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AimsCongestive heart failure (CHF) can be thought of as a state of chronic immune activation. Polymorphonuclear neutrophil (PMN) apoptosis is one of the mechanisms responsible for the resolution of inflammation. A reduced PMN apoptotic rate in CHF patients may generate a persistent inflammatory response and hence mediate tissue damage in this group of patients. We aimed to measure levels of spontaneous apoptosis of circulating PMNs in CHF patients and in controls, and to examine whether NYHA class, left ventricular ejection fraction (LV-EF), and laboratory parameters of inflammation, endothelial damage, and of liver and renal function, could predict the rate of PMN apoptosis in CHF patients. Methods and resultsA total of 29 CHF patients and 26 controls were studied. Propidium iodide and flow cytometry were used to assess PMN apoptosis. Delay in PMN apoptosis was expressed as percentage (expressed as median, first and third quartiles) of surviving PMNs in the study subjects. We found an increased percentage of surviving PMNs [38(27.1 -47.1)] in CHF patients compared with controls [19.4 (15.8-25.2)] (P , 0.05). The PMN survival rate in the CHF group was correlated to NYHA class, and plasma levels of C-reactive protein and alkaline phosphatase, while it was inversely correlated to LV-EF and protein levels. A positive relationship between PMN survival and increased ex vivo endothelial apoptosis was found. ConclusionIncreased PMN lifespan in patients with worsening CHF could be used as a novel measurement of tissue and endothelial damage in this group of patients.--

BackgroundInsulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.Methods and ResultsBesides inducing apoptosis, concentrations of doxorubicin comparable to those observed in patients after bolus infusion (0.1-1 µM) caused a progressive decrease in IGF-1R and increase in IGFBP-3 expression. Exogenous IGF-1 was capable to rescue cardiomyocytes from apoptosis triggered by 0.1 and 0.5 µM, but not 1 µM doxorubicin. The loss of response to IGF-1 was paralleled by a significant reduction in IGF-1 availability and signaling, as assessed by free hormone levels in conditioned media and Akt phosphorylation in cell lysates, respectively. Doxorubicin also dose-dependently induced p53, which is known to repress the transcription of IGF1R and induce that of IGFBP3. Pre-treatment with the p53 inhibitor, pifithrin-α, prevented apoptosis and the changes in IGF-1R and IGFBP-3 elicited by doxorubicin. The decrease in IGF-1R and increase in IGFBP-3, as well as apoptosis, were also antagonized by pre-treatment with the antioxidant agents, N-acetylcysteine, dexrazoxane, and carvedilol.ConclusionsDoxorubicin down-regulates IGF-1R and up-regulates IGFBP-3 via p53 and oxidative stress in H9c2 cells. This leads to resistance to IGF-1 that may contribute to doxorubicin-initiated apoptosis. Further studies are needed to confirm these findings in human cardiomyocytes and explore the possibility of manipulating the IGF-1 axis to protect against anthracycline cardiotoxicity.

BackgroundThe ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3′UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression.ResultsThe ACVR1/Alk-2 3′UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3′UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3′UTR sequence thereby modulating its expression.ConclusionIn this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3′UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3′UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches.