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Spinelli CC, Carrizzo A, Ferrario A, Villa F, Damato A, Ambrosio M, Madonna M, Frati G, Fucile S, Sciaccaluga M, Capunzo M, Calì G, Milanesi L, Maciag A, Puca AA, Vecchione C. LAV-BPIFB4 isoform modulates eNOS signalling through Ca2+/PKC-alpha-dependent mechanism. Cardiovasc Res 2018; 113:795-804. [PMID: 28419216 PMCID: PMC5437365 DOI: 10.1093/cvr/cvx072] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 04/12/2017] [Indexed: 12/15/2022] Open
Abstract
Aims Ageing is associated with impairment of endothelial nitric oxide synthase (eNOS) and progressive reduction in endothelial function. A genetic study on long-living individuals—who are characterized by delays in ageing and in the onset of cardiovascular disease—previously revealed I229V (rs2070325) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) as a longevity-associated variant (LAV); the LAV protein enhanced endothelial NO production and vasorelaxation through a protein kinase R–like endoplasmic reticulum kinase/14-3-3/heat shock protein 90 signal. Here, we further characterize the molecular mechanisms underlying LAV-BPIFB4-dependent enhancement of vascular function. Methods and results LAV-BPIFB4 upregulated eNOS function via mobilization of Ca2+ and activation of protein kinase C alpha (PKCα). Indeed, the overexpression of LAV-BPIFB4 in human endothelial cells enhanced ATP-induced Ca2+ mobilization and the translocation of PKCα to the plasma membrane. Coherently, pharmacological inhibition of PKCα blunted the positive effect of LAV-BPIFB4 on eNOS and endothelial function. In addition, although LAV-BPIFB4 lost the ability to activate PKCα and eNOS in ex vivo vessels studied in an external Ca2+-free medium and in vessels from eNOS−/− mice, it still potentiated endothelial activity, recruiting an alternative mechanism dependent upon endothelium-derived hyperpolarizing factor (EDHF). Conclusions We have identified novel molecular determinants of the beneficial effects of LAV-BPIFB4 on endothelial function, showing the roles of Ca2+ mobilization and PKCα in eNOS activation and of EDHF when eNOS is inhibited. These results highlight the role LAV-BPIFB4 can have in restoring signals that are lost during ageing.
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Affiliation(s)
| | | | - Anna Ferrario
- Institute for Biomedical Technologies-National Research Council, 20090 Segrate (MI), Italy
| | | | | | | | | | - Giacomo Frati
- IRCCS Neuromed, 86077 Pozzilli (IS), Italy.,Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome Polo Pontino, C.so della Repubblica 79, 04100 Latina, Italy
| | - Sergio Fucile
- IRCCS Neuromed, 86077 Pozzilli (IS), Italy.,Department of Physiology and Pharmacology, Sapienza University of Rome, 00185 Rome, Italy
| | | | - Mario Capunzo
- Department of Medicine and Surgery, University of Salerno, Via S. Allende, 84081 Baronissi (SA), Italy
| | - Gaetano Calì
- Institute of Experimental Endocrinology and Oncology, National Research Council, 80100 Naples, Italy
| | - Luciano Milanesi
- Institute for Biomedical Technologies-National Research Council, 20090 Segrate (MI), Italy
| | - Anna Maciag
- Ageing Unit, IRCCS MultiMedica, 20138 Milan, Italy
| | - Annibale Alessandro Puca
- Ageing Unit, IRCCS MultiMedica, 20138 Milan, Italy.,Department of Medicine and Surgery, University of Salerno, Via S. Allende, 84081 Baronissi (SA), Italy
| | - Carmine Vecchione
- IRCCS Neuromed, 86077 Pozzilli (IS), Italy.,Department of Medicine and Surgery, University of Salerno, Via S. Allende, 84081 Baronissi (SA), Italy
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In vitro cardiomyocyte differentiation of umbilical cord blood cells: crucial role for c-kit+ cells. Cytotherapy 2015; 17:1627-37. [DOI: 10.1016/j.jcyt.2015.07.012] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Revised: 07/16/2015] [Accepted: 07/20/2015] [Indexed: 11/22/2022]
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Zamilpa R, Navarro MM, Flores I, Griffey S. Stem cell mechanisms during left ventricular remodeling post-myocardial infarction: Repair and regeneration. World J Cardiol 2014; 6:610-620. [PMID: 25068021 PMCID: PMC4110609 DOI: 10.4330/wjc.v6.i7.610] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/28/2013] [Revised: 02/21/2014] [Accepted: 05/14/2014] [Indexed: 02/06/2023] Open
Abstract
Post-myocardial infarction (MI), the left ventricle (LV) undergoes a series of events collectively referred to as remodeling. As a result, damaged myocardium is replaced with fibrotic tissue consequently leading to contractile dysfunction and ultimately heart failure. LV remodeling post-MI includes inflammatory, fibrotic, and neovascularization responses that involve regulated cell recruitment and function. Stem cells (SCs) have been transplanted post-MI for treatment of LV remodeling and shown to improve LV function by reduction in scar tissue formation in humans and animal models of MI. The promising results obtained from the application of SCs post-MI have sparked a massive effort to identify the optimal SC for regeneration of cardiomyocytes and the paradigm for clinical applications. Although SC transplantations are generally associated with new tissue formation, SCs also secrete cytokines, chemokines and growth factors that robustly regulate cell behavior in a paracrine fashion during the remodeling process. In this review, the different types of SCs used for cardiomyogenesis, markers of differentiation, paracrine factor secretion, and strategies for cell recruitment and delivery are addressed.
