0232: A new role of the brain natriuretic peptide in the heart: Modulation of cardiac precursor cell proliferation and differentiation

The actual role of the brain natriuretic peptide (BNP) in the heart remains elusive despite its reported protective effect in ischemic animal hearts. Because recently BNP was shown to control the proliferation and differentiation of murine embryonic stem cells, we asked in this study whether BNP could influence the proliferation and differentiation of cardiac progenitor cells (CPC) in vitro and in vivo. We first identified a c-kit+ Sca-1+ cell population present in neonatal and adult hearts which expressed the NPR-A and NPR-B receptors. In vitro, these cells proliferated and in presence of BNP differentiated into CPCs (c-kit+ Sca-1+ Nkx2.5+) and into mature cardiomyocytes. In parallel, BNP was injected to newborn and adult healthy mice (n=6 mice per group). In the hearts of both neonatal and adult mice, BNP injection increased the number of newly formed cardiomyocytes (neonatal: + 23%, p= 0.009 and adult: +68%, p= 0.005) and the number of CPCs (neonatal: + 142%, p= 0.002 and adult: +134%, p= 0.04). BrdU injection to neonatal BNP treated mice demonstrated that BNP stimulated CPC proliferation. In anticipation that BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction (n=6-7 mice per group). Higher numbers of Nkx2.5+ cells were detected in both the infarcted (+38%, p=0.03) and non infarcted areas (+69%, p=0.02) of BNP treated hearts one week after surgery. Finally, by isolating neonatal cardiac cells from the hearts of NPR-A or NPR-B deficient mice, we demonstrated that BNP modulates the fate of CPCs via NPRB binding and that long term BNP treatment is correlated in vitro and in vivo with decreased Protein Kinase G activity. Our results highlight a new key role for BNP in the control of CPC proliferation and/or differentiation. This new function of BNP should be evaluated in therapies aimed to induce cardiac cell regeneration and should reopen the debate about the therapeutic use of BNP for patients suffering from heart diseases

Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organism

Brain natriuretic peptide is able to stimulate cardiac progenitor cell proliferation and differentiation in murine hearts after birth

Brain natriuretic peptide (BNP) contributes to heart formation during embryogenesis. After birth, despite a high number of studies aimed at understanding by which mechanism(s) BNP reduces myocardial ischemic injury in animal models, the actual role of this peptide in the heart remains elusive. In this study, we asked whether BNP treatment could modulate the proliferation of endogenous cardiac progenitor cells (CPCs) and/or their differentiation into cardiomyocytes. CPCs expressed the NPR-A and NPR-B receptors in neonatal and adult hearts, suggesting their ability to respond to BNP stimulation. BNP injection into neonatal and adult unmanipulated mice increased the number of newly formed cardiomyocytes (neonatal: +23%, p=0.009 and adult: +68%, p=0.0005) and the number of proliferating CPCs (neonatal: +142%, p=0.002 and adult: +134%, p=0.04). In vitro, BNP stimulated CPC proliferation via NPR-A and CPC differentiation into cardiomyocytes via NPR-B. Finally, as BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction. In pathological conditions, BNP treatment was cardioprotective by increasing heart contractility and reducing cardiac remodelling. At the cellular level, BNP stimulates CPC proliferation in the non-infarcted area of the infarcted hearts. In the infarcted area, BNP modulates the fate of the endogenous CPCs but also of the infiltrating CD45+ cells. These results support for the first time a key role for BNP in controlling the progenitor cell proliferation and differentiation after birth. The administration of BNP might, therefore, be a useful component of therapeutic approaches aimed at inducing heart regeneration