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Effect of 2, 4 dinitrophenol Preconditioning on the Expression Levels of Mesenchymal Markers in Neonatal Cardiac Progenitors Accepted Manuscript Effect of 2, 4 dinitrophenol Preconditioning on the Exp[.]
Accepted Manuscript Effect of 2, 4-dinitrophenol Preconditioning on the Expression Levels of Mesenchymal Markers in Neonatal Cardiac Progenitors Nazia Ahmed, Irfan Khan, Sumreen Begum, Asmat Salim PII: S1109-9666(17)30024-6 DOI: 10.1016/j.hjc.2017.01.007 Reference: HJC 116 To appear in: Hellenic Journal of Cardiology Received Date: February 2015 Accepted Date: 21 September 2015 Please cite this article as: Ahmed N, Khan I, Begum S, Salim A, Effect of 2, 4-dinitrophenol Preconditioning on the Expression Levels of Mesenchymal Markers in Neonatal Cardiac Progenitors, Hellenic Journal of Cardiology (2017), doi: 10.1016/j.hjc.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Effect of 2, 4-dinitrophenol Preconditioning on the Expression Levels of Mesenchymal Markers in Neonatal Cardiac Progenitors RI PT Nazia Ahmed#, Irfan Khan, Sumreen Begum# and Asmat Salim* Dr Panjwani Center for Molecular Medicine and Drug Research, International Center for Short title: Effect of DNP on Cardiac Progenitors M AN U *Corresponding author: SC Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Asmat Salim Dr Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University, Karachi, Karachi-75270, Pakistan Nazia Ahmed EP # Current Address: Email: asmat.salim@iccs.edu TE D Fax: (+92-21) 4819018-19 AC C Dow University of Health Sciences, Ojha Campus, Gulzar-e-Hijri,Suparco Road, KDA Scheme-33, Karachi, Pakistan Sumreen Begum Sindh Institute of Urology &Transplantation, Civil Hospital Karachi 74200, Pakistan ACCEPTED MANUSCRIPT KEYWORDS: RI PT Preconditioning; mesenchymal; cardiomyocytes, progenitor; adhesion INTRODUCTION The discovery of resident cardiac stem cells have destabilized the paradigm that adult heart is post mitotic organ without regeneration potential Several types of cardiac stem cells have been SC identified in adult heart which includes c-kit, Sca-1, LIM/homeodomain transcription factor islet1 (isl-1) and side population (SP) cells which express ATP-binding cassette transporters Abcg22 M AN U and CD34 positive epicardial cells.3 Several studies showed that cardiac progenitors also express mesenchymal markers e.g homing associated cell adhesion molecule or HCAM (CD44), Thy-1 (CD90), integrin β1(CD29), and endoglin SH-2 (CD105); which are routinely used for the identification of bone marrow derived mesenchymal stem cells (MSCs) These cardiac-derived TE D MSCs also co-express the stem cell marker, c-kit and have been shown angiomyogenic differentiation with reduced adipogenic and osteogenic gene expression as compared to the bone marrow derived MSCs.4 Besides characterization, the role of these markers has not been EP evaluated on cardiac precursors These mesenchymal markers are belong to the class of adhesion AC C proteins which generate cell-cell and cell-matrix adhesions and perform essential function of stemcell/progenitors nich maintainence, proliferation and differentiation The blocking studies against surface receptors integrin β1 and CD44 results in reduced bone marrow derived mesenchymal stem cell migration, engraftment and differentiation into cardiomyocytes and suggested their possible role in cardiomyogenesis.5, The activated leukocyte cell adhesion molecule (ALCAM or CD166) is another mesenchymal marker expressed on embryonic cardiac progenitors and play a role in cardiac differentiation during embryogenesis.7 The ACCEPTED MANUSCRIPT oxygen level in stem cell physiological niches is another important factor plays a critical role in controlling the stem cell proliferation and fate determination The approach of preconditioning or pre-culturing stem cells in low oxygen level reduces cell death and RI PT increased expression of cardiomyogenic and angiogenic markers, thereby enhancing the effectiveness of stem cell therapy.8 These observations led us to hypothesize that hypoxic