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Objectives This study sought to investigate the hypothesis that the favorable effects of mesenchymal stromal cells (MSCs) on infarct repair are mediated by macrophages. Background The favorable effects of MSC therapy in myocardial infarction (MI) are complex and not fully understood. Methods We induced MI in mice and allocated them to bone marrow MSCs, mononuclear cells, or saline injection into the infarct, with and without early (4 h before MI) and late (3 days after MI) macrophage depletion. We then analyzed macrophage phenotype in the infarcted heart by flow cytometry and macrophage secretome in vitro. Left ventricular remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging. Results The MSC therapy significantly increased the percentage of reparative M2 macrophages (F4/80+CD206+) in the infarcted myocardium, compared with mononuclear- and saline-treated hearts, 3 and 4 days after MI. Macrophage cytokine secretion, relevant to infarct healing and repair, was significantly increased after MSC therapy, or incubation with MSCs or MSC supernatant. Significantly, with and without MSC therapy, transient macrophage depletion increased mortality 30 days after MI. Furthermore, early macrophage depletion produced the greatest negative effect on infarct size and left ventricular remodeling and function, as well as a significant incidence of left ventricular thrombus formation. These deleterious effects were attenuated with macrophage restoration and MSC therapy. Conclusions Some of the protective effects of MSCs on infarct repair are mediated by macrophages, which are essential for early healing and repair. Thus, targeting macrophages could be a novel strategy to improve infarct healing and repair.

Previous studies have shown that pre-exposure of the heart to weak magnetic field reduces infarct size shortly after induction of myocardial ischemia. To investigate the role of AC magnetic field with a frequency of 15.95-16 Hz and 80 mT on left ventricular (LV) remodeling following chronic coronary occlusion and a short episode of ischemia followed by reperfusion (I/R). LV dimension and function were measured using echocardiography. Femur bone marrow was isolated and cells were phenotyped for endothelial linage and immuno stained for endothelial cells. The area at risk was measured using triphenyltetrazolium chloride staining. A significant reduction of 27% in shortening fraction (SF) was measured following acute myocardial infarction (AMI) compared with a 7% decrease in animals exposed to magnetic field (p < 0.04). A significantly higher number of colony forming units and endothelial progenitor cells were counted within the treated groups subjected to magnetic field (p < 0.02). Exposing the heart to magnetic field prior to reperfusion did not show any preservation either on SF or on infarct size. Magnetic field was protective in the AMI but not in the I/R model. The mechanisms underlying cardiac protection induced by AC magnetic field following chronic injury deserves further investigation.

Tissue engineering holds the promise of providing new solutions for heart transplant shortages and pediatric heart transplantation. The aim of this study was to evaluate the ability of a peritoneal-generated, tissue-engineered cardiac patch to replace damaged myocardium in a heterotopic heart transplant model. Fetal cardiac cells (1 × 106/scaffold) from syngeneic Lewis rats were seeded into highly porous alginate scaffolds. The cell constructs were cultured in vitro for 4 days and then they were implanted into the rat peritoneal cavity for 1 week. During this time the peritoneal-implanted patches were vascularized and populated with myofibroblasts. They were harvested and their performance in an infrarenal heterotopic abdominal heart transplantation model was examined ( n = 15). After transplantation and before reperfusion of the donor heart, a 5-mm left ( n = 6) or right ( n = 9) ventriculotomy was performed and the patch was sutured onto the donor heart to repair the defect. Echocardiographical studies carried out 1–2 weeks after transplantation showed normal LV function in seven of the eight hearts studied. After 1 month, visual examination of the grafted patch revealed no aneurysmal dilatation. Microscopic examination revealed, in most of the cardiac patches, a complete disappearance of the scaffold and its replacement by a consistent tissue composed of myofibroblasts embedded in collagen bundles. The cardiac patch was enriched with a relatively large number of infiltrating blood vessels. In conclusion, cardiac patches generated in the peritoneum were developed into consistent tissue patches with properties to seal and correct myocardial defects. Our study also offers a viable rat model for screening and evaluating new concepts in cardiac reconstruction and engineering.

