GPR17 is a dual receptor that is activated by both uracil nucleotides and cysteinylleukotrienes, two ligands families that are massively released in tissues undergoing hypoxia. Our previous data showed that GPR17 is highly expressed into organs susceptible to ischemic damage such as brain, heart and kidney. The heart contains cardiac resident stem cells (CSCs) that are involved in myocardium renewal and, possibly, myocardial repair after ischemia. It has been demonstrated that acute or chronic myocardial damage activates proliferation of CSCs; however ischemic damage has also a detrimental effect on self renewal of CSCs population by improving their senescence and limiting their self-renewal ability that ultimately leads to CSCs population exhaustion. On this basis, we made the hypothesis that GRP17 is involved in CSCs activation/failure following ischemia. We therefore investigated its expression by immunofluorescence and real-time RT-PCR in a mouse Stem Cell Antigen-1 (Sca-1) positive CSC primary cell line isolated in our laboratory from adult mouse hearts as well as by immunofluorescence followed by confocal imaging in myocardial tissue sections before and at various times following infarction (MI) by coronary artery ligation. Results on the CSCs primary line showed expression of GPR17 at high levels in undifferentiated Sca-1+ cells and co-expression with cardiac markers Nkx 2.5 and α- sarcomeric actin upon induction of cardiac differentiation (Figure 1). Results of ex vivo investigation in fixed myocardial sections showed the presence of GPR17 before and after ischemia (Figure 2). Before ischemia, GPR17 was localized in clusters of small cells (resembling CSCs) in close association with myocytes; after infarction it was expressed in small as well as larger cells infiltrating the ischemic tissue. Interestingly, these large cells (that were absent in the non-ischemic tissue) were labelled by Isolectin-B4, suggesting that they are blood-borne macrophages infiltrating the infarcted heart. Taken together, these results suggest that GPR17 is expressed in undifferentiated and differentiated CSCs in vitro and in vivo, and that it undergoes a dynamic regulation in cells associated to inflammatory response after MI. These dynamic changes closely resemble those found in the brain after induction of cerebral ischemia, when GPR17 is activated on residential adult stem-like cells in the peri-lesional brain parenchyma and is induced in microglia/macrophages entering the ischemic core to remodel it after injury (Ciana et al., 2006; Lecca et al., 2008). Current analyses are ongoing to characterize GPR17 intracellular signalling in CSCs by stimulation with specific agonists in vitro and reveal possible effects on modification of CSCs self-renewal/proliferation/differentiation; in addition, an immunofluorescence analysis on myocardial tissue is currently carried out in order to unveil the identity of GPR17+ cells before and after MI in vivo. FIGURE 1 FIGURE 2 References [1] Chimenti, C. et al. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res 93, 604-13 (2003).C.G. Dávila, P.P. Camanho, C.A. Rose, Failure criteria for FRP laminates. Journal of Composite Materials, 39, 323-345, 2005. [2] Ciana, P. et al. The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinylleukotrienes receptor. Embo J 25, 4615-27 (2006). [3] Lecca, D. et al. The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair. PLoS One 3, e3579 (2008). [4] Di Virgilio, F., Ceruti, S., Bramanti, P. & Abbracchio, M. P. Purinergic signalling in inflammation of the central nervous system. Trends Neurosci 32, 79-87 (2009).  Biochimica Biotecnologie  Cardiovascolare  Neuroscienze  Infiammazione Tossicologia Farmacognosia [5] Smits, A. M. et al. Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc 4, 232-43 (2009). [6] Goumans, M. J. et al. TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro. Stem Cell Res 1, 138-49 (2007). [7] Matsuura, K. et al. Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. J Clin Invest 119, 2204-17 (2009). [8] Oh, H. et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 100, 12313-8. (2003).

Expression and function of Uracil nucleotides/Cysteinyl-Leukotrienes dual GPR17 receptor in mouse cardiac derived stem cells / S. Cosentino, M. Pesce, E. Nobili, L. Sironi, L. Castiglioni, M.C. Capogrossi, E. Tremoli, M.P. Abbracchio. ((Intervento presentato al convegno Next step : la giovane ricerca avanza tenutosi a Milano nel 2010.

