The histone acetylase activator pentadecylidenemalonate 1b rescues proliferation and differentiation in the human cardiac mesenchymal cells of type 2 diabetic patients.

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Magnetic resonance imaging of human endothelial progenitors reveals opposite effects on vascular and muscle regeneration into ischaemic tissues.

AIMS: The assessment of progenitor cell survival and efficacy after transplantation is one of the major challenges in cardiovascular cell therapy. Translation of currently used imaging techniques to patients is not immediate. Possible options include iron oxide particle loading into cells to be tracked using magnetic resonance (MR) and by MR-based water diffusion anisotropy analysis. The aim of the present study was to assess, using these techniques, the localization and survival of human 'early' endothelial progenitor cells (EPCs) and their effects on vascular and skeletal muscle regeneration in a mouse model of hind limb ischaemia. METHODS AND RESULTS: A paramagnetic iron oxide particle loading protocol of human peripheral blood-derived early EPCs was devised. The iron+ EPCs maintained their phenotype and in vitro functional activity. In addition, the presence of iron+ cells was observed by MR until 7 days after injection into a pharmacologically immunosuppressed mouse model of hind limb ischaemia. Immunohistochemistry with human major histocompatibility complex antibodies revealed the absence of human cells at 7 days post-ischaemia. EPC death was confirmed by staining of iron+ cells with an anti-mouse CD68 antibody and by qPCR performed on DNA extracted from injected ischaemic limbs, at different times following injection. Surprisingly, early EPC injection enhanced arteriogenesis but caused a significant increase in ischaemic tissue inflammation and a retarded muscle regeneration, as evidenced by water diffusion anisotropy analysis and histology. CONCLUSION: In line with recent reports, our results show that the use of iron-based contrast agents does not allow detection of long-term EPC engraftment into ischaemic tissues. They further show that early EPCs exert a potent arteriogenic effect on ischaemic tissues that is not dependent on their prolonged survival. Unexpectedly, injection of these cells elicited a long-term inflammatory response that reflected a delayed muscle healing process.

GMP-based CD133(+) cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

SMC1 involvement in fragile site expression.

Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.