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Background Filamin C is a cytoskeletal protein expressed in cardiac cells. Nonsense variations in the filamin C gene (FLNC) were associated with dilated and arrhythmogenic cardiomyopathies. Methods and results We identified an intronic variation in FLNC gene (c.3791-1G > C) in three unrelated Ashkenazi Jewish families with variable expression of arrhythmia and cardiomyopathy. cDNA was prepared from a mutation carrier's cultured skin fibroblasts. Quantitative PCR demonstrated a reduction in total FLNC transcript, and no other FLNC splice variants were found. Single-nucleotide polymorphism (SNP) analysis revealed heterozygous variations in the genomic DNA that were not expressed in the messenger RNA. Immunohistochemical analysis of cardiac sections detected a normal distribution of filamin C protein in the heart ventricles. Conclusion The transcript that included the FLNC variant was degraded. Haploinsufficiency in filamin C underlies arrhythmogenic cardiomyopathy with variable symptoms.

Endothelial activation with up-regulation of E-selectin adhesion molecules mediates leukocyte rolling along the vascular wall and controls inflammation in many diseases including atherosclerosis and heart failure. Therefore, we aimed to test the hypothesis that inhibition of E-selectin-mediated interactions by a new E-selectin-targeted copolymer could inhibit the progression of atherosclerosis. To target E-selectin on activated endothelium, we developed a new N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer with or without dexamethasone (Dex) (designated P-(Esbp)-Dex and P-Esbp, respectively). To determine the effect of P-(Esbp)-Dex and P-Esbp on atherosclerosis, we allocated ApoE (−/−) mice on a high fat diet, to weekly intra-peritoneal injections of either 1) P-Esbp; 2) P-(Esbp)-Dex; 3) free Dex (1 mg/kg) or 4) saline, for four weeks. Aortic atherosclerosis and left ventricular (LV) remodeling and function were assessed by serial ultrasound studies and histology. Monocytes and macrophages were characterized by flow cytometry. After four weeks of treatment, P-Esbp effectively targeted aortic atherosclerotic lesions. Both P-Esbp and P-(Esbp)-Dex reduced wall thickening of the ascending aortas. However, only the drug-free copolymer (P-Esbp) significantly decreased the areas of necrotic core in the plaques and switched spleen macrophages toward an anti-inflammatory (M2) phenotype. Furthermore, P-Esbp attenuated adverse LV remodeling and dysfunction in ApoE (−/−) mice. In summary, P-Esbp copolymer targets activated endothelial cells, regresses and stabilizes atherosclerotic plaques, and prevents adverse LV remodeling and dysfunction in ApoE (−/−) mice. Our results suggest a new, drug-free macromolecular therapy to treat vascular inflammation.

The effectiveness of empagliflozin (EMPA), a sodium glucose cotransporter type 2 inhibitor, on the kidney, pancreas, and heart was investigated in the Cohen Rosenthal diabetic hypertensive rat model (CRDH rat). Six-week-old CRDH male rats were fed a sugar diet (SD) and treated with the compound EMPA (group Drug/SD) or respective comparator with vehicle (group Veh/SD). A control group was fed a regular diet without treatment (group Veh/P). Preventive treatment with EMPA was measured during 4 months of follow-up. The treatment effect was evaluated according to results observed after 4 months in group Drug/SD when compared to those in group Veh/SD. Significant effect resulted in the following parameters: enhancement of urinary glucose excretion in association with diuresis; amelioration of postprandial hyperglycemia and fasting blood glucose levels; and decrease in calculated Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) as well as lower systolic and diastolic blood pressures. At the end of treatment, EMPA preserved nephrin integrity in the kidney, reduced proteinuria, and prevented diabetes-induced damage to glomerular diaphragm structure. In the pancreas, EMPA demonstrated an impressive decrease in fatty infiltration and atrophy. Blood pressure was significantly reduced in the EMPA-treated group (15 ± 5.1 mm Hg, P < .05) in contrast to the vehicle and control groups. Finally, compared to controls, EMPA significantly reduced left ventricle (LV) mass and LV systolic dilatation, according to 2-dimensional echocardiography. The importance of the study lies in demonstrating the efficacy of an antidiabetic drug with beneficial effects on blood pressure, weight, kidney, and pancreas and a positive effect on the heart.

