Cristina Prat-Vidal

Human progenitor cells derived from cardiac adipose tissue ameliorate myocardial infarction in rodents.

Myocardial infarction caused by vascular occlusion results in the formation of nonfunctional fibrous tissue. Cumulative evidence indicates that cell therapy modestly improves cardiac function; thus, novel cell sources with the potential to repair injured tissue are actively sought. Here, we identify and characterize a cell population of cardiac adipose tissue-derived progenitor cells (ATDPCs) from biopsies of human adult cardiac adipose tissue. Cardiac ATDPCs express a mesenchymal stem cell-like marker profile (strongly positive for CD105, CD44, CD166, CD29 and CD90) and have immunosuppressive capacity. Moreover, cardiac ATDPCs have an inherent cardiac-like phenotype and were able to express de novo myocardial and endothelial markers in vitro but not to differentiate into adipocytes. In addition, when cardiac ATDPCs were transplanted into injured myocardium in mouse and rat models of myocardial infarction, the engrafted cells expressed cardiac (troponin I, sarcomeric α-actinin) and endothelial (CD31) markers, vascularization increased, and infarct size was reduced in mice and rats. Moreover, significant differences between control and cell-treated groups were found in fractional shortening and ejection fraction, and the anterior wall remained significantly thicker 30days after cardiac delivery of ATDPCs. Finally, cardiac ATDPCs secreted proangiogenic factors under in vitro hypoxic conditions, suggesting a paracrine effect to promote local vascularization. Our results indicate that the population of progenitor cells isolated from human cardiac adipose tissue (cardiac ATDPCs) may be valid candidates for future use in cell therapy to regenerate injured myocardium.

Idiopathic dilated cardiomyopathy exhibits defective vascularization and vessel formation

Ultrastructural findings of idiopathic dilated cardiomyopathy (IDCM) include myocyte atrophy and myofilament loss, yet little is known about the vascular abnormalities present in IDCM.

Human umbilical cord blood-derived mesenchymal stem cells promote vascular growth in vivo.

Stem cell therapies are promising strategies to regenerate human injured tissues, including ischemic myocardium. Here, we examined the acquisition of properties associated with vascular growth by human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs), and whether they promoted vascular growth in vivo. UCBMSCs were induced in endothelial cell-specific growth medium (EGM-2) acquiring new cell markers, increased Ac-LDL uptake, and migratory capacity as assessed by qRT-PCR, Western blotting, indirect immunofluorescence, and invasion assays. Angiogenic and vasculogenic potentials could be anticipated by in vitro experiments showing self organization into Matrigel-mediated cell networks, and activation of circulating angiogenic-supportive myeloid cells. In mice, following subcutaneous co-injection with Matrigel, UCBMSCs modified to co-express bioluminescent (luciferases) and fluorescent proteins were demonstrated to participate in the formation of new microvasculature connected with the host circulatory system. Response of UCBMSCs to ischemia was explored in a mouse model of acute myocardial infarction (MI). UCBMSCs transplanted using a fibrin patch survived 4 weeks post-implantation and organized into CD31+network structures above the infarcted myocardium. MI-treated animals showed a reduced infarct scar and a larger vessel-occupied area in comparison with MI-control animals. Taken together, the presented results show that UCBMSCs can be induced in vitro to acquire angiogenic and vasculogenic properties and contribute to vascular growth in vivo.

FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 ± 0.2 vs. 4.1 ± 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.

Abnormal calcium handling in atrial fibrillation is linked to up-regulation of adenosine A2A receptors