Background— Adverse cardiac remodeling and progression of heart failure after myocardial infarction are associated with excessive and continuous damage to the extracellular matrix. We hypothesized that injection of in situ–forming alginate hydrogel into recent and old infarcts would provide a temporary scaffold and attenuate adverse cardiac remodeling and dysfunction. Methods and Results— We developed a novel absorbable biomaterial composed of calcium-crosslinked alginate solution, which displays low viscosity and, after injection into the infarct, undergoes phase transition into hydrogel. To determine the outcome of the biomaterial after injection, calcium-crosslinked biotin-labeled alginate was injected into the infarct 7 days after anterior myocardial infarction in rat. Serial histology studies showed in situ formation of alginate hydrogel implant, which occupied up to 50% of the scar area. The biomaterial was replaced by connective tissue within 6 weeks. Serial echocardiography studies before and 60 days after injection showed that injection of alginate biomaterial into recent (7 days) infarct increased scar thickness and attenuated left ventricular systolic and diastolic dilatation and dysfunction. These beneficial effects were comparable and sometimes superior to those achieved by neonatal cardiomyocyte transplantation. Moreover, injection of alginate biomaterial into old myocardial infarction (60 days) increased scar thickness and improved systolic and diastolic dysfunction. Conclusions— We show for the first time that injection of in situ–forming, bioabsorbable alginate hydrogel is an effective acellular strategy that prevents adverse cardiac remodeling and dysfunction in recent and old myocardial infarctions in rat.

Objective: To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs) on cardiac function in a rat model of myocardial infarction (MI). Methods: Undifferentiated hESCs (0.5–1×10 6 ), human embryoid bodies (hEBs) (4–8 days; 0.5–1×10 6 ), 0.1 mm pieces of embryonic stem-derived beating myocardial tissue, and phosphate-buffered saline (control) were injected into the normal or infarcted myocardium of athymic nude rats (n = 58) by direct injection into the muscle or into preimplanted three-dimensional alginate scaffold. By 2–4 weeks after transplantation, heart sections were examined to detect the human cells and differentiation with fluorescent in situ hybridisation, using DNA probes specific for human sex chromosomes and HLA-DR or HLA-ABC immunostaining. Results: Microscopic examination showed transplanted human cells in the normal, and to a lesser extent in the infarcted myocardium (7/7 vs 2/6; p Conclusions: Undifferentiated hESCs and hEBs are not directed to form new myocardium after transplantation into normal or infarcted heart and may create teratoma. Nevertheless, this study shows that hESC-derived cardiomyocyte transplantation can attenuate post-MI scar thinning and left ventricular dysfunction.

We investigated the effect of low-intensity ultrasound (US) on tissue blood flow and angiogenesis after limb ischemia in vivo. Rats underwent surgical ligation of the femoral or the iliac arteries. Half the animals were exposed to low-intensity US (0.05 W/cm2) during three consecutive sessions. At 3 weeks postsurgery, limb perfusion was assessed using laser Doppler and angiography. Immunostaining and vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) expression were performed 7 d postsurgery. US irradiation significantly improved limb perfusion in both ischemic models (p = 0.04). Angiography showed increased blood vessels in the moderate ischemia (p = 0.01), but not in the severe ischemia (p = 0.19). Histology demonstrated a significantly higher number of blood vessels and proliferating cells in US-irradiated moderate and severe ischemia (p = 0.002 and p = 0.03, respectively). VEGF mRNA was significantly higher in moderate ischemia (p = 0.02). No differences in apoptotic cell death were evident in the models. Low-intensity US significantly improved tissue blood flow and angiogenesis, irrespective of the extent of the ischemia. (E-mail: ).

