幹細胞因具有可分化成多種細胞的能力，故近來被臨床試驗用於恢復缺氧-缺血所造成的組織傷害，目前已知受損組織所引起的缺氧環境會活化缺氧誘發因子(hypoxia inducible factor, HIF)並調控相關訊息傳遞。其中，受損組織所釋放的基質細胞衍生因子(Stromal cell-derived factor-1, SDF-1)在缺氧環境下被HIF調控，促使間質幹細胞(mesenchymal stem cell, MSC)或內皮前軀細胞(endothelial progenitor cell, EPC)移動到缺氧-缺血環境幫助組織修復及血管新生。過去研究中已得知缺氧可誘發Akt與NFκB的訊息並與幹細胞的抗凋亡(anti-apoptosis)與移動(migration)具相關性。此外，流體剪力(laminar shear stress, LSS)可增加成熟內皮細胞(endothelial cell, EC) NFκB的表現。本研究擬利用體外培養微環境(in vitro microenvironments)，比較缺氧下MSC與EPC抵抗缺氧環境及移動能力的差異，進而探討其可能之分子機轉，並了解流體剪力對其功能表現之影響。
本實驗使用三種細胞：MSC、EPC、及fibroblast。MSC係由6-8周大SD鼠的骨髓分離並培養；給予MSC 內皮細胞生長培養液可使其分化成EPC，而大鼠纖維母細胞株則是做為已分化成熟細胞的指標。體外缺氧微環境乃是藉由加入desferroxamine (DFO)模擬，其結果顯示MSC與EPC皆比fibroblast可抵抗缺氧造成的細胞凋亡，MSC表現較少磷酸化Akt (pAkt)及內生性NFκB蛋白，相對地，fibroblast具較高pAkt 與NFκB，且加入任一Akt或NFκB抑制劑皆會增加fibroblast在DFO引起的細胞凋亡。接著，本研究利用wound closure與transwell migration assays分析MSC與EPC在給予DFO下細胞移動的能力，缺氧會使MSC增加趨化因子受體CXCR4 mRNA與蛋白的表現，而EPC本身因具有較MSC高的CXCR4蛋白表現，並在DFO環境中表現較高的pAkt，故產生較MSC強的移動效果，抑制Akt與NFκB後發現EPC之細胞存活率與移動皆會下降。在給予LSS後發現，MSC無法因流體刺激產生型態學改變，而EPC可產生與成熟EC相同的反應，改變其方向性使其與血流方向平行。本研究發現EPC可為較優之細胞療法來源，而Akt-NFκB為調控此現象之可能機制。

Stem cells and progenitor cells have the potential to differentiate into different types of cells by chemical or mechanical stimulation. In clinical study, stem cells and progenitor cells were used to repair hypoxic/ischemic injury recently. Transient hypoxic microenvironments induced by injured tissue represent a cell niche through stabilizing the hypoxia inducible factor-1 (HIF-1). Stromal cell-derived factor-1 (SDF-1), as regulated by HIF-1, plays a critical role for mobilizing the mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) to the injured sites and promotes the angiogenesis. The hypoxia-induced anti-apoptosis and migration characteristics in stem cells are mediated by the Akt and NFκB signals. In addition, the laminar shear stress (LSS) enhanced the NFκB expression in mature endothelial cell (EC). The purpose of this study is to compare the anti-apoptosis and migration ability of MSC and EPC under hypoxia microenvironments in vitro. We will also investigate the underline molecular mechanism and functional responses to the LSS stimulation.
Three types of cells: MSC, EPC, and fibroblast were used in this study. The MSC was harvested from the bone marrow of 6-8 weeks-old SD rat and differentiated into EPC by treating with endothelial growth medium. The rat fibroblast cell line was used as the mature cells from same species. The hypoxic microenvironment was created by treating the cells with hypoxic mimetic desferroxamine (DFO) in vitro. Both the MSC and EPC were resisted to DFO-induced apoptosis as compared to fibroblast. MSC has lower level of phosphorylated Akt (pAkt) and less endogenous NFκB protein than fibroblast. Inhibition either Akt or NFκB activation in fibroblast by specific inhibitor enhanced the cell apoptosis from DFO induction. In MSC, the C-X-C chemokine receptor type 4 (CXCR4) gene and protein expressions were increased by DFO application. The EPC has higher CXCR4 protein expression and pAkt than MSC under DFO treatment. Thus, a higher cell migration ability which can be inhibited by Akt, NFκB, or CXCR4 inhibitors was discovered in EPC and confirmed the underline signal via Akt-NFκB pathway. The application of LSS showed no morphological change in MSC, but rearranged cell orientation parallel to flow direction in EPC as the same as mature EC. The Akt-NFκB signaling pathway plays an important role to regulate the anti-apoptosis and migration phenomenon in both MSCs and EPCs.

