For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic‐specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl‐2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl‐2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl‐2 proteins and the ways they regulate the apoptosis.

Stem cell-based therapy for myocardial regeneration has reported several functional improvements that are attributed mostly to the paracrine effects stimulating angiogenesis and cell survival. This study was conducted to comparatively evaluate the potential of factors secreted by mesenchymal stem cells (MSCs) in normoxic and hypoxic conditions to promote tissue repair by sustaining endothelial cell (EC) adhesion and proliferation and conferring protection against apoptosis. To this aim, a conditioned medium (CM) was generated from MSCs after 24-h incubation in a serum-free normal or hypoxic environment. MSCs exhibited resistance to hypoxia, which induced increased secretion of vascular endothelial growth factor (VEGF) and decreased levels of other cytokines, including stromal-derived factor-1 (SDF). The CM derived from normal (nMSC-CM) and hypoxic cells (hypMSC-CM) induced similar protective effects on H9c2 cells in hypoxia. Minor differences were noticed in the potential of normal versus hypoxic CM to promote angiogenesis, which were likely connected to SDFa and VEGF levels: the nMSC-CM was more effective in stimulating EC migration, whereas the hypMSC-CM had an enhanced effect on EC adhesion. However, the factors secreted by MSCs in normoxic or hypoxic conditions supported adhesion, but not proliferation, of ECs in vitro, as revealed by impedance-based dynamic assessments. Surprisingly, factors secreted by other stem/progenitor cells, such as endothelial progenitor cells (EPCs), had complementary effects to the MSC-CM. Thus, the EPC-CM, in either a normal or hypoxic environment, supported EC proliferation, but did not sustain EC adhesion. Combined use of the MSC-CM and EPC-CM promoted both EC adhesion and proliferation, suggesting that the local angiogenesis at the site of ischemic injury might be better stimulated by simultaneous releasing of factors secreted by multiple stem/progenitor cell populations.

Objective— Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN ) as a member of several coronary artery disease–associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results— In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr −/− mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN’s role in vascular endothelial function, we administered α-1-PDX to Apoe −/− mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions— We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Mesenchymal stromal cells (MSC) are attractive tools for cell-based therapy, yet the mechanisms underlying their migration and survival post-transplantation are unclear. Accumulating evidence indicates that MSC apoptosis modulates both innate and adaptive immune responses which impact on MSC therapeutic effects. Using a dual tracking system, namely the Luciferase expression and VivoTrack680 labelling, and in vivo optical imaging, we investigated the survival and migration of MSC transplanted by various routes (intravenous, subcutaneous, intrapancreatic and intrasplenic) in order to identify the best delivery approach that provides an accumulation of therapeutic cells to the injured pancreas in the non-obese diabetic (NOD) mouse. The results showed that transplanted MSC had limited migration capacity, irrespective of the administration route, and were short-lived with almost total disappearance at 7 days after transplantation. Within one day after transplantation, cells activated hypoxia signalling pathways, followed by Caspase 3-mediated apoptosis. These were subsequently followed by local recruitment of immune cells at the transplantation site, and the engulfment of apoptotic MSC by macrophages. Our results argue for a “hit and die” mechanism of transplanted MSC. Further investigations will elucidate the molecular crosstalk between the inoculated and the host-immune cells.