Introduction and Hypothesis: Mesenchymal stem/stromal cells (MSCs) are appealing candidates for cardiac cell therapy due to their ease of preparation, immunoprivilege properties, reparative potential and safety. We aimed to test the hypothesis that the inflammatory environment following myocardial infarction (MI) and left ventricular (LV) dysfunction could modulate the reparative properties of resident and transplanted MSCs. Methods and Results: To test this hypothesis, we isolated and cultured resident cardiac and subcutaneous (SC) fat tissue MSCs from mice, 28 days after MI (LVEF: 27.9%±3.8) or sham operation (LVEF: 54.9%±3.8). Proliferation rate was highest in cardiac MSCs after MI, compared with other groups (pTLR4 ) deficient and wild-type mice, with the secretome of LPS-activated macrophages. Significantly, there was less inflammatory cytokine secretion in TLR4-deficient MSCs, compared with control suggesting that cardiac MSC polarization is mediated by TLR4 activation. Conclusions: Post MI inflammatory environment switches resident cardiac and implanted MSCs toward a pro-inflammatory phenotype and impair their reparative properties. This effect is mediated by TLR4 . Our findings are relevant to the pathogenesis and progression of LV remodeling, and could guide the selection and engineering of MSCs for heart repair.

Aim: Despite clear evidence of immune system involvement in the pathogenesis of myocarditis, the treatment of myocarditis remains nonspecific and supportive. We sought to test the hypothesis that injection of a collagen-based implant into the inflamed myocardium would stabilize the left ventricular (LV) wall and prevent adverse remodeling and dysfunction. Methods and Results: Autoimmune myocarditis was induced in 42 male Lewis rats. Development of myocarditis was evaluated and confirmed by serial echocardiography and cardiac magnetic resonance scans, LV wall thickening, global and regional LV wall motion abnormalities, and in some cases pericardial effusion. Sick animals were randomized to either injectable collagen implantation or saline injection into the anterior inflamed myocardium 14 days after immunization. Significantly, injectable collagen implantation improved 31-day survival compared with controls (85.7% vs 50%; P = .03). Furthermore, although injectable collagen significantly attenuated LV systolic and diastolic dilatation and preserved LV geometry and function, control animals developed significant LV dilatation and dysfunction. These favorable effects on LV remodeling were confirmed by postmortem morphometry. Significantly, the injectable collagen implant attenuated cardiomyocyte hypertrophy and infiltration of macrophages and lymphocytes into the myocardium. Conclusions: The present study shows, for the first time, that injectable collagen biomaterial improves survival and attenuates cardiac inflammation, cardiomyocyte hypertrophy, LV remodeling, and dysfunction in the early period after myocarditis in rats. Our findings suggest a new biomaterial-based strategy to ameliorate the devastating effects of myocarditis.

Background Human mesenchymal stromal cells ( hMSC s) from adipose cardiac tissue have attracted considerable interest in regard to cell‐based therapies. We aimed to test the hypothesis that hMSC s from the heart and epicardial fat would be better cells for infarct repair. Methods and Results We isolated and grew hMSC s from patients with ischemic heart disease from 4 locations: epicardial fat, pericardial fat, subcutaneous fat, and the right atrium. Significantly, hMSC s from the right atrium and epicardial fat secreted the highest amounts of trophic and inflammatory cytokines, while hMSC s from pericardial and subcutaneous fat secreted the lowest. Relative expression of inflammation‐ and fibrosis‐related genes was considerably higher in hMSC s from the right atrium and epicardial fat than in subcutaneous fat hMSC s. To determine the functional effects of hMSC s, we allocated rats to hMSC transplantation 7 days after myocardial infarction. Atrial hMSC s induced greatest infarct vascularization as well as highest inflammation score 27 days after transplantation. Surprisingly, cardiac dysfunction was worst after transplantation of hMSC s from atrium and epicardial fat and minimal after transplantation of hMSC s from subcutaneous fat. These findings were confirmed by using hMSC transplantation in immunocompromised mice after myocardial infarction. Notably, there was a correlation between tumor necrosis factor‐α secretion from hMSC s and posttransplantation left ventricular remodeling and dysfunction. Conclusions Because of their proinflammatory properties, hMSC s from the right atrium and epicardial fat of cardiac patients could impair heart function after myocardial infarction. Our findings might be relevant to autologous mesenchymal stromal cell therapy and development and progression of ischemic heart disease.