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Musa S, Xin LZ, Govindasamy V, Fuen FW, Kasim NHA. Global search for right cell type as a treatment modality for cardiovascular disease. Expert Opin Biol Ther 2013; 14:63-73. [DOI: 10.1517/14712598.2014.858694] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Ramkisoensing AA, De Vries AAF, Schalij MJ, Atsma DE, Pijnappels DA. Brief report: Misinterpretation of coculture differentiation experiments by unintended labeling of cardiomyocytes through secondary transduction: delusions and solutions. Stem Cells 2013; 30:2830-4. [PMID: 22987287 DOI: 10.1002/stem.1236] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2012] [Revised: 08/21/2012] [Accepted: 08/29/2012] [Indexed: 11/10/2022]
Abstract
Cardiomyogenic differentiation of stem cells can be accomplished by coculture with cardiomyocytes (CMCs). To facilitate their identification, stem cells are often labeled through viral transduction with a fluorescent protein. A second marker to distinguish stem cell-derived CMCs from native CMCs is rarely used. This study aimed to investigate the occurrence of secondary transduction of unlabeled neonatal rat (nr) CMCs after coculture with human cells that had been transduced 0, 7, or 14 days earlier with a vesicular stomatitis virus (VSV) G protein-pseudotyped lentiviral vector (LV) encoding enhanced green fluorescent protein (GFP). To reduce secondary LV transfer, GFP-labeled cells were incubated with non-heat-inactivated human serum (NHI) or with VSV-neutralizing rabbit serum (αVSV). Heat-inactivated human serum and normal rabbit serum were used as controls. Immunostaining showed substantial GFP gene transfer to nrCMCs in cocultures started at the day of transduction indicated by the presence of GFP-positive/human lamin A/C-negative nrCMCs. The extent of secondary transduction was significantly reduced in cocultures initiated 7 days after GFP transduction, while it was completely abolished when human cells were added to nrCMCs 14 days post-transduction. Both NHI and αVSV significantly reduced the occurrence of secondary transduction compared to their controls. However, under all circumstances, GFP-labeled human cells had to be passaged for 14 days prior to coculture initiation to prevent any horizontal GFP gene transfer to the nrCMCs. This study emphasizes that differentiation experiments involving the use of viral vector-marked donor cells should be interpreted with caution and describes measures to reduce/prevent secondary transduction.
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Affiliation(s)
- Arti A Ramkisoensing
- Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
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Qiu P, Song W, Niu Z, Bai Y, Li W, Pan S, Peng S, Hua J. Platelet-derived growth factor promotes the proliferation of human umbilical cord-derived mesenchymal stem cells. Cell Biochem Funct 2012; 31:159-65. [PMID: 22961649 DOI: 10.1002/cbf.2870] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2012] [Revised: 08/06/2012] [Accepted: 08/08/2012] [Indexed: 01/01/2023]
Abstract
This study was designed to investigate the effect of platelet-derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC-MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC-MSCs. The human UC-MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT-PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real-time (QRT-PCR). The results showed that PDGF could promote the proliferation of UC-MSCs in vitro in a dose-dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC-MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC-MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF-treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation-related genes C-MYC, PCNA and TERT and cell cycle-related genes cyclin A, cyclin 1 and CDK2 were up-regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT-PCR. The PDGF could promote the proliferation of human UC-MSCs in vitro.
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Affiliation(s)
- Pubin Qiu
- College of Veterinary Medicine, Shaanxi Stem Cell Engineering and Technology Research Center, Key Laboratory of Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling 712100, China
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Teo A, Mantalaris A, Lim M. Hydrodynamics and bioprocess considerations in designing bioreactors for cardiac tissue engineering. ACTA ACUST UNITED AC 2012. [DOI: 10.7243/2050-1218-1-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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Avitabile D, Crespi A, Brioschi C, Parente V, Toietta G, Devanna P, Baruscotti M, Truffa S, Scavone A, Rusconi F, Biondi A, D'Alessandra Y, Vigna E, Difrancesco D, Pesce M, Capogrossi MC, Barbuti A. Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process. Am J Physiol Heart Circ Physiol 2011; 300:H1875-84. [PMID: 21357510 DOI: 10.1152/ajpheart.00523.2010] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.