AIMS Atrial fibrillation (AF) is associated with abnormal sarcoplasmic reticulum (SR) calcium release, which is promoted by adenosine A(2A) receptor (A(2A)R) activation. Here, we tested the hypothesis that abnormal calcium release in AF is linked to A(2A)R remodelling. METHODS AND RESULTS Western blotting and quantitative real-time PCR were used to determine A(2A)R mRNA and protein levels in right atrial samples from patients with and without AF. Effects of A(2A)R activation on calcium handling were assessed with patch-clamp technique and confocal calcium imaging. A(2A)R mRNA levels and functional A(2A)Rs were moderately up-regulated in patients with atrial dilation and markedly up-regulated in those with AF. Accordingly, A(2A)R stimulation significantly increased ryanodine receptor phosphorylation in AF patients, and spontaneous calcium waves increased moderately in myocytes from patients with atrial dilation and strongly in patients with AF (2.2 ± 2.1 to 14.3 ± 8.8 min(-1), n = 6, P = 0.01). Moreover, the high baseline level of calcium waves in AF was reduced by A(2A)R antagonists (3.5 ± 2.0 to 1.3 ± 1.3 min(-1), n = 6, P = 0.007) or adenosine deaminase (1.7 ± 1.5 to 0.5 ± 0.6 min(-1), n = 10, P = 0.02) suggesting that A(2A)Rs are activated by endogenous adenosine. Indeed, intracellular perfusion with adenosine significantly increased the calcium wave frequency (1.1 ± 0.8 to 8.2 ± 3.3 min(-1), n = 8), whereas adenosine removal from the cytosol decreased it (2.1 ± 0.9 to 0.3 ± 0.3 min(-1), n = 8, P = 0.04). CONCLUSIONS Atrial fibrillation patients show increased A(2A)R expression that may account for the high baseline level of spontaneous SR calcium release seen in myocytes from these patients, and the ability of A(2A)R antagonists to reduce this abnormal calcium release points to the A(2A)R as a novel molecular target in AF.

Transposition of a pericardial-derived vascular adipose flap for myocardial salvage after infarct.

AIMS Coronary artery occlusion is associated with the risk of ventricular remodelling, heart failure, and cardiogenic shock. Novel strategies are sought to treat these ominous complications. We examined the effect of a pericardial-derived fat flap secured over an acute infarct caused by coronary occlusion. METHODS AND RESULTS A novel intervention consisting of the pericardial isolation of a vascularized adipose flap and its transposition fully covering acute infarcted myocardium was developed in the swine model of coronary artery ligation (n= 52). Left ventricular (LV) ejection fraction and LV end-diastolic and end-systolic volumes were assessed using magnetic resonance imaging (MRI). Infarct size and gene expression analysis were performed on Day 6 and 1 month. Histological changes, collagen volume fraction (CVF), and vascular density were also evaluated on postmortem sections. One month after the intervention, a 18.8% increase in LV ejection fraction (P= 0.007), and significant reductions in LV end-systolic (P= 0.009) and LV end-diastolic volumes (P= 0.03) were found in treated animals compared with the control-MI group. At Day 6, histopathology confirmed a significant infarct size reduction (P= 0.018), the presence of vascular connections at the flap-myocardium interface, and less apoptosis in the infarct border zone compared with control animals (P< 0.001). Up-regulation of genes involved in cell cycle progression, cellular growth and proliferation, and angiogenesis were identified within the flap. CONCLUSIONS Our results indicate that a vascular fat flap exerts beneficial effects on LV function and limits myocardial remodelling. Future studies must confirm whether these findings provide an alternative therapeutic approach for myocardial salvage after infarction.

Chimerism and microchimerism of the human heart: evidence for cardiac regeneration.

For decades, it has been widely accepted that the heart is a terminally differentiated organ that is unable to regenerate. Studies of recipients of hearts donated by other humans have shed light on the regenerative potential of the human heart. Investigators have been able to trace the Y chromosome by fluorescence in situ hybridization or polymerase chain reaction, or both, in sex-mismatched heart recipients. Cardiac chimerism has been reported, with concentrations of chimeric cells ranging from 0.04% to 10.0%. Cardiac chimerism after bone marrow or progenitor cell transplantation has also been reported to a low extent (∼0.20%), suggesting that a fraction of the extracardiac cells that colonize the myocardium are of bone marrow origin. Cardiac chimerism after pregnancy with male offspring (fetal cell microchimerism) has also been demonstrated. Cells of fetal origin have been shown to be capable of differentiating into myocardial cells. Collectively, we show that chimerism studies provide a proof of concept of a process that it is likely to be part of normal cardiac homeostasis in humans but apparently insufficient for cardiac repair in diseased hearts.