Background The immune system plays a pivotal role in myocardial homeostasis and response to injury. Interleukins‐4 and ‐13 are anti‐inflammatory type‐2 cytokines, signaling via the common interleukin‐13 receptor α1 chain and the type‐2 interleukin‐4 receptor. The role of interleukin‐13 receptor α1 in the heart is unknown. Methods and Results We analyzed myocardial samples from human donors (n=136) and patients with end‐stage heart failure (n=177). We found that the interleukin‐13 receptor α1 is present in the myocardium and, together with the complementary type‐2 interleukin‐4 receptor chain Il4ra , is significantly downregulated in the hearts of patients with heart failure. Next, we showed that Il13ra1 ‐deficient mice develop severe myocardial dysfunction and dyssynchrony compared to wild‐type mice (left ventricular ejection fraction 29.7±9.9 versus 45.0±8.0; P =0.004, left ventricular end‐diastolic diameter 4.2±0.2 versus 3.92±0.3; P =0.03). A bioinformatic analysis of mouse hearts indicated that interleukin‐13 receptor α1 regulates critical pathways in the heart other than the immune system, such as extracellular matrix (normalized enrichment score=1.90; false discovery rate q=0.005) and glucose metabolism (normalized enrichment score=−2.36; false discovery rate q=0). Deficiency of Il13ra1 was associated with reduced collagen deposition under normal and pressure‐overload conditions. Conclusions The results of our studies in humans and mice indicate, for the first time, a role of interleukin‐13 receptor α1 in myocardial homeostasis and heart failure and suggests a new therapeutic target to treat heart disease.

Aims Ischaemic damage is associated with up-regulation of pro-inflammatory cytokines, as well as invasion of leucocytes and lymphocytes to the injured muscle. Regulatory T cells (Tregs) exert suppressive effects on several immune and non-immune cellular elements. We hypothesized that adoptive Treg cell transfer and depletion will influence re-establishment of flow in the hindlimb ischaemia model, and that this effect would be mediated by the cytokine interleukin (IL)-10. Methods and results To study the functional role of Tregs in hindlimb ischaemia, we either adoptively transferred Tregs or functionally blocked Tregs by antibodies to CD25. Initially, we showed that the number and function of Tregs is altered after the induction of ischaemia. Treg ablation resulted in reduced blood flow by laser Doppler at Day 7 that became more robust at Day 14. Adoptive Treg transfer led to a significant improvement of flow in the ligated limb. Treg-mediated improvement in flow was abolished by employing blocking anti-IL-10 antibodies. Conclusions These results show that Tregs play an important role in processes that control flow re-establishment after inducible hindlimb ischaemia, and that IL-10 plays a requisite role mediating these effects.

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.

Aims The aim of this study was to assess the use of a 3 T clinical cardiac magnetic resonance (CMR) scanner to detect injury to the heart in experimental autoimmune myocarditis (EAM). Methods and results The use of 3 T CMR for the detection of cardiac injury was assessed in EAM ( n = 55) and control ( n = 10) male Lewis rats. Animals were evaluated with serial CMR imaging studies, using a 3 T scanner, and with 2D echocardiography before, and at 2 and 5 weeks after EAM induction. By CMR, regional wall motion abnormalities were noted in seven out of eight rats with myocarditis 5 weeks after induction. Subsequently, the rats developed significant left ventricular (LV) dilatation, wall thickening, and pericardial effusion. Average LV systolic and diastolic volumes increased from 131 ± 10 to 257 ± 20 µL ( P = 0.0008), and from 309 ± 14 to 412 ± 24 µL ( P < 0.0001), and ejection fraction markedly deteriorated (from 58 ± 2 to 37 ± 5%; P = 0.0003). Areas of fibrosis were located by late gadolinium enhancement (LGE) CMR at the subepicardium, mainly within the anterior, lateral, and inferior walls. The extent and location of LGE were highly correlated ( r = 0.94; P < 0.0001) with areas of myocardial fibrosis by histopathology, with 85% sensitivity and 86% specificity. Conclusion A clinical 3 T CMR scanner enables accurate detection, quantification, and monitoring of experimental myocarditis in rats, and could be used for translational research to study the pathophysiology of the disease and evaluate novel therapies.