Angelos MG, Brown MA, Satterwhite LL, Levering VW, Shaked NT, Truskey GA. 2010. Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress. Biophys J 99(11):3545-3554.
Annabi B, Lee YT, Turcotte S, Naud E, Desrosiers RR, Champagne M, Eliopoulos N, Galipeau J, Beliveau R. 2003. Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation. Stem Cells 21(3):337-347.
Aoki M, Nata T, Morishita R, Matsushita H, Nakagami H, Yamamoto K, Yamazaki K, Nakabayashi M, Ogihara T, Kaneda Y. 2001. Endothelial apoptosis induced by oxidative stress through activation of NF-kappaB: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension 38(1):48-55.
Asahara T, Kawamoto A. 2004. Endothelial progenitor cells for postnatal vasculogenesis. Am J Physiol Cell Physiol 287(3):C572-579.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275(5302):964-967.
Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. 1999. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18(14):3964-3972.
Bai SW, Herrera-Abreu MT, Rohn JL, Racine V, Tajadura V, Suryavanshi N, Bechtel S, Wiemann S, Baum B, Ridley AJ. 2011. Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration. BMC Biol 9:54.
Baksh D, Song L, Tuan RS. 2004. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 8(3):301-316.
Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S, Smulson M. 1999. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem 274(33):22932-22940.
Brown MA, Wallace CS, Angelos M, Truskey GA. 2009. Characterization of umbilical cord blood-derived late outgrowth endothelial progenitor cells exposed to laminar shear stress. Tissue Eng Part A 15(11):3575-3587.
Culver C, Sundqvist A, Mudie S, Melvin A, Xirodimas D, Rocha S. 2010. Mechanism of hypoxia-induced NF-kappaB. Mol Cell Biol 30(20):4901-4921.
Deng J, Han Y, Yan C, Tian X, Tao J, Kang J, Li S. 2010. Overexpressing cellular repressor of E1A-stimulated genes protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt. Apoptosis 15(4):463-473.
Elmore S. 2007. Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495-516.
Harris AL. 2002. Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2(1):38-47.
Harrison JS, Rameshwar P, Chang V, Bandari P. 2002. Oxygen saturation in the bone marrow of healthy volunteers. Blood 99(1):394.
Herzog EL, Chai L, Krause DS. 2003. Plasticity of marrow-derived stem cells. Blood 102(10):3483-3493.
Hung SC, Pochampally RR, Chen SC, Hsu SC, Prockop DJ. 2007. Angiogenic effects of human multipotent stromal cell conditioned medium activate the PI3K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase survival, and stimulate angiogenesis. Stem Cells 25(9):2363-2370.
Hur J, Yoon CH, Lee CS, Kim TY, Oh IY, Park KW, Kim JH, Lee HS, Kang HJ, Chae IH, Oh BH, Park YB, Kim HS. 2007. Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle. Stem Cells 25(7):1769-1778.
Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418(6893):41-49.
Lee SH, Lee YJ, Song CH, Ahn YK, Han HJ. 2010. Role of FAK phosphorylation in hypoxia-induced hMSCS migration: involvement of VEGF as well as MAPKS and eNOS pathways. Am J Physiol Cell Physiol 298(4):C847-856.
Lee SP, Youn SW, Cho HJ, Li L, Kim TY, Yook HS, Chung JW, Hur J, Yoon CH, Park KW, Oh BH, Park YB, Kim HS. 2006. Integrin-linked kinase, a hypoxia-responsive molecule, controls postnatal vasculogenesis by recruitment of endothelial progenitor cells to ischemic tissue. Circulation 114(2):150-159.
Li H, Tan J, Zou Z, Huang CG, Shi XY. 2011. Propofol post-conditioning protects against cardiomyocyte apoptosis in hypoxia/reoxygenation injury by suppressing nuclear factor-kappa B translocation via extracellular signal-regulated kinase mitogen-activated protein kinase pathway. Eur J Anaesthesiol 28(7):525-534.
