Alexandrina Burlacu

Regulation of apoptosis by Bcl‐2 family proteins

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.

Factors secreted by mesenchymal stem cells and endothelial progenitor cells have complementary effects on angiogenesis in vitro.

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 SDFα 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.

Effect of 5-azacytidine: evidence for alteration of the multipotent ability of mesenchymal stem 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.

Remote transplantation of mesenchymal stem cells protects the heart against ischemia-reperfusion injury.

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 naïve 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 naïve 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 naïve‐ 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 2014;32:2123–2134

Controlled intramyocardial release of engineered chemokines by biodegradable hydrogels as a treatment approach of myocardial infarction.

Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time‐span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time‐span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met‐CCL5 over 24 hrs and a slow degradable hydrogel (SDH) for a gradual release of protease‐resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time‐controlled release using Met‐CCL5‐FDH and CXCL12 (S4V)‐SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time‐controlled, biopolymer‐mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell‐based therapies.

Noninvasive Molecular Ultrasound Monitoring of Vessel Healing After Intravascular Surgical Procedures in a Preclinical Setup

Objective—Cardiovascular interventions induce damage to the vessel wall making antithrombotic therapy inevitable until complete endothelial recovery. Without a method to accurately determine the endothelial status, many patients undergo prolonged anticoagulation therapy, denying them any invasive medical procedures, such as surgical operations and dental interventions. Therefore, we aim to introduce molecular ultrasound imaging of the vascular cell adhesion molecule (VCAM)-1 using targeted poly-n-butylcyanoacrylate microbubbles (MBVCAM-1) as an easy accessible method to monitor accurately the reendothelialization of vessels. Approach and Results—ApoE−/− mice were fed with an atherogenic diet for 1 and 12 weeks and subsequently, endothelial denudation was performed in the carotid arteries using a guidewire. Molecular ultrasound imaging was performed at different time points after denudation (1, 3, 7, and 14 days). An increased MBVCAM-1 binding after 1 day, a peak after 3 days, and a decrease after 7 days was found. After 12 weeks of diet, MBVCAM-1 binding also peaked after 3 days but remained high until 7 days, indicating a delay in endothelial recovery. Two-photon laser scanning microscopy imaging of double fluorescence staining confirmed the exposure of VCAM-1 on the superficial layer after arterial injury only during the healing phase. After complete reendothelialization, VCAM-1 expression persisted in the subendothelial layer but was not reachable for the MBVCAM-1 anymore. Conclusion—Molecular ultrasound imaging with MBVCAM-1 is promising to assess vascular damage and to monitor endothelial recovery after arterial interventions. Thus, it may become an important diagnostic tool supporting the development of adequate therapeutic strategies to personalize anticoagulant and anti-inflammatory therapy after cardiovascular intervention.

Isolation of a mouse bone marrow population enriched in stem and progenitor cells by centrifugation on a Percoll gradient.

Given the complex composition of bone marrow, a cell separation technique that results in populations enriched in progenitor cells is required for cellular differentiation and transplantation studies. In the present study, we designed a method that allows for the isolation of a progenitor‐enriched population of bone marrow by exploiting the physical properties of these cells. Bone marrow aspirate was separated on a discontinuous Percoll gradient (ranging from 1.050 to 1.083 g/cm3) that resulted in the recovery of six cell fractions. The fractions were characterized by FACS and RT–PCR (reverse transcription–PCR) analyses and evaluated for their capacity to differentiate into haematopoietic and mesenchymal cells. Fraction IV, including cells with a density of 1.070–1.076 g/ml, contained 11.68% of total bone marrow cells and was enriched in c‐kit+ and Sca‐1+ (stem cell antigen‐1) progenitor cells as compared with total bone marrow. This fraction demonstrated an increase in clonogenic capacity under specific conditions as well as a potential to generate a mesenchymal stem cell culture in a shorter period than that using bone marrow aspirate. Furthermore, this fraction lacked differentiated cell types and contained cells positive for endothelial markers, which further increases its value in cellular transplant. In conclusion, a bone marrow subpopulation that is enriched in progenitor cells and may be valuable in cellular transplant therapy can be isolated by exploiting the physical properties of these cells.

Combinatorial approach for improving the outcome of angiogenic therapy in ischemic tissues.

Two major populations of endothelial progenitor cells (EPC), namely endothelial colony forming cells (ECFC, or late outgrowth EPC) and circulating angiogenic cells (CAC, or early outgrowth EPC) have been reported to play important roles in vasculogenesis in numerous pathological conditions. However, the poor retention of cells into the ischemic tissue and neovessel fragility are two major flaws that need to be overcome for successful angiogenic therapy. The objective of this study was to explore and exploit the functional properties of EPC populations in order to increase the effectiveness of post-ischemic cell therapy. The results indicate different, still complementary, effects of the two EPC populations on adherence and proliferation of vascular endothelial cells. Matrigel plug assay and mouse hind limb ischemia model showed that concomitant administration of CAC-secreted factors and ECFC resulted in three-fold increase in local cell retention and improved muscle perfusion, vessel maturation and hind limb regeneration, in comparison to either treatment alone. By concluding, factors secreted by CAC co-administered at the time of ECFC transplantation improve tissue regeneration and vascular repair through stabilization of newly-derived blood vessels.

Cardiomyocyte apoptosis in ischaemia‐reperfusion due to the exogenous oxidants at the time of reperfusion

Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic‐reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia‐reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25‐OH‐cholesterol (25‐hydroxycholesterol) for 1–3 days. Both ischaemia‐reperfusion and exogenous oxidants increased the Bax/Bcl‐2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25‐OH‐cholesterol promoted CMC apoptosis by a caspase 3‐dependent mechanism that involved the transcriptional activation of the pro‐apoptotic protein, Bax and post‐translational degradation of the anti‐apoptotic protein, Bcl‐2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored.

Synergic effects of VEGF-A and SDF-1 on the angiogenic properties of endothelial progenitor cells.

Here we investigated the impact of hypoxic environment on the angiogenic properties of early‐outgrowth endothelial progenitor cells (EPCs), with particular focus on the role of secreted vascular endothelial growth factor‐A (VEGF‐A) and stromal derived factor‐1 (SDF‐1) in mediating these effects. We found that cultured EPCs secreted factors with paracrine effects on chemotaxis, migration, proliferation and tube formation of mature endothelial cells (ECs), and these properties were not affected by hypoxia. Depletion of VEGF‐A did not change the ability of EPC‐conditioned medium (CM) to promote EC migration and tube formation in vitro, suggesting that the pro‐angiogenic paracrine effects of EPCs did not totally rely on the presence of VEGF‐A. These findings were confirmed by in vivo experiments, on a mouse model of hind limb ischaemia, which showed that VEGF‐depleted EPC‐CM sustained tissue perfusion at the same level as complete EPC‐CM. However, concomitant deletion of VEGF‐A and SDF‐1 in EPC‐CM impaired the pro‐angiogenic properties of EPC‐CM, by inhibition of EC spreading in culture, tube‐like structure formation on Matrigel support, in vivo neovessels formation and ischaemic hind limb regeneration. Taken together, our data demonstrate that: (i) hypoxia does not affect the capacity of EPCs to support the angiogenic process; (ii) the absence of either VEGF‐A or SDF‐1 from EPC‐CM can be rescued by the presence of the other one, so that the overall angiogenic effects remain unchanged; and (iii) and the concomitant deletion of VEGF‐A and SDF‐1 from EPC‐CM impairs its pro‐angiogenic effect, both in vitro and in vivo. Copyright