Uncontrolled activation of pro-inflammatory macrophages after myocardial infarction (MI) accelerates adverse left ventricular (LV) remodeling and dysfunction. Hemin, an iron-containing porphyrin, activates heme oxygenase-1 (HO-1), an enzyme with anti-inflammatory and cytoprotective properties. We sought to determine the effects of hemin formulated in a macrophage-targeted lipid-based carrier (denoted HA-LP) on LV remodeling and function after MI. Hemin encapsulation efficiency was ~ 100% at therapeutic dose levels. In vitro, hemin/HA-LP abolished TNF-α secretion from macrophages, whereas the same doses of free hemin and drug free HA-LP had no effect. Hemin/HA-LP polarized peritoneal and splenic macrophages toward M2 anti-inflammatory phenotype. We next induced MI in mice and allocated them to IV treatment with hemin/HA-LP (10 mg/kg), drug free HA-LP, free hemin (10 mg/kg) or saline, one day after MI. Active in vivo targeting to infarct macrophages was confirmed with HA-LP doped with PE-rhodamine. LV remodeling and function were assessed by echocardiography before, 7, and 30 days after treatment. Significantly, hemin/HA-LP effectively and specifically targets infarct macrophages, switches infarct macrophages toward M2 anti-inflammatory phenotype, improves angiogenesis, reduces scar expansion and improves infarct-related regional function. In conclusion, macrophage-targeted lipid-based drug carriers with hemin switch macrophages into an anti-inflammatory phenotype, and improve infarct healing and repair. Our approach presents a novel strategy to modulate inflammation and improve infarct repair.

Background: Little is known about the potentially unfavorable effects of mesenchymal stromal cell (MSC) activation on the heart. MSCs can respond to tissue injury by anti- or proinflammatory activation. We aimed to study the potential negative interaction between left ventricular dysfunction (LVD) and MSC activation. Methods: We isolated MSCs from cardiac and subcutaneous fat tissues of mice with LVD 28 days after myocardial infarction or sham operation. To evaluate the effect of LVD on MSCs, we characterized cardiac MSCs and subcutaneous MSCs in vitro. Subsequently, we injected MSCs or saline into the infarcted myocardium of mice and evaluated LV remodeling and function 28 days after myocardial infarction. To test the hypothesis that toll-like receptor 4 ( TLR4 ) mediates proinflammatory polarization of MSCs, we characterized cardiac MSCs from TLR4 -/- and wild-type (WT) mice after inflammatory stimulation in vitro. Next, we transplanted cardiac MSCs from TLR4 -/- and WT male mice into the infarcted myocardium of female WT mice and evaluated infarct size, MSC retention, inflammation, remodeling, and function after 7 days. Results: LVD switched cardiac MSCs toward an inflammatory phenotype, with increased secretion of inflammatory cytokines as well as chemokines. The effect of LVD on subcutaneous MSCs was less remarkable. Although transplantation of cardiac MSCs and subcutaneous MSCs from LVD and sham hearts did not improve LV remodeling and function, cardiac MSCs from LVD exacerbated anterior wall thinning 28 days after myocardial infarction. The inflammatory polarization of cardiac MSCs by LVD was mediated by TLR4 , as we found less secretion of inflammatory cytokines and higher secretion of anti-inflammatory cytokines from activated cardiac MSCs of TLR4 -deficient mice, compared with WT cardiac MSCs. Significantly, TLR4 deficiency preserved the expression of CD47 (don’t eat me signal) on cardiac MSCs after both TLR4 stimulation in vitro and transplantation into the infarcted heart. Compared with WT cardiac MSCs and saline, TLR4 -/- cardiac MSCs survived in the cardiac tissue and maintained their reparative properties, reduced infarct size, increased scar thickness, and attenuated LV dilatation 7 days after myocardial infarction. Conclusions: The environment of the failing and infarcted myocardium drives resident and transplanted MSCs toward a proinflammatory phenotype and restricts their survival and reparative effects in a mechanism mediated by TLR4 .