Expression and function of Uracil nucleotides/Cysteinyl-Leukotrienes dual GPR17 receptor in mouse cardiac derived stem cells

S. Cosentino
Primo
;
E. Nobili;L. Sironi;L. Castiglioni;E. Tremoli
Penultimo
;
M.P. Abbracchio
Ultimo
2010-07-01

Abstract

GPR17 is a dual receptor that is activated by both uracil nucleotides and cysteinylleukotrienes, two ligands families that are massively released in tissues undergoing hypoxia. Our previous data showed that GPR17 is highly expressed into organs susceptible to ischemic damage such as brain, heart and kidney. The heart contains cardiac resident stem cells (CSCs) that are involved in myocardium renewal and, possibly, myocardial repair after ischemia. It has been demonstrated that acute or chronic myocardial damage activates proliferation of CSCs; however ischemic damage has also a detrimental effect on self renewal of CSCs population by improving their senescence and limiting their self-renewal ability that ultimately leads to CSCs population exhaustion. On this basis, we made the hypothesis that GRP17 is involved in CSCs activation/failure following ischemia. We therefore investigated its expression by immunofluorescence and real-time RT-PCR in a mouse Stem Cell Antigen-1 (Sca-1) positive CSC primary cell line isolated in our laboratory from adult mouse hearts as well as by immunofluorescence followed by confocal imaging in myocardial tissue sections before and at various times following infarction (MI) by coronary artery ligation. Results on the CSCs primary line showed expression of GPR17 at high levels in undifferentiated Sca-1+ cells and co-expression with cardiac markers Nkx 2.5 and α- sarcomeric actin upon induction of cardiac differentiation (Figure 1). Results of ex vivo investigation in fixed myocardial sections showed the presence of GPR17 before and after ischemia (Figure 2). Before ischemia, GPR17 was localized in clusters of small cells (resembling CSCs) in close association with myocytes; after infarction it was expressed in small as well as larger cells infiltrating the ischemic tissue. Interestingly, these large cells (that were absent in the non-ischemic tissue) were labelled by Isolectin-B4, suggesting that they are blood-borne macrophages infiltrating the infarcted heart. Taken together, these results suggest that GPR17 is expressed in undifferentiated and differentiated CSCs in vitro and in vivo, and that it undergoes a dynamic regulation in cells associated to inflammatory response after MI. These dynamic changes closely resemble those found in the brain after induction of cerebral ischemia, when GPR17 is activated on residential adult stem-like cells in the peri-lesional brain parenchyma and is induced in microglia/macrophages entering the ischemic core to remodel it after injury (Ciana et al., 2006; Lecca et al., 2008). Current analyses are ongoing to characterize GPR17 intracellular signalling in CSCs by stimulation with specific agonists in vitro and reveal possible effects on modification of CSCs self-renewal/proliferation/differentiation; in addition, an immunofluorescence analysis on myocardial tissue is currently carried out in order to unveil the identity of GPR17+ cells before and after MI in vivo. FIGURE 1 FIGURE 2 References [1] Chimenti, C. et al. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res 93, 604-13 (2003).C.G. Dávila, P.P. Camanho, C.A. Rose, Failure criteria for FRP laminates. Journal of Composite Materials, 39, 323-345, 2005. [2] Ciana, P. et al. The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinylleukotrienes receptor. Embo J 25, 4615-27 (2006). [3] Lecca, D. et al. The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair. PLoS One 3, e3579 (2008). [4] Di Virgilio, F., Ceruti, S., Bramanti, P. & Abbracchio, M. P. Purinergic signalling in inflammation of the central nervous system. Trends Neurosci 32, 79-87 (2009).  Biochimica Biotecnologie  Cardiovascolare  Neuroscienze  Infiammazione Tossicologia Farmacognosia [5] Smits, A. M. et al. Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc 4, 232-43 (2009). [6] Goumans, M. J. et al. TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro. Stem Cell Res 1, 138-49 (2007). [7] Matsuura, K. et al. Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. J Clin Invest 119, 2204-17 (2009). [8] Oh, H. et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A 100, 12313-8. (2003).
GPR17 ; mouse ; Cardiac Stem Cell ; Sca1 ; Purinergic/Cysteinyl-Leukotriene receptors
Settore BIO/14 - Farmacologia
Centro di Ricerche Farmacologiche per lo Studio e la Prevenzione delle Malattie Cardiovascolari CRF
Expression and function of Uracil nucleotides/Cysteinyl-Leukotrienes dual GPR17 receptor in mouse cardiac derived stem cells / S. Cosentino, M. Pesce, E. Nobili, L. Sironi, L. Castiglioni, M.C. Capogrossi, E. Tremoli, M.P. Abbracchio. ((Intervento presentato al convegno Next step : la giovane ricerca avanza tenutosi a Milano nel 2010.
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