The treatment of cardiac diseases by cell therapy continues to be challenged by a limited supply of appropriate cells. Although stem cells can generate myocytes after local delivery into the heart, this is often accompanied by the generation of several other cell types as a consequence of environment-driven differentiation. One strategy for overcoming dysregulated differentiation is the pretreatment of stem cells with the demethylation agent 5-azacytidine. The effects of 5-azacytidine on various stem cell types vary from cardiomyogenic differentiation to failure of differentiation or from adipogenic and chondrogenic differentiation to uncontrollable expression of a variety of genes. The underlying mechanisms remain poorly understood, and the effect of 5-azacytidine on the multipotent capacity of stem cells has never been addressed. This study was designed to investigate the changes induced by 5-azacytidine in mesenchymal stem cells (MSC), with particular focus on multipotency maintenance and the capacity of 5-azacytidine to boost myogenic differentiation. Our results show that MSCs retained their multipotent capacity after one pulse with 5-azacytidine, whereas additional pulses resulted in a restricted differentiation potential with concomitant increased ability to accomplish chondrogenic commitment. The induction of cardiac differentiation of MSCs was not observed unless the transcriptional activation of several genes was induced by random hypomethylation. Nevertheless, 5-azacytidine treatment promoted cell response to subsequent stimuli and generation of myogenic differentiation under permissive environmental conditions. Therefore, we assume that one pulse with 5-azacytidine might similarly promote the subsequent cardiac differentiation of MSCs, but it is dependent on the finding of adequate conditions for myocardial differentiation.

Cardioprotection can be evoked through extracardiac approaches. This prompted us to investigate whether remote transplantation of stem cells confers protection of the heart against ischemic injury. The cardioprotective effect of subcutaneous transplantation of na€ ıve versus heme oxygenase-1 (HMOX-1)-overexpressing mouse mesenchymal stem cells (MSC) to mice was investigated in hearts subjected to ischemia-reperfusion in a Langendorff perfusion system. Mice were transplanted into the interscapular region with na€ ıve or HMOX-1 transfected MSC isolated from transgenic luciferase reporter mice and compared to sham-treated animals. The fate of transplanted cells was followed by in vivo bioluminescence imaging, revealing that MSC proliferated, but did not migrate detectably from the injection site. Ex vivo analysis of the hearts showed that remote transplantation of mouse adipose-derived MSC (mASC) resulted in smaller infarcts and improved cardiac function after ischemia-reperfusion compared to sham-treated mice. Although HMOX-1 overexpression conferred cytoprotective effects on mASC against oxidative stress in vitro, no additive beneficial effect of HMOX-1 transfection was noted on the ischemic heart. Subcutaneous transplantation of MSC also improved left ventricular function when transplanted in vivo after myocardial infarction. Plasma analysis and gene expression profile of na€ ıve-and HMOX-1-mASC after transplantation pointed toward pentraxin 3 as a possible factor involved in the remote cardioprotective effect of mASC. These results have significant implications for understanding the behavior of stem cells after transplantation and development of safe and noninvasive cellular therapies with clinical applications. Remote transplantation of MSC can be considered as an alternative procedure to induce cardioprotection. STEM CELLS

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The role of reactive oxygen species (ROS) in the pathogenesis of vascular diseases is well established, but few data exist on the mechanisms by which ROS induce endothelial cell (EC) death. We examined the conditions and the mechanisms by which oxidative stress induces EC death, using cultured confluent bovine aortic ECs exposed for 30 min to different concentrations of hydroxyl radicals (HO*) generated by hydrogen peroxide (H(2)O(2)) in the presence of 100 microM ferrous sulfate (FeSO(4)). Cell viability assays, Hoechst DNA staining, TUNEL (TDT-mediated dUTP-biotin nick end-labeling) analysis, agarose gel electrophoresis and annexin V assay were used to determine the effect of HO* on the viability of ECs, and to distinguish between apoptosis and necrosis. The results showed that at concentrations of up to 0.1 mM H(2)O(2)/FeSO(4), the large majority of cells are viable, except for approximately 12.5% death, which occurs by apoptosis. At a concentration of 0.2 mM H(2)O(2), the cell viability is reduced to 66%, while EC apoptosis remained at comparable values (14%). At high oxidative stress (0.5 mM H(2)O(2)), the cell viability was drastically reduced (approximately 39%), and the prevalent form of death was necrosis; apoptosis accounted for only approximately 17%. Together, these data indicate that: (1) HO* induce EC death either by apoptosis or necrosis and (2) the mechanisms of EC death differ as a function of the concentration of HO. Thus, the same insult can cause apoptosis and/or necrosis, as a function of the intensity rather than the nature of the insult.