Apelin/APJ AT1 (putative receptor protein related to the angiotensin. Chinese Journal of Tissue Engineering Research November 28, 2017 Vol.21, No.

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1 Chinese Journl of Tissue Engineering Reserch November 28, 217 Vol.21, No.33 HIF-1α/pelin/APJ ( 5112). HIF-1α/pelin/APJ [J] (33): DOI:1.3969/j.issn ORCID: () HIF-1α/pelin/APJ HIF-1α/pelin/APJ α α β α β 1α 1α 1α 1β Apelin/APJ AT1 (puttive receptor protein relted to the ngiotensin receptor AT1, APJ) G AT1 Apelin APJ APJ pelin/apj Apelin APJ Apelin/APJ :R394.2 :B : (217) HIF-1α/pelin/APJ ( 1% O 2) ( 2% O 2) 24 h MTS Western blot 1α pelin APJ 5 24 h 2 Western blot 1α pelin APJ ctnt ctnt 24 h A 49 (P <.1) 24 h 2 1α pelin APJ (P <.1) 2 ctnt (P <.1) ctnt (P <.1) 1α/pelin/APJ 1α AT1 AT1 1 α (817242) lncrna-h19/mir-199-5p/vegf pelin MSCs (217A ) Apelin PI3K/AKT Wng Lei, Studying for mster s degree, Deprtment of Emergency, Sun Yt-Sen Memoril Hospitl, Sun Yt-Sen University, Gungzhou 5112, Gungdong Province, Chin Corresponding uthor: Hou Jing-ying, Mster, Attending physicin, Deprtment of Emergency, Sun Yt-Sen Memoril Hospitl, Sun Yt-Sen University, Gungzhou 5112, Gungdong Province, Chin 54 P.O. Box 12, Shenyng 1118

2 . HIF-1α/pelin/APJ lncrna-h19/mir-199-5p/vegf MSCs (A216264) HIF-1α/Apelin/APJ/ACE2 ANGII (A2171) Apelin PI3K/AKT/miR-199-5p/VEGF 1α hypoxi induced fctor-1α HIF-1α AT1 puttive receptor protein relted to the ngiotensin receptor AT1 APJ The role of hypoxi induced fctor-1lph/pelin/apj pthwy in crdic stem cell prolifertion nd crdiogenic differentition fter hypoxi preconditioning Wng Lei, Hou Jing-ying, Long Hui-bo, Wu Ho, Zhou Chng-qing, Guo Tin-zhu, Wu Qun-hu, Zhong Ting-ting, Chen Xu-xing, Wng Tong (Deprtment of Emergency Sun Yt-Sen Memoril Hospitl, Sun Yt-Sen University, Gungzhou 5112, Gungdong Province, Chin) Abstrct BACKGROUND: Our previous studies demonstrted tht crdic stem cells (CSCs) trnsplnttion could improve crdic function in rts with myocrdil infrction (MI). However, the overll survivl nd crdic differentition of CSCs were low. OBJECTIVE: To investigte the effect of hypoxi preconditioning on CSCs prolifertion nd crdiogenic differentition nd the role of hypoxi induced fctor-1lph (HIF-1α)/pelin/puttive receptor protein relted to the ngiotensin receptor AT1 (APJ) pthwy in the procedure. METHODS: Cells cultured in vitro experienced exposure to hypoxi (1% O 2) for 24 hours. Crdiogenic differentition ws induced by using 5-zcytidine for nother 24 hours. Then, cells were cultured in norml condition for 2 weeks. Normoxi (2% O 2) ws used s negtive control during the whole process. Cell prolifertion ws detected using MTS method nd expressions of HIF-1α, pelin, ctnt nd APJ were detected using western blot ssy fter 24 hours of preconditioning nd 2 weeks fter the induction of differentition; the percentge of ctnt-positive crdiomyocyte-like cells ws observed by immunofluorescence stining. RESULTS AND CONCLUSION: Compred with the normoxi group, the hypoxi group presented higher prolifertion rte nd higher bsorbnce vlue t 49 nm (P <.1); the protein expressions of HIF-1α, pelin nd APJ were ll enhnced fter hypoxi exposure for 24 hours nd 2 weeks fter the induction of differentition (P <.1); the percentge of ctnt-positive cells ws gretly incresed in the hypoxi group (P <.1), nd the expression of ctnt ws lso significntly intensified (P <.1). To conclude, hypoxi preconditioning could promote the prolifertion nd crdiogenic differentition of CSCs, nd the ctivtion of HIF-1α/Apelin/APJ pthwy might be involved in this process. Subject hedings: Myocytes, Crdic; Cell Hypoxi; Hypoxi-Inducible Fctor 1, lph Subunit; Tissue Engineering Funding: the Ntionl Nturl Science Foundtion of Chin, No ; the Science nd Technology Pln of Gungdong Province, No. 217A ; the Medicl Reserch Foundtion of Gungdong Province, No. A216264, A2171 Cite this rticle: Wng L, Hou JY, Long HB, Wu H, Zhou CQ, Guo TZ, Wu QH, Zhong TT, Chen XX, Wng T. The role of hypoxi induced fctor-1lph/pelin/apj pthwy in crdic stem cell prolifertion nd crdiogenic differentition fter hypoxi preconditioning. Zhongguo Zuzhi Gongcheng Ynjiu. 217;21(33): Introduction [1] [2] [2-4] [5] [6] 1α (hypoxi induced fctor-1α HIF-1α) HIF-1α [7-8] AT1 (puttive receptor protein relted to the ngiotensin receptor AT1 APJ) G AT1Apelin APJ APJ pelin/apjapelin APJ [9] Apelin/APJ kf @sin.com P.O.Box 12,Shenyng