Liekens S, Schols D, Hatse S. 2010. CXCL12-CXCR4 axis in angiogenesis, metastasis and stem cell mobilization. Curr Pharm Des 16(35):3903-3920.
Liu C, Tsai AL, Chen YC, Fan SC, Huang CH, Wu CC, Chang CH. 2012. Facilitation of human osteoblast apoptosis by sulindac and indomethacin under hypoxic injury. J Cell Biochem 113(1):148-155.
Lopaczynski W, Zeisel SH. 2001. Antioxidants, programmed cell death, and cancer. Nutrition research (New York, NY) 21(1):295-307.
Losordo DW, Dimmeler S. 2004. Therapeutic angiogenesis and vasculogenesis for ischemic disease: part II: cell-based therapies. Circulation 109(22):2692-2697.
Luo W, Xiong W, Zhou J, Fang Z, Chen W, Fan Y, Li F. 2011. Laminar shear stress delivers cell cycle arrest and anti-apoptosis to mesenchymal stem cells. Acta Biochim Biophys Sin (Shanghai) 43(3):210-216.
Maeng YS, Choi HJ, Kwon JY, Park YW, Choi KS, Min JK, Kim YH, Suh PG, Kang KS, Won MH, Kim YM, Kwon YG. 2009. Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCbeta2 axis. Blood 113(1):233-243.
Matsushita H, Morishita R, Nata T, Aoki M, Nakagami H, Taniyama Y, Yamamoto K, Higaki J, Yasufumi K, Ogihara T. 2000. Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: in vivo evidence of the importance of NF-kappaB in endothelial cell regulation. Circ Res 86(9):974-981.
Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. 2010. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 7(2):150-161.
Morgan MJ, Liu ZG. 2011. Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell Res 21(1):103-115.
Oswald J, Boxberger S, Jørgensen B, Feldmann S, Ehninger G, Bornhäuser M, Werner C. 2004. Mesenchymal Stem Cells Can Be Differentiated Into Endothelial Cells In Vitro. Stem Cells 22(3):377-384.
Petzold T, Orr AW, Hahn C, Jhaveri KA, Parsons JT, Schwartz MA. 2009. Focal adhesion kinase modulates activation of NF-kappaB by flow in endothelial cells. Am J Physiol Cell Physiol 297(4):C814-822.
Pittenger MF, Martin BJ. 2004. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 95(1):9-20.
Rafii S, Lyden D. 2003. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 9(6):702-712.
Raheja LF, Genetos DC, Wong A, Yellowley CE. 2011. Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1alpha. Cell Biol Int 35(10):981-989.
Ridder DA, Schwaninger M. 2009. NF-kappaB signaling in cerebral ischemia. Neuroscience 158(3):995-1006.
Rosova I, Dao M, Capoccia B, Link D, Nolta JA. 2008. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 26(8):2173-2182.
Saito A, Maier CM, Narasimhan P, Nishi T, Song YS, Yu F, Liu J, Lee YS, Nito C, Kamada H, Dodd RL, Hsieh LB, Hassid B, Kim EE, Gonzalez M, Chan PH. 2005. Oxidative stress and neuronal death/survival signaling in cerebral ischemia. Mol Neurobiol 31(1-3):105-116.
Schroder K, Kohnen A, Aicher A, Liehn EA, Buchse T, Stein S, Weber C, Dimmeler S, Brandes RP. 2009. NADPH oxidase Nox2 is required for hypoxia-induced mobilization of endothelial progenitor cells. Circ Res 105(6):537-544.
Semenza GL. 2009. Regulation of oxygen homeostasis by hypoxia-inducible factor 1. Physiology (Bethesda) 24:97-106.
Shaik SS, Soltau TD, Chaturvedi G, Totapally B, Hagood JS, Andrews WW, Athar M, Voitenok NN, Killingsworth CR, Patel RP, Fallon MB, Maheshwari A. 2009. Low intensity shear stress increases endothelial ELR+ CXC chemokine production via a focal adhesion kinase-p38{beta} MAPK-NF-{kappa}B pathway. J Biol Chem 284(9):5945-5955.
Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, Vela D, Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira EM, He R, Geng YJ, Willerson JT, Perin EC. 2005. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 111(2):150-156.
Song YS, Narasimhan P, Kim GS, Jung JE, Park EH, Chan PH. 2008. The role of Akt signaling in oxidative stress mediates NF-kappaB activation in mild transient focal cerebral ischemia. J Cereb Blood Flow Metab 28(12):1917-1926.