pre-conditioning may increase the SC expression of various cell surface glycoproteins and adhesion molecules which may contribute in better engraftment and homing of cardiac progenitors to the desired tissues or organs, across the M AN U extracellular matrix and differentiation into the desired cell type by making cell-cell and cellmatrix adhesions The present study was conducted to analyze whether DNP preconditioning modulates the expression of cell surface glycoproteins and cell adhesion molecules and coexpression of mesenchymal markers on neonatal cardiac progenitors Keeping in view the role of TE D these molecules in cardiomyogenesis, this study will help to see if the change in their expression METHODS Cell Culture EP levels has any effect during ischemic cardiac injury AC C Cardiac progenitors were isolated from 1-2 days old neonatal NMRI mice The excised ventricles were incubated in 0.5% trypsin/EDTA solution at 37°C in thermal rocker for complete digestion The cells were cultured in cardiomyocyte growth medium (CGM) having Dulbecco’s modified Eagle’s medium/F12; DMEM/F12 (1:1) supplemented with 20% FBS, 100units/ml penicillin, 100µg/ml streptomycin, mM sodium pyruvate, mM L-glutamine and 1µg/ml insulin The cells were incubated at 37°C in CO2 incubator at 37°C and maintained till passage Characterization of Cardiac progenitors ACCEPTED MANUSCRIPT Flow Cytometry CPs were characterized by using primary antibodies against surface epitopes, CD34, GATA4, myosin heavy chain, and actin Cells were stained with Alexa fluor 546 goat anti mouse RI PT secondary antibodies analyzed by flow cytometer (BD Facs calibur, Becton Dickinson, USA) DNP Treatment The cultured cardiac progenitors were given chemical hypoxia using 2, dinitrophenol (DNP) SC For optimization, different concentrations (0.05-2 mM) of DNP were used for 10 minutes The number of dead cells was counted by JC-1 cytotoxicity assay through flow cytometry The M AN U corresponding concentration of DNP with least cytotoxicity was used in all experiments Cells were treated with an optimized dose of 0.1mM DNP in deaerated glucose free DMEM for 10 The medium was replaced with normal DMEM/F12 and then the cells were incubated for 24 hr at 37oC in humidified chamber with 5% CO2 for reoxygenation TE D Reverse-Transcriptase PCR After 24 hr reoxygenation, total RNA was extracted from untreated and DNP treated cardiac progenitors First strand cDNA was synthesized and amplified by performing PCR reaction EP Gene primers are shown in Table The densitometeric analysis of PCR bands were carried out The integrated density of each band was calculated and band density was normalized to AC C GAPDH RESULTS Isolation, Expansion and Phenotypic Characteristics of Cardiac Progenitors CPs adhered to cell culture flasks after 24 hr of isolation At passage and 2, majority of the cells grew larger in size More striations were seen and cells were more flattened and octagonal shaped with various cellular projections (Supplementary Fig S1) ACCEPTED MANUSCRIPT Identification of Cardiac progenitors CPs were analyzed for the expression of various epitopes through flow cytometry More than 60% of the cells showed positive staining for monoclonal antibodies against CD34 and cardiac RI PT proteins including GATA4, myosin heavy chain (MHC) and actin (Fig 1) Effect of DNP Preconditioning on CPs Chemical hypoxia was given to adherent CPs using 2, dinitrophenol (DNP) Cells were treated SC with an optimized dose of 0.1mM DNP for 10 During hypoxia, some of the cells were shrunken and lose their connections from neighboring cells and only few cells became rounded M AN U and detached from culture flask (Fig 2b) However, cells gained their normal morphology after 24 hr when normal conditions were restored (Fig 2c) JC-1 cytotoxicity assay showed no difference between normoxic and hypoxic CPs (Supplementary Fig S2) Expression of Mesenchymal Markers in CPs TE D Change in the expression of mesenchymal markers was determined in CPs after DNP treatment and 24 hr reoxygenation Except CD90 whose expression levels were significantly increased (p≤0.05), hypoxic stress did not bring about any significant change in the gene expression of DISCUSSION EP other mesenchymal markers CD29, CD44 and ALCAM (Fig 3) AC C Heart reserves some population of progenitors having stemness i.e they are able to divide and self renew with the potential to differentiate into beating cardiomyocytes and rescue the heart tissue from damage In the present study, the CPs were isolated from neonatal hearts of NMRI mice The cells were not fully differentiated showing the presence of progenitor markers along with cardiac specific transcription factors and cardiac specific proteins Similar results were observed by Itzhaki-Alfia and coworkers who isolated the CPs from adult human heart.9 The ACCEPTED MANUSCRIPT isolated CPs were positive for haematopoetic marker, CD34 which suggest that they are originated from epicardial derived CD34+ stem cells.3 Although the heart is made up of contractile myocytes, smooth muscle cells, endothelial cells, RI PT cardiac stem/progenitors cells, the cardiac fibroblast and myocytes contribute the most abundant cellular constituent of adult heart These fibroblast cells were acquired either from bone marrow derived cells through blood circulation or from epicardium derived cardiac progenitors.10 The SC discovery of epicardial derived CPs supports the hypothesis that cardiac progenitors infiltrate into myocardium and contribute to development of stem cell niches Recently, the mesenchymal M AN U markers have been reported in cardiac progenitors isolated from embryonic, fetal and adult heart The expression of mesenchymal markers has been demonstrated in c-kit cardiac progenitors cells, Sca-1 cells, cardiosphere derived cells from human cardiac explants and CD34+ epicardial cells.3, 11-13 In our study, primary neonatal mice ventricular cardiac progenitors were used to TE D investigate the expression of mesenchymal markers including CD29, CD44, CD90 and ALCAM The effects of short term chemical hypoxia and reoxygenation on the expression of mesenchyme surface glycoproteins and cell adhesion proteins involve in homing and cardiac differentiation EP were analyzed in cardiac progenitors The sublethal hypoxia dose has not shown any detrimental effects on cell morphology Cells regained normal morphology with only few dead cells when AC C cells were reoxygenated for 24 hr This suggests that the optimized hypoxia was not toxic to the cells The effect of hypoxic stress on mesenchymal markers has not been studied so far We have found that hypoxic stress had no effect on the expression of CD29 and CD44 as well as on ALCAM However, CD90 expression was significantly increased CD90 is an important regulator of inflammation and wound repair and participate in cell-matrix and cell-cell adhesion during inflammatory events which results in increased migration to inflammatory sites and ACCEPTED MANUSCRIPT secretion of various cytokines and growth factors which contribute to healing the injured tissue.14 CD90 also plays an important role in generating the neuronal network during postnatal development and neuronal regeneration.15 These finding suggest that CD90 may contribute to RI PT maintain cardiac stem cell pool residing in hypoxic niches in postnatal heart tissue and is also associated with increased infiltration of cardiac progenitors for repair of damaged heart myocardium SC CONCLUSION Our results indicate that neonatal CPs possesses mesenchymal markers with probable roles in M AN U maintenance, differentiation and adhesion CD90 is one of the important mesenchymal markers having suggestive role during low oxygen supply to the heart Future studies should be done to determine the role of these mesenchymal markers in the development, maintenance and TE D regeneration of cardiac stem cell niches CONFLICT OF INTEREST EP None declared ACKNOWLEDGEMENT AC C The financial support for this study was provided by the Pakistan Science Foundation ACCEPTED MANUSCRIPT REFERENCES Beltrami AP, UBrbanek K, Kajstura J, et al Yan SM, Finato N, Bussani R, Nadal Ginard B, Silvestri F, Leri A, Beltrami CA, Anversa P