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Affiliation(s)
- Daniele Avitabile
- Department of Biomolecular Sciences and Biotechnology, University of Milan, Milan, Italy
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Di Stefano V, Giacca M, Capogrossi MC, Crescenzi M, Martelli F. Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle. J Biol Chem 2011; 286:8644-8654. [PMID: 21209082 DOI: 10.1074/jbc.m110.184549] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Proliferation of mammalian cardiomyocytes stops rapidly after birth and injured hearts do not regenerate adequately. High cyclin-dependent kinase inhibitor (CKI) levels have been observed in cardiomyocytes, but their role in maintaining cardiomyocytes in a post-mitotic state is still unknown. In this report, it was investigated whether CKI knockdown by RNA interference induced cardiomyocyte proliferation. We found that triple transfection with p21(Waf1), p27(Kip1), and p57(Kip2) siRNAs induced both neonatal and adult cardiomyocyte to enter S phase and increased the nuclei/cardiomyocyte ratio; furthermore, a subpopulation of cardiomyocytes progressed beyond karyokynesis, as assessed by the detection of mid-body structures and by straight cardiomyocyte counting. Intriguingly, cardiomyocyte proliferation occurred in the absence of overt DNA damage and aberrant mitotic figures. Finally, CKI knockdown and DNA synthesis reactivation correlated with a dramatic change in adult cardiomyocyte morphology that may be a prerequisite for cell division. In conclusion, CKI expression plays an active role in maintaining cardiomyocyte withdrawal from the cell cycle.
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Affiliation(s)
- Valeria Di Stefano
- From the Molecular Cardiology Laboratory, IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy
| | - Mauro Giacca
- Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, 34149 Trieste, Italy
| | - Maurizio C Capogrossi
- Vascular Pathology Laboratory, Istituto Dermopatico dell'Immacolata-IRCCS, 00167 Rome, Italy, and
| | - Marco Crescenzi
- the Department of Environment and Primary Prevention, Istituto Superiore di Sanità, 00161 Rome, Italy
| | - Fabio Martelli
- Vascular Pathology Laboratory, Istituto Dermopatico dell'Immacolata-IRCCS, 00167 Rome, Italy, and.
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Dynamics of progenitor cells and ventricular assist device intervention. J Cardiovasc Transl Res 2010; 3:147-52. [PMID: 20560028 DOI: 10.1007/s12265-009-9141-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/25/2009] [Accepted: 10/05/2009] [Indexed: 10/20/2022]
Abstract
Experimental and clinical evidence suggests that a heterogeneous group of bone-marrow-derived circulating progenitor cells, with variations in phenotype and function, provide an endogenous repair mechanism, contributing to vascular healing and remodeling under physiological and pathological conditions, such as cancer, atherosclerosis, myocardial infarction, and end-stage heart failure. Implantation of ventricular assist devices (VADs) for circulatory support is indicated in selected patients with end-stage heart failure as a bridge to heart transplantation, however seldom; improvement of ventricular contractility has been well documented with prolonged cardiac unloading. The current review summarizes recent findings from in vitro and in vivo studies, focusing on the biological features and the possible role of progenitor cells in the transient myocardial recovery, occasionally seen after VAD implantation, and speculates on their clinical utilities for the treatment of the failing human heart.
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Abstract
The use of stem cells for cardiac regeneration is a revolutionary, emerging research area. For proper function as replacement tissue, stem cell-derived cardiomyocytes (SC-CMs) must electrically couple with the host cardiac tissue. Electrophysiological mapping techniques, including microelectrode array (MEA) and optical mapping, have been developed to study cardiomyocytes and cardiac cell monolayers, and these can be applied to study stem cells and SC-CMs. MEA recordings take extracellular measurements at numerous points across a small area of cell cultures and are used to assess electrical propagation during cell culture. Optical mapping uses fluorescent dyes to monitor electrophysiological changes in cells, most commonly transmembrane potential and intracellular calcium, and can be easily scaled to areas of different sizes. The materials and methods for MEA and optical mapping are presented here, together with detailed notes on their use, design, and fabrication. We also provide examples of voltage and calcium maps of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs), obtained in our laboratory using optical mapping techniques.
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Affiliation(s)
- Seth Weinberg
- Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, USA
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Jovin IS, Giordano FJ. Differentiation by association: is a cell's fate determined by the company it keeps? Am J Physiol Heart Circ Physiol 2008; 294:H1503-4. [PMID: 18296559 DOI: 10.1152/ajpheart.00138.2008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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