Tang YL, Zhu W, Cheng M, Chen L, Zhang J, Sun T, Kishore R, Phillips MI, Losordo DW, Qin G. 2009. Hypoxic preconditioning enhances the benefit of cardiac progenitor cell therapy for treatment of myocardial infarction by inducing CXCR4 expression. Circ Res 104(10):1209-1216.
Taylor CT, Cummins EP. 2009. The role of NF-kappaB in hypoxia-induced gene expression. Ann N Y Acad Sci 1177:178-184.
Tille JC, Pepper MS. 2002. Mesenchymal cells potentiate vascular endothelial growth factor-induced angiogenesis in vitro. Exp Cell Res 280(2):179-191.
Tsubokawa T, Yagi K, Nakanishi C, Zuka M, Nohara A, Ino H, Fujino N, Konno T, Kawashiri MA, Ishibashi-Ueda H, Nagaya N, Yamagishi M. 2010. Impact of anti-apoptotic and anti-oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia. Am J Physiol Heart Circ Physiol 298(5):H1320-1329.
Urbich C, Dimmeler S. 2004. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 95(4):343-353.
Wang GL, Semenza GL. 1993. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82(12):3610-3615.
Wang Y, Flores L, Lu S, Miao H, Li YS, Chien S. 2009. Shear Stress Regulates the Flk-1/Cbl/PI3K/NF-kappaB Pathway Via Actin and Tyrosine Kinases. Cell Mol Bioeng 2(3):341-350.
Weidemann A, Johnson RS. 2008. Biology of HIF-1alpha. Cell Death Differ 15(4):621-627.
Woo KJ, Lee TJ, Park JW, Kwon TK. 2006. Desferrioxamine, an iron chelator, enhances HIF-1alpha accumulation via cyclooxygenase-2 signaling pathway. Biochem Biophys Res Commun 343(1):8-14.
Wu CC, Chao YC, Chen CN, Chien S, Chen YC, Chien CC, Chiu JJ, Linju Yen B. 2008. Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells. J Biomech 41(4):813-821.
Wu CC, Li YS, Haga JH, Kaunas R, Chiu JJ, Su FC, Usami S, Chien S. 2007. Directional shear flow and Rho activation prevent the endothelial cell apoptosis induced by micropatterned anisotropic geometry. Proc Natl Acad Sci U S A 104(4):1254-1259.
Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, Murasawa S, Bosch-Marce M, Masuda H, Losordo DW, Isner JM, Asahara T. 2003. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation 107(9):1322-1328.
Yamamoto K, Takahashi T, Asahara T, Ohura N, Sokabe T, Kamiya A, Ando J. 2003. Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress. J Appl Physiol 95(5):2081-2088.
Yin Q, Jin P, Liu X, Wei H, Lin X, Chi C, Liu Y, Sun C, Wei Y. 2011. SDF-1alpha inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways. Mol Biol Rep 38(1):9-16.
Yoon CH, Hur J, Park KW, Kim JH, Lee CS, Oh IY, Kim TY, Cho HJ, Kang HJ, Chae IH, Yang HK, Oh BH, Park YB, Kim HS. 2005. Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases. Circulation 112(11):1618-1627.
Youn SW, Lee SW, Lee J, Jeong HK, Suh JW, Yoon CH, Kang HJ, Kim HZ, Koh GY, Oh BH, Park YB, Kim HS. 2011. COMP-Ang1 stimulates HIF-1{alpha}-mediated SDF-1 overexpression and recovers ischemic injury through BM-derived progenitor cell recruitment. Blood 117(16):4376-4386.
Zaruba MM, Franz WM. 2010. Role of the SDF-1-CXCR4 axis in stem cell-based therapies for ischemic cardiomyopathy. Expert Opin Biol Ther 10(3):321-335.
Zemani F, Silvestre JS, Fauvel-Lafeve F, Bruel A, Vilar J, Bieche I, Laurendeau I, Galy-Fauroux I, Fischer AM, Boisson-Vidal C. 2008. Ex vivo priming of endothelial progenitor cells with SDF-1 before transplantation could increase their proangiogenic potential. Arterioscler Thromb Vasc Biol 28(4):644-650.
Zheng H, Fu G, Dai T, Huang H. 2007. Migration of endothelial progenitor cells mediated by stromal cell-derived factor-1alpha/CXCR4 via PI3K/Akt/eNOS signal transduction pathway. J Cardiovasc Pharmacol 50(3):274-280.