Nowadays, combination therapy became a standard in oncology. In this study, we compare the activity of two polymeric carriers bearing a combination of the anticancer drugs paclitaxel (PTX) and doxorubicin (DOX), which differ mainly in their architecture and supramolecular assembly. Drugs were covalently bound to a linear polymer, polyglutamic acid (PGA) or to a dendritic scaffold, polyglycerol (PG) decorated with poly(ethylene glycol) (PEG), forming PGA-PTX-DOX and PG-PTX-bz-DOX-PEG, respectively. We explored the relationship between the polymeric architectures and their performance with the aim to augment the pharmacological benefits of releasing both drugs simultaneously at the tumor site at a synergistic ratio. We recently designed and characterized a PGA-PTX-DOX conjugate. Here, we describe the synthesis and characterization of PG dendritic scaffold bearing the combination of PTX and DOX. The performance of both conjugates was evaluated in a murine model of mammary adenocarcinoma in immunocompetent mice, to investigate whether the activity of the treatments is affected by the immune system. Drug conjugation to a nano-sized polymer enabled preferred tumor accumulation by extravasation-dependent targeting, making use of the enhanced permeability and retention (EPR) effect. Both PGA-PTX-DOX and PG-PTX-bz-DOX-PEG nano-sized conjugates exhibited superior anti-tumor efficacy and safety compared to the combination of the free drugs, at equivalent concentrations. However, while PGA-PTX-DOX was more efficient than a mixture of each drug conjugated to a separate PGA chain, as was previously shown, PG-PTX-bz-DOX-PEG had similar activity to the mixture of the PG-PTX-bz-PEG and PG-DOX-PEG conjugates. Our results show that both conjugates are potential candidates as precision combination nanomedicines for the treatment of breast cancer.

Background Irreversible electroporation (IRE) is a non-thermal cell ablation approach that induces selective damage to cell membranes only. The purpose of the current study was to evaluate and optimize its use for in-vivo myocardial decellularization. Methods Forty-two Sprague-Dawley rats were used to compare myocardial damage of seven different IRE protocols with anterior myocardial infarction damage. An in-vivo open thoracotomy model was used, with two-needle electrodes in the anterior ventricular wall. IRE protocols included different combinations of pulse lengths (70 vs. 100 μseconds), frequency (1, 2, 4 Hz), and number (10 vs. 20 pulses), as well as voltage intensity (50, 250 and 500 Volts). All animals underwent baseline echocardiographic evaluation. Degree of myocardial ablation was determined using repeated echocardiography measurements (days 7 and 28) as well as histologic and morphometric analysis at 28 days. Results All animals survived 28 days of follow-up. Compared with 50V and 250V, electroporation with 500V was associated with significantly increased myocardial scar and reduction in ejection fraction (67.4%±4% at baseline vs. 34.6%±20% at 28 days; p