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

Adverse cardiac remodeling and dysfunction after myocardial infarction (MI) is associated with excessive damage to the extracellular matrix. Biomaterials, such as the in situ-forming alginate hydrogel (BioLineRx, BL-1040 myocardial implant), provide temporary support and attenuate these processes. Here, we tested the effects of decorating alginate biomaterial with cell adhesion peptides, containing the sequences RGD and YIGSR, or a non-specific peptide (RGE), in terms of therapeutic outcome soon after MI. The biomaterial (i.e., both unmodified and peptide-modified alginate) solutions retained the ability to flow after cross-linking with calcium ions, and could be injected into 7-day infarcts, where they underwent phase transition into hydrogels. Serial echocardiography studies performed before and 60 days after treatment showed that alginate modification with the peptides reduced the therapeutical effects of the hydrogel, as revealed by the extent of scar thickness, left ventricle dilatation and function. Histology and immunohistochemistry revealed no significant differences in blood vessel density, scar thickness, myofibroblast or macrophage infiltration or cell proliferation between the experimental groups BioLineRx BL-1040 myocardial implant. Our studies thus reveal that the chemical and physical traits of the biomaterial can affect its therapeutical efficacy in attenuating left ventricle remodeling and function, post-MI.

Background— Cell labeling with superparamagnetic iron oxide (SPIO) nanoparticles enables noninvasive MRI and tracking of transplanted stem cells. We sought to determine whether mesenchymal stem cell (MSC) outcome is affected by SPIO labeling in a rat model of myocardial infarction. Methods and Results— Rat MSCs were labeled with SPIO (ferumoxides; Endorem; Guerbet, Villepinte, France). By trypan-blue exclusion assay, almost 100% of the cells remained viable after labeling. Seven days after MI, rats were randomized to injections of 2×10 6 SPIO-labeled MSCs, 2×10 6 unlabeled MSCs, or saline. Labeled cells were visualized in the infarcted myocardium as large black spots by serial MRI studies throughout the 4-week follow-up. The presence of labeled cells was confirmed by iron staining and real-time polymerase chain reaction on postmortem specimens. At 4 weeks after transplantation, the site of cell injection was infiltrated by inflammatory cells. Costaining for iron and ED1 (resident macrophage marker) showed that the iron-positive cells were cardiac macrophages. By real-time polymerase chain reaction, the Y-chromosome-specific SRY DNA of MSCs from male donors was not detected in infarcted hearts of female recipients. Serial echocardiography studies at baseline and 4 weeks after cell transplantation showed that both unlabeled and labeled MSCs attenuated progressive left ventricular dilatation and dysfunction compared with controls. Conclusions— At 4 weeks after transplantation of SPIO-labeled MSCs, the transplanted cells are not present in the scar and the enhanced MRI signals arise from cardiac macrophages that engulfed the SPIO nanoparticles. However, both labeled and unlabeled cells attenuate left ventricular dilatation and dysfunction after myocardial infarction.

Background— Activated macrophages have a significant role in wound healing and damaged tissue repair. We sought to explore the ability of ex vivo activated macrophages to promote healing and repair of the infarcted myocardium. Methods and Results— Human activated macrophage suspension (AMS) was prepared from a whole blood unit obtained from young donors in a closed sterile system and was activated by a novel method of hypo-osmotic shock. The AMS (&4×10 5 cells) included up to 43% CD14-positive cells and was injected into the ischemic myocardium of rats (n=8) immediately after coronary artery ligation. The control group (n=9) was treated with saline injection. The human cells existed in the infarcted heart 4 to 7 days after injection, as indicated by histology, human growth hormone-specific polymerase chain reaction, and magnetic resonance imaging (MRI) tracking of iron oxide–nanoparticle-labeled cells. After 5 weeks, scar vessel density (±SE) (25±4 versus 10±1 per mm 2 ; P P P P Conclusions— Early after myocardial infarction, injection of AMS accelerates vascularization, tissue repair, and improves cardiac remodeling and function. Our work suggests a novel clinically relevant option to promote the repair of ischemic tissue.