3 . HIF-1α/pelin/APJ [1] pelin/apj HIF-1α pelin/apj [11-12] pelin/apj HIF-1α HIF-1α/pelin/ APJ 1Mterils nd methods d SPFSD 1 25 g SCXK( ) CO 2 (Thermo) (Eppendorff) ( )(NSS-2) (Leic)PCR SYBRPCR ABI PRISM 75(Invitrogen) (HP ) ( JET BIOFIL ) (HERACELL15i)( Thermo Scientific ) (Eppendorf/Glxy 48 R)( Eppendorf/Glxy ) DMEM PBS(Hyclone ) HIF-1α(Genetx ) pelin(abcm) APJ (LifeSpn) ctnt(abcm) ( ) (Southern Biotech) (Millpore) celltiter96aq (Promeg ) BCA (Abcm) TRIZOL RNA (Snt cruz ) 5 (Sigm) [3-4 13] 3 SD [ ] ( 2% O 2 ) ( 1%O 2 ) 24 h 1 d3 25 cm 2 9% 95% 1% O 2 5% CO 2 94% N 2 Glxy 48R 24 h [18] MTS 24 h L µl celltiter96aq µl 1 µl 4 h multi-scn MK3 A 49 [18] h / 6 24 h1 µmol/l 5 2 µl 2 ctnt ctnt 4 g/l15 min 1% 3 min (ctnt 1 1) 4 PBS 3 5 min (1 1 ) 1 h PBS 3 5 min DAPI5 min PBS 1 5 min [19] Western blot 24 h 2 HIF-1α/pelin/APJ 2cTnT 1 ml 1 µl PMSF(1 mmol/l) 1 µl Cocktil 1 µl PMSF Cocktil 3 min 4 14 r/min 1 min BCA SDS-PAGEPVDF 5% 1 h 4 TBST 5 min h TBST 5 min h TBST 5 min 3 2 min ml/cm 2 5 min 1.6 x _ ±s SPSS 17. for windows x _ ±s t P < P.O. Box 12, Shenyng 1118

stem cells prolifertion detected by MTS 24 h A 49 P <.

.5 B HIF-1α pelin APJ β-ctin HIF-1α 2. 1.

the determinnts in HIF-1α/pelin/APJ pthwy 24 hours fter hypoxi

h 2 HIF-1α/pelin/APJ 24 h 2 HIF-1α pelin APJ P <.

4 . HIF-1α/pelin/APJ A (%) h 24 h h 24 h MTS Figure 1 Comprison of crdic stem cells prolifertion detected by MTS 24 h A 49 P <.1 A HIF-1α pelin APJ β-ctin HIF-1α pelin 1..5 APJ 1..5 B HIF-1α pelin APJ β-ctin HIF-1α pelin APJ h 2 HIF-1α/pelin/APJ Figure 2 Comprison of expressions of the determinnts in HIF-1α/pelin/APJ pthwy 24 hours fter hypoxi preconditioning nd 2 weeks fter induction of differentition A B 24 h 2 HIF-1α/pelin/APJ 24 h 2 HIF-1α pelin APJ P <.1 HIF-1α 1α A b c ctnt (%) B ctnt β-ctin CTnT Figure 3 Comprison of crdiogenic differentition of crdic stem cells between the two groups A 2 ctnt ( 2) ctnt( ) DAPI b ctnt c b B Western blot 2 ctnt ctnt P <.1 ISSN CN /R CODEN: ZLKHAH 543

