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

RIPK1 protects from TNF-α-mediated liver damage during hepatitis

Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-alpha) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-alpha-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1K45A) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1LPC-KO), to characterize the role of RIPK1 and TNF-alpha in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1LPC-KO mice challenged with ConA, TNF-alpha triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-kappa B activation, as well as TNF-dependent, but canonical NF-kappa B-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases

Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

Aims High-mobility group box 1 (HMGB1) is a nuclear protein actively secreted by immune cells and passively released by necrotic cells that initiates pro-inflammatory signalling through binding to the receptor for advance glycation end-products. HMGB1 has been established as a key inflammatory mediator during myocardial infarction, but the proximal mechanisms responsible for myocardial HMGB1 expression and release in this setting remain unclear. Here, we investigated the possible involvement of peroxynitrite, a potent cytotoxic oxidant formed during myocardial infarction, on these processes. Methods and results The ability of peroxynitrite to induce necrosis and HMGB1 release in vitro was evaluated in H9c2 cardiomyoblasts and in primary murine cardiac cells (myocytes and non-myocytes). In vivo, myocardial HMGB1 expression and nitrotyrosine content (a marker of peroxynitrite generation) were determined following myocardial ischaemia and reperfusion in rats, whereas peroxynitrite formation was inhibited by two different peroxynitrite decomposition catalysts: 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III) (FeTPPS) or Mn(III)-tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). In all types of cells studied, peroxynitrite (100 μM) elicited significant necrosis, the loss of intracellular HMGB1, and its passive release into the medium. In vivo, myocardial ischaemia-reperfusion induced significant myocardial necrosis, cardiac nitrotyrosine formation, and marked overexpression of myocardial HMGB1. FeTPPS reduced nitrotyrosine, decreased infarct size, and suppressed HMGB1 overexpression, an effect that was similarly obtained with MnTBAP. Conclusion These findings indicate that peroxynitrite represents a key mediator of HMGB1 overexpression and release by cardiac cells and provide a novel mechanism linking myocardial oxidative/nitrosative stress with post-infarction myocardial inflammatio

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

Cyclin-Dependent Kinase 5 Promotes Pancreatic β-Cell Survival via Fak-Akt Signaling Pathways

ObjectiveCyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1-like 1 has recently been linked to type 2 diabetes by genome-wide association studies. While CDK5 and its regulatory protein p35 are both expressed and display enzymatic activity in pancreatic β-cells, their precise role in the β-cell remains unknown. Because type 2 diabetes is characterized by a deficit in β-cell mass and increased β-cell apoptosis, we investigated the role of CDK5 in β-cell survival.Research design and methodsWe used INS 832/13 cells, rat islets isolated from wild-type or human islet amyloid polypeptide (h-IAPP) transgenic rats, and pancreatic tissue from rats and humans with and without type 2 diabetes and investigated the effect of CDK5/p35 inhibition (by small interfering RNA or by chemical inhibition) as well as CDK5/p35 overexpression on β-cell vulnerability to apoptosis.ResultsCDK5 inhibition led to increased β-cell apoptosis. To identify the mechanisms involved, we examined the phosphorylation state of focal adhesion kinase (Fak)(Ser732), a known target of CDK5. Following CDK5 inhibition, the phosphorylation of Fak(Ser732) decreased with resulting attenuation of phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway. Conversely, CDK5 overexpression increased Fak(Ser732) phosphorylation and protected β-cells against apoptosis induced by the inhibition of the β-1 integrin signaling pathway. Also, Fak(Ser732) phosphorylation was less abundant in β-cells in both h-IAPP transgenic rats and humans with type 2 diabetes.ConclusionsThis study shows that by regulating Fak phosphorylation and subsequently PI3K/Akt survival pathway, CDK5 plays a previously unrecognized role in promoting β-cell survival

Pathogenic Mouse Hepatitis Virus or Poly(I:C) Induce IL-33 in Hepatocytes in Murine Models of Hepatitis.

International audienceThe IL-33/ST2 axis is known to be involved in liver pathologies. Although, the IL-33 levels increased in sera of viral hepatitis patients in human, the cellular sources of IL-33 in viral hepatitis remained obscure. Therefore, we aimed to investigate the expression of IL-33 in murine fulminant hepatitis induced by a Toll like receptor (TLR3) viral mimetic, poly(I:C) or by pathogenic mouse hepatitis virus (L2-MHV3). The administration of poly(I:C) plus D-galactosamine (D-GalN) in mice led to acute liver injury associated with the induction of IL-33 expression in liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells (VEC), while the administration of poly(I:C) alone led to hepatocyte specific IL-33 expression in addition to vascular IL-33 expression. The hepatocyte-specific IL-33 expression was down-regulated in NK-depleted poly(I:C) treated mice suggesting a partial regulation of IL-33 by NK cells. The CD1d KO (NKT deficient) mice showed hepatoprotection against poly(I:C)-induced hepatitis in association with increased number of IL-33 expressing hepatocytes in CD1d KO mice than WT controls. These results suggest that hepatocyte-specific IL-33 expression in poly(I:C) induced liver injury was partially dependent of NK cells and with limited role of NKT cells. In parallel, the L2-MHV3 infection in mice induced fulminant hepatitis associated with up-regulated IL-33 expression as well as pro-inflammatory cytokine microenvironment in liver. The LSEC and VEC expressed inducible expression of IL-33 following L2-MHV3 infection but the hepatocyte-specific IL-33 expression was only evident between 24 to 32h of post infection. In conclusion, the alarmin cytokine IL-33 was over-expressed during fulminant hepatitis in mice with LSEC, VEC and hepatocytes as potential sources of IL-33

The pancreatic beta cell surface proteome

The pancreatic beta cell is responsible for maintaining normoglycaemia by secreting an appropriate amount of insulin according to blood glucose levels. The accurate sensing of the beta cell extracellular environment is therefore crucial to this endocrine function and is transmitted via its cell surface proteome. Various surface proteins that mediate or affect beta cell endocrine function have been identified, including growth factor and cytokine receptors, transporters, ion channels and proteases, attributing important roles to surface proteins in the adaptive behaviour of beta cells in response to acute and chronic environmental changes. However, the largely unknown composition of the beta cell surface proteome is likely to harbour yet more information about these mechanisms and provide novel points of therapeutic intervention and diagnostic tools. This article will provide an overview of the functional complexity of the beta cell surface proteome and selected surface proteins, outline the mechanisms by which their activity may be modulated, discuss the methods and challenges of comprehensively mapping and studying the beta cell surface proteome, and address the potential of this interesting subproteome for diagnostic and therapeutic applications in human disease

Bio-inspired computation: where we stand and what's next

In recent years, the research community has witnessed an explosion of literature dealing with the adaptation of behavioral patterns and social phenomena observed in nature towards efficiently solving complex computational tasks. This trend has been especially dramatic in what relates to optimization problems, mainly due to the unprecedented complexity of problem instances, arising from a diverse spectrum of domains such as transportation, logistics, energy, climate, social networks, health and industry 4.0, among many others. Notwithstanding this upsurge of activity, research in this vibrant topic should be steered towards certain areas that, despite their eventual value and impact on the field of bio-inspired computation, still remain insufficiently explored to date. The main purpose of this paper is to outline the state of the art and to identify open challenges concerning the most relevant areas within bio-inspired optimization. An analysis and discussion are also carried out over the general trajectory followed in recent years by the community working in this field, thereby highlighting the need for reaching a consensus and joining forces towards achieving valuable insights into the understanding of this family of optimization techniques