Inflammation has been implicated in the initiation, progression and manifestation of hypertensive heart disease. We sought to determine the role of monocytes/macrophages in hypertension and pressure overload induced left ventricular (LV) remodeling.We used two models of LV hypertrophy (LVH). First, to induce hypertension and LVH, we fed Sabra salt-sensitive rats with a high-salt diet. The number of macrophages increased in the hypertensive hearts, peaking at 10 weeks after a high-salt diet. Surprisingly, macrophage depletion, by IV clodronate (CL) liposomes, inhibited the development of hypertension. Moreover, macrophage depletion reduced LVH by 17% (p0.05), and reduced cardiac fibrosis by 75%, compared with controls (p=0.001). Second, to determine the role of macrophages in the development and progression of LVH, independent of high-salt diet, we depleted macrophages in mice subjected to transverse aortic constriction and pressure overload. Significantly, macrophage depletion, for 3 weeks, attenuated LVH: a 12% decrease in diastolic and 20% in systolic wall thickness (p0.05), and a 13% in LV mass (p=0.04), compared with controls. Additionally, macrophage depletion reduced cardiac fibrosis by 80% (p=0.006). Finally, macrophage depletion down-regulated the expression of genes associated with cardiac remodeling and fibrosis: transforming growth factor beta-1 (by 80%) collagen type III alpha-1 (by 71%) and atrial natriuretic factor (by 86%).Macrophages mediate the development of hypertension, LVH, adverse cardiac remodeling, and fibrosis. Macrophages, therefore, should be considered as a therapeutic target to reduce the adverse consequences of hypertensive heart disease.

Background The neonatal heart possesses the unique power to regenerate in response to resection of the left ventricular apex. We sought to determine whether the type of injury affects the mode of repair and regeneration. Methods and Results Apical resection, or permanent left anterior descending coronary artery ligation, was induced in neonatal 1‐day‐old mice. Echocardiography was used to confirm and monitor cardiac injury and remodeling. Histological and immunohistochemical examinations of the resected and infarcted neonatal hearts revealed inflammation and granulation tissue formation. From day 3, early regeneration was identified at the injured sites and was characterized by dedifferentiation and proliferation of cardiomyocytes around the injured areas. The young cardiomyocytes infiltrated the granulation tissue and replaced it with a new myocardium. The ability of neonatal cardiomyocytes to proliferate was confirmed in neonatal heart organ cultures. Notably, myocardial infarction in neonatal mouse produced incomplete regeneration with a residual small infarct and, sometimes, aneurysm at 28 days after myocardial infarction. We then repeated the same experiments in the adult heart. Remarkably, myocardial infarction in the adult mouse heart produced a typical thin scar, whereas apical resection revealed an abnormal, epicardial, hemorrhagic scar 21 days after injury. Conclusions Our findings suggest that the type of injury, resection, or infarction affects the mode of repair in both neonatal and adult mouse hearts. Identifying the differences in the mechanisms or repair of these 2 types of injuries could help to develop novel regenerative therapies relevant to human patients.

Ligand-receptor mediated targeting may affect differently the performance of supramolecular drug carriers depending on the nature of the nanocarrier. In this study, we compare the selectivity, safety and activity of doxorubicin (Dox) entrapped in liposomes versus Dox conjugated to polymeric nanocarriers in the presence or absence of a folic acid (FA)-targeting ligand to cancer cells that overexpress the folate receptor (FR). Two pullulan (Pull)-based conjugates of Dox were synthesized, (FA-PEG)-Pull-(Cyst-Dox) and (NH2-PEG)-Pull-(Cyst-Dox). The other delivery systems are Dox loaded PEGylated liposomes (PLD, Doxil®) and the FR-targeted version (PLD-FA) obtained by ligand post-insertion into the commercial formulation. Both receptor-targeted drug delivery systems (DDS) were shown to interact in vitro specifically with cells via the folate ligand. Treatment of FR-overexpressing human cervical carcinoma KB tumor-bearing mice with three-weekly injections resulted in slightly enhanced anticancer activity of PLD-FA compared to PLD and no activity for both pullulan-based conjugates. When the DDS were administered intravenously every other day, the folated-Pull conjugate and the non-folated-Pull conjugate displayed similar and low antitumor activity as free Dox. At this dosing regimen, the liposome-based formulations displayed enhanced antitumor activity with an advantage to the non-folated liposome. However, both liposomal formulations suffered from toxicity that was reversible following treatment discontinuation. Using a daily dosing schedule, with higher cumulative dose, the folated-Pull conjugate strongly inhibited tumor growth while free Dox was toxic at this regimen. For polymeric constructs, increasing dose intensity and cumulative dose strongly affects the therapeutic index and reveals a major therapeutic advantage for the FR-targeted formulation. All DDS were able to abrogate doxorubicin-induced cardiotoxicity. This study constitutes the first side-by-side comparison of two receptor-targeted ligand-bearing systems, polymer therapeutics versus nanoparticulate systems, evaluated in the same mouse tumor model at several dosing regimens.