5 . HIF-1α/pelin/APJ 2Results 2.1 MTS 24 hmts (14.56±.17)% (95.32±.85)%(P <.1) A ± ±.7 A 49 ( 1 P <.1) 2.2 HIF-1α/pelin/APJ 24 h 2HIF-1α/pelin/APJ 24 h ( 2A)2 ( 2B) HIF-1α pelin APJ ( 2 P <.1) 2.3 ctnt 2 ctnt ctnt (P <.1 3A) Western blot ctnt (P <.1 3B) 3Discussion [1 2] [3-4] [2-4] [3-4] [21] [5 8 22] 544 / [23] HIF-1α HIF-1α HIF-1α [24] HIF-1α [25] c-kit(+) [26-27] 24 h A 49 HIF-1αHIF-1α HIF-1α HIF-1α HIF-1α [6-7] HIF-1α HIF-1α [28] HIF-1α HIF-1α TGF-β1 Smd4 Nkx2.5 GATA4 SMA ctnt [29-3] HIF-1α HIF-1α 2 HIF-1α ctnt ctnt HIF-1α pelin [31] Apelin Ccspse [18 32] PI3K/AKT MAPK/ERK1/2 [18 33] pelin [12] pelin [34] pelin/apj Apelin/APJApelin [35] Wng [1] pelin P.O. Box 12, Shenyng 1118

6 . HIF-1α/pelin/APJ pelin ERK1/2 ERK5 pelin/apj pelin pelin APJ 2pelin APJ ctnt ctnt pelin/apj HIF-1α pelin/apj [11-12] HIF-1αpelin (HER)pelin [36] HIF-1α pelin APJ [36] HIF-1α pelin/apj HIF-1αpelin/APJ [12] HIF-α pelin/apj [37] pelin/apj HIF-1α HIF-1α HIF-1α pelin APJ 2 HIF-1α pelin APJ HIF-1α/pelin/APJ HIF-1α/pelin/APJ HIF-1α/pelin/APJ CNKI 3 ( ) 3. 4 References [1] Leong YY, Ng WH, Ellison-Hughes GM, et l. Crdic Stem Cells for Myocrdil Regenertion: They Are Not Alone. Front Crdiovsc Med. 217;4:47. [2] Zwetsloot PP, Végh AM, Jnsen of Lorkeers SJ, et l. Crdic Stem Cell Tretment in Myocrdil Infrction: A Systemtic Review nd Met-Anlysis of Preclinicl Studies. Circ Res. 216;118(8): [3] Kulndvelu S, Krntlis V, Fritsch J, et l. Pim1 Kinse Overexpression Enhnces ckit+ Crdic Stem Cell Crdic Repir Following Myocrdil Infrction in Swine. J Am Coll Crdiol. 216;68(22): [4] Zheng SX, Weng YL, Zhou CQ, et l. Comprison of crdic stem cells nd mesenchyml stem cells trnsplnttion on the crdic electrophysiology in rts with myocrdil infrction. Stem Cell Rev. 213;9(3): [5] Lee JH, Yoon YM, Lee SH. Hypoxic Preconditioning Promotes the Bioctivities of Mesenchyml Stem Cells vi the HIF-1α-GRP78-Akt Axis. Int J Mol Sci. 217;18(6): E132. [6] Ng KM, Lee YK, Chn YC, et l. Exogenous expression of HIF-1 lph promotes crdic differentition of embryonic stem cells. J Mol Cell Crdiol. 21;48(6): [7] Kudová J, Všíček O, Číž M, et l. Meltonin promotes crdiomyogenesis of embryonic stem cells vi inhibition of HIF-1α stbiliztion. J Pinel Res. 216;61(4): [8] Lv B, Hu T, Li F, et l. Hypoxi-inducible fctor 1 α protects mesenchyml stem cells ginst oxygen-glucose deprivtion-induced injury vi utophgy induction nd PI3K/AKT/mTOR signling pthwy. Am J Trnsl Res. 217;9(5): [9] Dlzell JR, Rocchiccioli JP, Weir RA, et l. The Emerging Potentil of the Apelin-APJ System in Hert Filure. J Crd Fil. 215;21(6): [1] Wng L, Zhu ZM, Zhng NK, et l. Apelin: n endogenous peptide essentil for crdiomyogenic differentition of mesenchyml stem cells vi ctivting extrcellulr signl-regulted kinse 1/2 nd 5. Cell Biol Int. 216;4(5): [11] Kong HL, Li ZQ, Zho SM, et l. Apelin-APJ effects of ginsenoside-rb1 depending on hypoxi-induced fctor 1α in hypoxi neontl crdiomyocytes. Chin J Integr Med. 215;21(2): [12] Li L, Li L, Zhng Z, et l. Hypoxi promotes bone mrrow-derived mesenchyml stem cell prolifertion through pelin/apj/utophgy pthwy. Act Biochim Biophys Sin (Shnghi). 215;47(5): [13] Hou J, Yn P, Guo T, et l. Crdic stem cells trnsplnttion enhnces the expression of connexin 43 vi the ANG II/AT1R/TGF-bet1 signling pthwy in rt model of myocrdil infrction. Exp Mol Pthol. 215;99(3): ISSN CN /R CODEN: ZLKHAH 545