Cardiac adipose tissue: A new frontier for cardiac regeneration?

The human heart has limited regenerative capacity. We focused on cardiac adipose tissue as a source of progenitor cells and biological matrix material for salvaging injured myocardium. First, a population of human adult mesenchymal-like progenitors derived from cardiac adipose tissue, with inherent cardiac and endothelial cell potential, was identified and characterized. Next, a salvage strategy was tested, where a pericardial-derived, vascularized, adipose flap was used to cover oxygen-deprived myocardium in a porcine model. The fat flap reduced the myocardial scar size, in both acute and chronic infarcts. A human clinical trial to examine this novel intervention is currently underway.

Online monitoring of myocardial bioprosthesis for cardiac repair

BACKGROUND/OBJECTIVES The aim of this study was to develop a myocardial bioprosthesis for cardiac repair with an integrated online monitoring system. Myocardial infarction (MI) causes irreversible myocyte loss and scar formation. Tissue engineering to reduce myocardial scar size has been tested with variable success, yet scar formation and modulation by an engineered graft is incompletely characterized. METHODS Decellularized human pericardium was embedded using self-assembling peptide RAD16-I with or without GFP-labeled mediastinal adipose tissue-derived progenitor cells (MATPCs). Resulting bioprostheses were implanted over the ischemic myocardium in the swine model of MI (n=8 treated and n=5 control animals). For in vivo electrical impedance spectroscopy (EIS) monitoring, two electrodes were anchored to construct edges, covered by NanoGold particles and connected to an impedance-based implantable device. Histological evaluation was performed to identify and characterize GFP cells on post mortem myocardial sections. RESULTS Pluripotency, cardiomyogenic and endothelial potential and migratory capacity of porcine-derived MATPCs were demonstrated in vitro. Decellularization protocol efficiency, biodegradability, as well as in vitro biocompatibility after recellularization were also verified. One month after myocardial bioprosthesis implantation, morphometry revealed a 36% reduction in infarct area, Ki67(+)-GFP(+)-MATPCs were found at infarct core and border zones, and bioprosthesis vascularization was confirmed by presence of Griffonia simplicifolia lectin I (GSLI) B4 isolectin(+)-GFP(+)-MATPCs. Electrical impedance measurement at low and high frequencies (10 kHz-100 kHz) allowed online monitoring of scar maturation. CONCLUSIONS With clinical translation as ultimate goal, this myocardial bioprosthesis holds promise to be a viable candidate for human cardiac repair.

Post-infarction scar coverage using a pericardial-derived vascular adipose flap. Pre-clinical results

BACKGROUND Myocardial salvage after coverage with a fat flap was recently demonstrated in acute coronary occlusion. The effect of this novel therapeutic strategy on a chronic myocardial scar is unknown. METHODS Myocardial infarction (MI) was induced by coil deployment in the mid circumflex artery in the swine model. Two weeks after infarction, a pericardial-derived adipose flap was transposed, fully covering the scar, in the treated group. Infarct size and histopathology were analyzed on post mortem sections. To assess cell migration, adenoviral eGFP vectors were injected in the adipose flap and expression was evaluated upon sacrifice both at the flap and myocardium. Magnetic resonance imaging (MRI) was used to measure left ventricular (LV) ejection fraction and ventricular volumes at baseline, 2 weeks post-MI, and at 6 weeks. RESULTS One month after flap transposition, histopathology confirmed a 34% reduction in infarct size (8.7% vs. 5.7%; P=0.04) and the presence of vascular connections at the flap-myocardium interface. High eGFP expression was detected at the infarct core both at the gene and protein level (negligible signal was detected at the flap on sacrifice). At the functional level, changes in LV ejection fraction and volumes (end-systolic and end-diastolic) were not significantly different between groups (all P values>0.1). CONCLUSIONS Our data support the use of post-infarction scar coverage with a pericardial-derived fat flap to reduce infarct size, due partly to neovascular connections and cell trafficking at the flap-myocardium interface. Further studies are needed to validate the functional and clinical relevance of this intervention.