Hypertension and Diabetes commonly coexist and have been implicated in deterioration of kidneys, heart and impairment of pancreas. Empagliflozin (Empa), a selective SGLT2 inhibitor, is a new agent for treatment of diabetes via enhancing glucosuria, independent of insulin resistance. This study evaluates Empa's prophylactic beneficial effect on glomerular, cardiac and pancreatic integrity, in CRDH animals. Cohen Rosenthal Diabetic Hypertensive rats (CRDH) were divided into 3 groups: A- Sugar diet (SD) + Empa, B-Sugar Diet + Veh, C-Regular Chow + Veh (Control). Drug was given for 4 months via drinking water at a dose of 10mg/kg/day. Hyperglycemia, hypertension, and additional biochemical parameters were monitored. Immunofluorescence analysis of glomerular nephrin and H&E in pancreas was performed in different experimental groups and their sham controls. To assess left ventricular (LV) remodeling and function, a special small animal echocardiography scanner was used. Blood pressure was significantly (P

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.

Introduction: Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest for cell-based therapies. However, the best source of hMSCs for infarct repair is still unknown. Hypothesis: We aimed to test the hypothesis that, because of their proximity to the heart, hMSCs from the heart and epicardial fat would be better cells for infarct repair. Methods and Results: We isolated and grew hMSCs from patients with ischemic heart disease (IHD) from four different sources: 1) epicardial fat 2) pericardial fat 3) subcutaneous fat and 4) the right atrium. hMSCs from the right atrium and epicardial fat secreted the highest amounts of trophic and inflammatory cytokines, while hMSCs from pericardial and subcutaneous fat secreted the lowest (pin vivo , we allocated rats to hMSC transplantation 7 days after myocardial infarction (MI) (n=8-10 per group). Atrial hMSCs induced the highest number of vessels in the infarct, together with the highest inflammation score and macrophage accumulation 27 days after their injection. However, left ventricular (LV) dysfunction was worst after therapy with atrium and epicardial fat hMSCs, and minimal after subcutaneous fat hMSC therapy. Notably, there was a correlation between levels of tumor necrosis factor-α and hepatocyte growth factor, in vitro , and post-transplantation LV systolic dilatation. These surprising findings were supported by gene expression analysis in hMSCs from different sources of a patient with IHD. We found that the relative expression of inflammation and fibrosis-related genes was considerably higher in hMSCs from the right atrium and epicardial fat than in subcutaneous fat hMSCs. Conclusions: The origin of hMSCs affects their reparative and immunomodulatory properties. Because of their pro-inflammatory properties, hMSCs from the right atrium and epicardial fat of IHD patients could impair heart function after MI. Our findings might be relevant to autologous MSC therapy and pathogenesis and progression of IHD.

PURPOSE: Recent reports have demonstrated complete cardiac regeneration in newborn mice following resection of the cardiac apex. However, different types of injury could affect the mechanism of regeneration and repair. HYPOTHESIS: We aimed to test the hypothesis that the course of repair and regeneration after MI is different from apical resection in both neonatal and adult mouse heart. Methods and Results: Apical resection or permanent LAD coronary artery ligation was induced in 1-day-old or 12-week-old ICR mice. Echocardiography was used to confirm and monitor cardiac injury and remodeling. Mice were euthanized at different time points after operation, and hearts were harvested, processed, immunostained and compared with sham operated neonatal and adult hearts. Histological and immunohistochemical examination of both resected and infarcted neonatal hearts revealed inflammation and granulation tissue formation within 3 to 5 days. In the resected hearts, early regeneration was identified at the injured sites, and marked dedifferentiation of cardiomyocytes, represented by sarcomeric disassembly and marginalization, was evident around the injured areas. In addition, we noticed intensive proliferation of young cardiomyocytes which infiltrated the granulation tissue and formed a new myocardium within 21 days. In contrast, incomplete regeneration with residual small infract was detected 28 days after coronary occlusion. Echocardiography at 2,7,14 and 28 days after MI confirmed deteriorating LV function and LV remodeling with apical aneurysm formation. Surprisingly, 21 days after cardiac injury in adult mice, MI produced typical, thin, fibrotic scar whereas apical resection produced an apical tumor-like thick scar. Conclusions: The mode of injury, whether resection or infarction, affect regeneration and repair in both neonatal and adult mouse heart. Particularly, after apical resection, the newborn heart almost completely regenerates whereas regeneration is incomplete after MI, suggesting that infarction and subsequent inflammation might inhibit complete regeneration. Understanding these differences could be translated to development of new approaches to induce myocardial regeneration in adult heart.