The aim of conducting this study was to assess the clinical relevance of matrix metalloproteinase (MMP) inhibition by doxycycline, an effective MMP inhibitor, in a rat model of extensive myocardial infarction (MI) and left ventricular (LV) dysfunction. Rats (n = 22) were subjected to extensive anterior MI. Doxycycline (25 mg SC, daily) or saline (control) injections were started for nine days thereafter. The effect of doxycycline on MMP activity in the infarcted and remote myocardium was measured by zymography, in another subgroup (n = 8), nine days after MI. Echocardiography and magnetic resonance imaging (MRI) studies were performed at one and thirty days after MI to assess LV remodeling and function. After 4 weeks, hearts were fixed, and subjected to morphometric and histological analysis. Compared with control, doxycycline treatment attenuated MMP-9 and -2 activity in both infarcted and remote myocardium. Serial echocardiography studies showed that doxycycline failed to attenuate scar thinning, LV dilatation and dysfunction. MRI study showed that doxycycline impaired LV compensatory hypertrophy. Furthermore, compared with control, doxycycline reduced vessel density (/mm(2) +/- SEM) in the infarcted myocardium (84 +/- 16 vs. 46 +/- 9/mm(2), respectively; p < 0.05). Our work suggest that effective MMPs' inhibition in the infarcted and remote myocardium by doxycycline does not prevent LV remodeling and dysfunction but impairs angiogenesis and compensatory LV hypertrophy. Our findings caution against aggressive, non-selective inhibition of MMPs in the early healing phase after MI.

The use of adult stem cells for myocardial tissue repair might be limited in elderly and sick people because their cells are depleted and exhausted. The present study was conducted to explore the potential of human umbilical cord blood (UCB) CD133+ progenitor cells for myocardial tissue repair in a model of extensive myocardial infarction (MI). CD133+ progenitor cells were isolated from newborn UCB. Cells (1.2–2 × 106) or saline (control) was infused intravenously 7 days after permanent coronary artery ligation in athymic nude rats. Left ventricular (LV) function was assessed before and 1 month after infusion by echocardiography. Tracking of human cells was performed by fluorescent in situ hybridization for human X and Y chromosomes or by immunostaining for HLA-DR or HLA-ABC. One month after delivery, LV fractional shortening improved by 42 ± 17% in cell-treated hearts and decreased by 39 ± 10% in controls (p = .001). Anterior wall thickness decreased significantly in controls but not in treated hearts. Microscopic examination revealed that the UCB cells were able to migrate, colonize, and survive in the infarcted myocardium. Human cells were identified near vessel walls and LV cavity and were occasionally incorporated into endothelial cells in six of nine cell-treated animals but not in controls. Scar tissue from cell-treated animals was significantly populated with autologous myofibroblasts as indicated by colocalization of HLA-DR and α-smooth muscle actin staining. In conclusion, the present work suggests that, after MI, intravenous delivery of human UCB-derived CD133+ cells can produce functional recovery by preventing scar thinning and LV systolic dilatation.

We review an experimental protocol for investigating concepts and methods for myocardial repair using fetal cardiomyocyte transplantation. We describe methods of cell isolation, culture, labeling, and assessment of the influence of the engrafted cells on left ventricular remodeling and function.

Purpose: Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of leukocytes and lymphocytes into the heart and to injured muscle. Regulatory T cells (Tregs) exert suppressive effects on several immune and non-immune cellular elements. We aimed hypothesized that Tregs improve cardiac function after myocardial infarction (MI) with potential alteration of postischemic vessel growth. Methods and results: The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental MI (n=40), and to hindlimb ischemia (n=24). The numbers of splenocyte-derived Tregs were significantly higher after the injury, both in MI mice (16±0.6% vs. 12±0.7%, p

qualitative and quantitative techniques. Between January 1, 2003 and April 30, 2007, 175 patients underwent PCI for STEMI with a high thrombus grade (TIMI grade ≥4). Fifty-three underwent RT followed by balloons or stents, and 122 underwent a nonthrombectomy approach (NRT). A comparative cohort of 223 patients with STEMI and a low thrombus grade (TIMI grade b4) underwent nonthrombectomy PCI. An angiographic adverse event was defined as the occurrence of no-reflow, slow flow, distal embolization, perforation, or abrupt closure. Results: Baseline clinical characteristics of the RTandNRT groups were similar. Table 1 shows the clinical and angiographic outcomes of the 3 study groups. Conclusion: (1) RT reduces the angiographic adverse event rates of highly thrombotic lesions in STEMI patients. (2) RT in such a high-risk group tended to be associated with a lower 30-day mortality. Further data analysis with additional patients is ongoing and will be announced at a later date.