Evidence that human cardiac myocytes RI PT divide after myocardial infarction N Engl J Med 2001; 344:1750-7 Barile L, Messina E, Giacomello A, Marbán E Endogenous cardiac stem cells Prog Cardiovasc Dis 2007; 50(1): 31-48 SC Limana F, Zacheo A, Mocini D, Mangoni A, Borsellino G, Diamantini A, De Mori R, Battistini L, Vigna E, Santini M, Loiaconi V, Pompilio G, Germani A, Capogrossi MC M AN U Identification of myocardial and vascular precursor cells in human and mouse epicardium Circ Res 2007; 101:1255-65 Gambini E, Pompilio G, Biondi A et al C-kit+ cardiac progenitors exhibit mesenchymal markers and preferential cardiovascular commitment Cardiovasc Res 2011; 89: 362-73 TE D Ip JE, Wu Y, Huang J, Zhang L, Pratt RE, Dzau VJ Mesenchymal Stem Cells Use Integrin β1 Not CXC Chemokine Receptor for Myocardial Migration and EP Engraftment Mol Biol Cell 2007; 18: 2873-82 Hirata H, Murakami Y, Miyamoto Y, et al ALCAM (CD166) is a surface marker for AC C early murine cardiomyocytes Cells Tissues Organs 2006; 184:172-80 Rust W, Balakrishnan T, Zweigerdt R Cardiomyocyte enrichment from human embryonic stem cell cultures by selection of ALCAM surface expression Regen Med 2009; 4: 225-37 Song H, Song BW, Cha MJ, Choi IJ, Hwang KC Modification of mesenchymal stem cells for cardiac regeneration Expert Opin Biol Ther 2010; 10: 309-19 ACCEPTED MANUSCRIPT Itzhaki-Alfia A, Leor J, Raanani E, et al Patient characteristics and cell source determine the number of isolated human cardiac progenitor cells Circulation 2009; 120: 2559-66 10 Wu SM, Fujiwara Y, Cibulsky SM, et al Developmental origin of a bipotential RI PT myocardial and smooth muscle cell precursor in the mammalian heart Cell 2006; 127: 1137-50 11 Davis DR, Kizana E, Terrovitis J, et al Isolation and expansion of functionally- SC competent cardiac progenitor cells directly from heart biopsies J Mol Cell Cardiol 2010; 49: 312-21 M AN U 12 Smith RR, Barile L, Cho HC, et al Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens Circulation 2007; 115: 896-908 13 Naga GV, Tripathy NK, Nityanand S Fetal cardiac mesenchymal stem cells express TE D embryonal markers and exhibit differentiation into cells of all three germ layers World J Stem Cells 2013; 5: 26-33 14 Saalbach A, Haustein U, Anderegg U A ligand of human Thy-1 is localized on EP polymorphonuclear leukocytes and monocytes and mediates the binding to activated Thy1-positive microvascular endothelial cells and fibroblasts J Invest Dermatol 2000; 115: AC C 882-8 15 Rege TA, Hagood JS Thy-1 as a regulator of cell–cell and cell–matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis FASEB J 2006; 20: 1045-54 ACCEPTED MANUSCRIPT Figure Legends: Fig 1: Characterization of mice neonatal cardiac progenitors (CPs) through flow cytometry showing controls, (a) unstained cells, and (b) cells stained only with secondary antibody (Alexa GATA4, (e) myosin heavy chain and (f) actin RI PT fluor 546 goat anti mouse) and cells stained with primary antibodies against (c) CD34, (d) Fig 2: Cardiac progenitors (CPs) showing (a) normal morphology in untreated CPs, (b) shrunken SC cells which lose their connections with each other after DNP treatment, and (c) normal morphology after 24 hr reoxygenation M AN U Fig 3: Bar diagrams showing densitometry analysis of mesenchymal marker genes in neonatal cardiac progenitors (CPs) of (a) CD29, (b) CD44, (c) CD90 and (d) ALCAM in normoxic and hypoxic cardiomyocytes Representative gel images are also shown Data is presented as mean ± standard error Value of p ...ACCEPTED MANUSCRIPT Effect of 2, 4- dinitrophenol Preconditioning on the Expression Levels of Mesenchymal Markers in Neonatal Cardiac Progenitors RI PT Nazia Ahmed#, Irfan Khan,... of the cells showed positive staining for monoclonal antibodies against CD 34 and cardiac RI PT proteins including GATA4, myosin heavy chain (MHC) and actin (Fig 1) Effect of DNP Preconditioning. .. cell surface glycoproteins and cell adhesion molecules and coexpression of mesenchymal markers on neonatal cardiac progenitors Keeping in view the role of TE D these molecules in cardiomyogenesis,