Introduction: The mechanism of myocarditis is complex and poorly understood. Monocytes and macrophages comprise the majority of infiltrates in myocarditis. Hypothesis: We aimed to test the hypothesis that macrophage subsets have a key role in the pathogenesis and progression of myocarditis, and that targeting macrophages would modulate disease progression and outcome. Methods and Results: Experimental autoimmune myocarditis (EAM) was induced in 67 male Lewis rats. Of them, 27 were sacrificed at different time points to assess M1 and M2- macrophage subsets by flow cytometry. The remaining rats were randomized either to early macrophage depletion (from day 8-14 after EAM induction), by intravenous injections of clodronate-liposomes (CL), or late depletion (from day 15-35 after induction of EAM), or PBS-liposome injections (control). Left ventricula (LV) remodeling and function were assessed by echocardiography; before induction of EAM, before CL treatment, and 35 days after induction of EAM. Macrophages in the heart peaked at 21 days after induction of EAM (25±7%,p=0.0008).The number of M1 macrophages in the heart was highest at 14 days after EAM induction (16.3±2%), while M2 macrophages peaked at day 21 (15.5±2.7%,p=0.003). Early macrophage depletion (8-14d) decreased LV diastolic dimension and volume by 5.3±3.9% and 10.4±8% (p=0.04). Late macrophage depletion (15-35d) increased LV wall thickness and decreased LV systolic dimension and volume by 10.2±4% and 21±8% (p=0.03). Furthermore, late macrophage depletion increased LV ejection fraction and fractional shortening by 14.6±6% (p=0.01) and 22±10% (p=0.02) 35 days after EAM. Conclusions: Our study describes, for the first time, the kinetics of M1 and M2 macrophages in ocarditis and shows that macrophages contribute to the progression of adverse remodeling and dysfunction. Thus, targeting macrophages could be a new therapeutic strategy to improve cardiac remodeling and function in myocarditis.

Introduction: Inflammation has been implicated in the initiation, progression and manifestation of hypertension. However, the role of macrophages in hypertension-induced LV remodeling has yet to be determined. Hypothesis: We assessed the hypothesis that macrophages play a significant role in the initiation and progression of hypertension-induced LV remodeling. Methods and Results: Hypertension was induced in male Sabra salt-sensitive (SBH/y) rats with high salt diet (8%NaCl) over 6 weeks. Hypertensive SBH/y developed LV hypertrophy, hyper-contractility and a higher LV ejection fraction (EF). Notably, the number of macrophages, particularly cardiac macrophages was significantly greater in hypertensive SBH/y than in controls. Macrophage depletion was induced after induction of hypertension by intravenous administration of clodronate liposomes at 3-day intervals over 4 weeks (n=9) and was validated by FACS analysis and ED1 (CD68) staining of heart sections. Control hypertensive rats (n=9) were treated with PBS liposomes. Surprisingly, macrophage depletion attenuate the progression of hypertension in the clodronate-treated group (from 177±2 to 179±2 mmHg) compared with controls (from 179±2 to 184 ± 5mmHg; p=0.01).Serial echocardiography studies before and 30 days after initiation of macrophage depletion or control showed that LV systolic diameter and volume were smaller; anterior wall thickening, fractional shortening and EF were higher in the macrophage-depletion group (p Conclusion: Our findings suggest a significant role of macrophages in the initiation and progression of salt-sensitive hypertension and the related heart disease. Targeting macrophages, therefore, could be a potential therapeutic target for the treatment of hypertension and prevention of LV remodeling and dysfunction.