7 . HIF-1α/pelin/APJ [14],,,. [J]., 215, 19(28): [15],,,.12 [J]., 215, 19(1): [16],,,.CSCs MSCs [J]., 215, 24(9): [17],,,. ANG /AT1R/TGF-β1/SMAD/CX43 [J].,216,2(28): [18],,,. pelin [J]., 217, 21(1): [19] Hou J, Long H, Zhou C, et l. Long noncoding RNA Brvehert promotes crdiogenic differentition of mesenchyml stem cells in vitro. Stem Cell Res Ther. 217;8(1):4. [2] Micheu MM, Scf-Udrişte A, DorobnŢu M.Bringing crdic stem cell therpy from bench to bedside: lessons from the pst nd future perspectives. Rom J Morphol Embryol. 216;57(2): [21] Liu S, Zhou J, Zhng X, et l. Strtegies to Optimize Adult Stem Cell Therpy for Tissue Regenertion. Int J Mol Sci. 216;17(6): E982. [22] Hn YS, Lee JH, Yoon YM, et l. Hypoxi-induced expression of cellulr prion protein improves the therpeutic potentil of mesenchyml stem cells. Cell Deth Dis. 216;7(1):e2395. [23] RjendrnNir DS, Krunkrn J, Nir RR. Sub-physiologicl oxygen levels optiml for growth nd survivl of humn tril crdic stem cells. Mol Cell Biochem. 217;432(1-2): [24] Kimur W, Xio F, Cnseco DC, et l. Hypoxi fte mpping identifies cycling crdiomyocytes in the dult hert. Nture ;523(7559): [25] Ong SG, Lee WH, Hung M, et l. Cross tlk of combined gene nd cell therpy in ischemic hert disese: role of exosoml microrna trnsfer. Circultion. 214;13 (11 Suppl 1):S6-69. [26] Bellio MA, Rodrigues CO, Lndin AM, et l. Physiologicl nd hypoxic oxygen concentrtion differentilly regultes humn c-kit+ crdic stem cell prolifertion nd migrtion. Am J Physiol Hert Circ Physiol. 216;311(6): H159-H1519. [27] Hung M, Nguyen P, Ji F, et l. Double knockdown of prolyl hydroxylse nd fctor-inhibiting hypoxi-inducible fctor with nonvirl minicircle gene therpy enhnces stem cell mobiliztion nd ngiogenesis fter myocrdil infrction. Circultion. 211;124(11 Suppl):S [28] Lee HJ, Jeong CH, Ch JH, et l. PKC-delt inhibitors sustin self-renewl of mouse embryonic stem cells under hypoxi in vitro. Exp Mol Med. 21;42(4): [29] Wng Y, Feng C, Xue J, et l. Adenovirus-medited hypoxi-inducible fctor 1lph double-mutnt promotes differentition of bone mrrow stem cells to crdiomyocytes. J Physiol Sci. 29;59(6): [3] Wng Y, Sun A, Xue J, et l. Adenovirus-medited expression of hypoxi-inducible fctor 1α double mutnt converts neontl crdic fibroblsts into (crdio)myocyte phenotype. Cell Biochem Funct. 212;3(1): [31] Hou J, Zhong T, Guo T, et l. Apelin promotes mesenchyml stem cells survivl nd vsculriztion under hypoxic-ischemic condition in vitro involving the upregultion of vsculr endothelil growth fctor. Exp Mol Pthol. 217;12(2): [32] Zeng X, Yu SP, Tylor T, et l. Protective effect of pelin on cultured rt bone mrrow mesenchyml stem cells ginst poptosis. Stem Cell Res. 212;8(3): [33] Mottghi S, Lrijni B, Shrifi AM. Apelin 13: novel pproch to enhnce efficcy of hypoxic preconditioned mesenchyml stem cells for cell therpy of dibetes. Med Hypotheses. 212;79(6): [34] Zhng NK, Co Y, Zhu ZM, et l. Activtion of Endogenous Crdic Stem Cells by Apelin-13 in Infrcted Rt Hert. Cell Trnsplnt. 216;25(9): [35] Wng IN, Wng X, Ge X, et l. Apelin enhnces directed crdic differentition of mouse nd humn embryonic stem cells. PLoS One. 212;7(6):e [36] Kong HL, Li ZQ, Zho SM, et l. Apelin-APJ effects of ginsenoside-rb1 depending on hypoxi-induced fctor 1α in hypoxi neontl crdiomyocytes. Chin J Integr Med. 215;21(2): [37] Zhng J, Liu Q, Fng Z, et l. Hypoxi induces the prolifertion of endothelil progenitor cells vi upregultion of Apelin/APLNR/MAPK signling. Mol Med Rep. 216; 13(2): P.O. Box 12, Shenyng 1118

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