen, B. K. (2010) The role of exercise-induced myokines in muscle homeostasis and the defense against chronic diseases. Journal of biomedicine & biotechnology 2010, 520258
9. Henriksen, T., Green, C., and Pedersen, B. K. (2012) Myokines in myogenesis and health. Recent patents on biotechnology 6, 167-171
10. Bostrom, P., Wu, J., Jedrychowski, M. P., Korde, A., Ye, L., Lo, J. C., Rasbach, K. A., Bostrom, E. A., Choi, J. H., Long, J. Z., Kajimura, S., Zingaretti, M. C., Vind, B. F., Tu, H., Cinti, S., Hojlund, K., Gygi, S. P., and Spiegelman, B. M. (2012) A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481, 463-468
11. Puigserver, P. (2005) Tissue-specific regulation of metabolic pathways through the transcriptional coactivator PGC1-alpha. International journal of obesity 29 Suppl 1, S5-9
12. Roca-Rivada, A., Castelao, C., Senin, L. L., Landrove, M. O., Baltar, J., Belen Crujeiras, A., Seoane, L. M., Casanueva, F. F., and Pardo, M. (2013) FNDC5/irisin is not only a myokine but also an adipokine. PloS one 8, e60563
13. Novelle, M. G., Contreras, C., Romero-Pico, A., Lopez, M., and Dieguez, C. (2013) Irisin, Two Years Later. International journal of endocrinology 2013, 746281
14. Hoeben, A., Landuyt, B., Highley, M. S., Wildiers, H., Van Oosterom, A. T., and De Bruijn, E. A. (2004) Vascular endothelial growth factor and angiogenesis. Pharmacological reviews 56, 549-580
15. Li, F., Chong, Z. Z., and Maiese, K. (2006) Winding through the WNT pathway during cellular development and demise. Histology and histopathology 21, 103-124
16. Cheresh, D. A., and Stupack, D. G. (2008) Regulation of angiogenesis: apoptotic cues from the ECM. Oncogene 27, 6285-6298
17. Kuo, H. M., Lin, C. Y., Lam, H. C., Lin, P. R., Chan, H. H., Tseng, J. C., Sun, C. K., Hsu, T. F., Wu, C. C., Yang, C. Y., Hsu, C. M., and Tai, M. H. (2012) PTEN overexpression attenuates angiogenic processes of endothelial cells by blockade of endothelin-1/endothelin B receptor signaling. Atherosclerosis 221, 341-349
18. Lam, H. C., Kuo, S. M., Chuang, M. J., Keng, H. M., Lin, P. R., Liu, G. S., Hsu, C. M., Howng, S. L., and Tai, M. H. (2006) Blockade of endothelin-1 release contributes to the anti-angiogenic effect by pro-opiomelanocortin overexpression in endothelial cells. Experimental biology and medicine 231, 782-788
19. Kumagai, T., Ishino, T., and Nakagawa, Y. (2012) Acidic sphingomyelinase induced by electrophiles promotes proinflammatory cytokine production in human bladder carcinoma ECV-304 cells. Archives of biochemistry and biophysics 519, 8-16
20. Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., and van der Schoot, C. E. (2000) Immortalisation of human bone marrow endothelial cells: characterisation of new cell lines. European journal of clinical investigation 30, 618-629
21. Wang, W., Lin, S., Xiao, Y., Huang, Y., Tan, Y., Cai, L., and Li, X. (2008) Acceleration of diabetic wound healing with chitosan-crosslinked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired STZ diabetic rats. Life sciences 82, 190-204
22. Sheu, S. J., Bee, Y. S., Ma, Y. L., Liu, G. S., Lin, H. C., Yeh, T. L., Liou, J. C., and Tai, M. H. (2009) Inhibition of choroidal neovascularization by topical application of angiogenesis inhibitor vasostatin. Molecular vision 15, 1897-1905
23. Tai, M. H., Kuo, S. M., Liang, H. T., Chiou, K. R., Lam, H. C., Hsu, C. M., Pownall, H. J., Chen, H. H., Huang, M. T., and Yang, C. Y. (2006) Modulation of angiogenic processes in cultured endothelial cells by low density lipoproteins subfractions from patients with familial hypercholesterolemia. Atherosclerosis 186, 448-457
24. Srivastava, K., Kundumani-Sridharan, V., Zhang, B., Bajpai, A. K., and Rao, G. N. (2007) 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires STAT3-dependent expression of VEGF. Cancer research 67, 4328-4336
25. Weng, W. T., Huang, S. C., Ma, Y. L., Chan, H. H., Lin, S. W., Wu, J. C., Wu, C. Y., Wen, Z. H., Wang, E. M., Wu, C. L., and Tai, M. H. (2014) alpha-Melanocyte-stimulating hormone inhibits angiogenesis through attenuation of VEGF/VEGFR2 signaling pathway. Biochimica et biophysica acta 1840, 1850-1860
26. Yang, X., Cui, W., Yu, S., Xu, C., Chen, G., Gu, A., Li, T., Cui, Y., Zhang, X., and Bian, X. (2014) A synthetic dl-nordihydroguaiaretic acid (Nordy), inhibits angiogenesis, invasion and proliferation of glioma stem cells within a zebrafish xenotransplantation model. PloS one 9, e85759
27. Lawson, N. D., and Weinstein, B. M. (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Developmental biology 248, 307-318
28. Agren, M. S., Mertz, P. M., and Franzen, L. (1997) A comparative study of three occlusive dressings in the treatment of full-thickness wounds in pigs. Journal of the American Academy of Dermatology 36, 53-58
29. De Vries, H. J., Zeegelaar, J. E., Middelkoop, E., Gijsbers, G., Van Marle, J., Wildevuur, C. H., and Westerhof, W. (1995) Reduced wound contraction and scar formation in punch biopsy wounds. Native collagen dermal substitutes. A clinical study. The British journal of dermatology 132, 690-697
30. Hung, L. Y., Tseng, J. T., Lee, Y. C., Xia, W., Wang, Y. N., Wu, M. L., Chuang, Y. H., Lai, C. H., and Chang, W. C. (2008) Nuclear epidermal growth factor receptor (EGFR) interacts with signal transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene expression. Nucleic acids research 36, 4337-4351
31. Pankov, R., and Yamada, K. M. (2002) Fibronectin at a glance. Journal of cell science 115, 3861-3863
32. Williams, C. M., Engler, A. J., Slone, R. D., Galante, L. L., and Schwarzbauer, J. E. (2008) Fibronectin expression modulates mammary epithelial cell proliferation during acinar differentiation. Cancer research 68, 3185-3192
33. Schumacher, M. A., Chinnam, N., Ohashi, T., Shah, R. S., and Erickson, H. P. (2013) The structure of irisin reveals a novel intersubunit beta-sheet fibronectin type III (FNIII) dimer: implications for receptor activation. The Journal of biological chemistry 288, 33738-33744