The extension in human lifespan in the last century results in a significant increase in incidence of age related diseases. It is therefore crucial to identify key factors that control elderly healthspan. Similar to dietary restriction, mice overexpressing the NAD+ dependent protein deacylase SIRT6 (MOSES) live longer and have reduced IGF-1 levels. However, it is as yet unknown whether SIRT6 also affects various healthspan parameters. Here, a range of age related phenotypes was evaluated in MOSES mice. In comparison to their wild-type (WT) littermates, old MOSES mice showed amelioration of a variety of age-related disorders, including: improved glucose tolerance, younger hormonal profile, reduced age-related adipose inflammation and increased physical activity. The increased activity was accompanied with increased muscle AMP-activated protein kinase (AMPK) activity. Altogether, these results indicate that overexpression of SIRT6 in mice retards important aspects of the aging process and suggest SIRT6 to be a potential therapeutic target for the treatment of a set of age-related disorders.

Background: Myocarditis is a life-threatening heart disease characterized by myocardial inflammation, necrosis, and chronic fibrosis. While mast cell inhibition has been suggested to prevent fibrosis in rat myocarditis, little is known about its effectiveness in attenuating cardiac remodeling and dysfunction in myocarditis. Thus, we sought to test the hypothesis that mast cell inhibition will attenuate the inflammatory reaction and associated left ventricular (LV) remodeling and dysfunction after fulminant autoimmune myocarditis. Methods and Results: To induce experimental autoimmune myocarditis, we immunized 30 rats with porcine cardiac myosin (PCM) twice at a 7-day interval. On day 8 animals were randomized into treatment with either an intraperitoneal (IP) injection of 25mg/kg of cromolyn sodium (n = 13) or an equivalent volume (∼0.5 mL IP) of normal saline (n = 11). All animals were scanned by serial echocardiography studies before treatment (baseline echocardiogram) and after 20 days of cromolyn sodium (28 days after immunization). Furthermore, serial cardiac magnetic resonance was performed in a subgroup of 12 animals. After 20 days of treatment (28 days from first immunization), hearts were harvested for histopathological analysis. By echocardiography, cromolyn sodium prevented LV dilatation and attenuated LV dysfunction, compared with controls. Postmortem analysis of hearts showed that cromolyn sodium reduced myocardial fibrosis, as well as the number and size of cardiac mast cells in the inflamed myocardium, compared with controls. Conclusions: Our study suggests that mast cell inhibition with cromolyn sodium attenuates adverse LV remodeling and dysfunction in myocarditis. This mechanism-based therapy is clinically relevant and could improve the outcome of patients at risk for inflammatory cardiomyopathy and heart failure.

Methods and results Male Lewis rats (n=11) were subjected to myosin immunization and developed autoimmune myocarditis. Approximately 3 weeks later, rats with myocarditis underwent CMR scan and subsequently, histopathological evaluation. Rats with myocarditis showed pericardial thickening, effusion, and LV wall motion abnormalities with septal hypokinesis. Short axis views showed patchy delayed enhancement of epicardial segments, with distribution mainly located within the inferolateral LV wall including the septum. This increased signal/hyperenhancement defines focal areas of myocardial fibrosis/ edema and/or necrosis, highly suggestive of inflammation. Additionally, the presence of large pericardial effusion provides supportive evidence for the existence of perimyocarditis. Indeed, high correlation was found

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.

We appreciate the comments of Drs Sadek and Garry on our study.1 We agree that ‘the competent immune system of the rat may have recognized the mesenchymal cells as foreign and eliminated those cells via cellular rejection.‘ We also agree that studies with autologous stem cells or delivery …

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.

Purpose A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3–5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI. Methods Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofloerescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted. Results The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3–5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced. Conclusions TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions.

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