Regenerative Potential of a Suspension and Spheroids of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord on the Model of Myocardial Infarction in Rats.

Regenerative potential of multipotent mesenchymal stromal cells from the human umbilical cord (MMSC-UC) in the suspension and spheroid form was revealed during the progression of experimental small focal myocardial infarction in rats. In isoproterenol-induced myocardial infarction, foci of necrosis and inflammatory infiltrate and at later terms fibrosis foci were found mainly in the left ventricle of rat heart. In rats receiving MMSC-UC, destructive changes in the myocardium, fibrous scars, and inflammatory process were less pronounced. MMSC-UC also contributed to normalization of the morphofunctional parameters of the heart. Spheroids exhibited higher efficiency in comparison with cell suspension.

MiR-18a-5p inhibits endothelial-mesenchymal transition and cardiac fibrosis through the Notch2 pathway.

Hyperglycemia plays a crucial role in the pathogenesis of diabetic complications; however, the mechanisms underlying diabetic cardiac fibrosis remain unclear. Endothelial cells are known to contribute to cardiac fibrosis through endothelial-mesenchymal transition (EndMT) under high glucose stimulation. Here we investigated the expression of miR-18a-5p and examined its functional role in human aortic valvular endothelial cells (HAVECs). Using HAVECs, we revealed that miR-18a-5p regulated high glucose-induced EndMT. Moreover, high glucose levels induced Notch2 expression, which promoted EndMT, resulting in the downregulation of vascular endothelial cadherin and CD31 and upregulation of fibroblast-specific protein-1, α-smooth muscle actin, fibronectin, and vimentin. Furthermore, Notch2 was identified as a target of miR-18a-5p. Our data showed that the overexpression of miR-18a-5p could downregulate Notch2 expression and subsequently suppress EndMT. In conclusion, our findings demonstrated that miR-18a-5p/Notch2 signaling pathway participates in the regulation of high glucose-induced EndMT, and may act as a novel promising target for myocardial fibrosis in diabetic cardiomyopathy.

Inflammatory and fibrotic processes are involved in the cardiotoxic effect of sunitinib: Protective role of L-carnitine.

Sunitinib (Su) is currently approved for treatment of several malignances. However, along with the benefits of disease stabilization, cardiovascular toxicities have also been increasingly recognized. The aim of this study was to analyze which mechanisms are involved in the cardiotoxicity caused by Su, as well as to explore the potential cardioprotective effects of l-carnitine (LC). To this end, four groups of Wistar rats were used: (1) control; (2) rats treated with 400mg LC/kg/day; (3) rats treated with 25mg Su/kg/day; and (4) rats treated with LC+Su simultaneously. In addition, cultured rat cardiomyocytes were treated with an inhibitor of nuclear factor kappa B (NF-κB), in order to examine the role of this transcription factor in this process. An elevation in the myocardial expression of pro-inflammatory cytokines, together with an increase in the mRNA expression of NF-κB, was observed in Su-treated rats. These results were accompanied by an increase in the expression of pro-fibrotic factors, nitrotyrosine and NOX 2 subunit of NADPH oxidase; and by a decrease in that of collagen degradation factor. Higher blood pressure and heart rate levels were also found in Su-treated rats. All these alterations were inhibited by co-administration of LC. Furthermore, cardiotoxic effects of Su were blocked by NF-κB inhibition. Our results suggest that: (i) inflammatory and fibrotic processes are involved in the cardiac toxicity observed following treatment with Su; (ii) these processes might be mediated by the transcription factor NF-κB; (iii) LC exerts a protective effect against arterial hypertension, cardiac inflammation and fibrosis, which are all observed after Su treatment.

Myocardial fibrosis induced by exposure to subclinical lipopolysaccharide is associated with decreased miR-29c and enhanced NOX2 expression in mice.

BACKGROUND: Exposure to subclinical levels of lipopolysaccharide (LPS) occurs commonly and is seemingly well tolerated. However, recurrent LPS exposure induces cardiac fibrosis over 2 to 3 months in a murine model, not mediated by the renin-angiotensin system. Subclinical LPS induces cardiac fibrosis by unique mechanisms. METHODS: In C57/Bl6 mice, LPS (10 mg/kg) or saline (control) were injected intraperitoneally once a week for 1-4 weeks. Mice showed no signs of distress, change in activity, appetite, or weight loss. Mice were euthanized after 3 days, 1, 2, or 4 weeks to measure cardiac expression of fibrosis-related genes and potential mediators (measured by QRT-PCR), including micro-RNA (miR) and NADPH oxidase (NOX). Collagen fraction area of the left ventricle was measured with picrosirius red staining. Cardiac fibroblasts isolated from adult mouse hearts were incubated with 0, 0.1, 1.0 or 10 ng/ml LPS for 48 hours. RESULTS: Cardiac miR expression profiling demonstrated decreased miR-29c after 3 and 7 days following LPS, which were confirmed by QRT-PCR. The earliest changes in fibrosis-related genes and mediators that occurred 3 days after LPS were increased cardiac expression of TIMP-1 and NOX-2 (but not of NOX-4). This persisted at 1 and 2 weeks, with additional increases in collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, TIMP2, and periostin. There was no change in TGF-ß or connective tissue growth factor. Collagen fraction area of the left ventricle increased after 2 and 4 weeks of LPS. LPS decreased miR-29c and increased NOX-2 in isolated cardiac fibroblasts. CONCLUSIONS: Recurrent exposure to subclinical LPS induces cardiac fibrosis after 2-4 weeks. Early changes 3 days after LPS were decreased miR-29c and increased NOX2 and TIMP1, which persisted at 1 and 2 weeks, along with widespread activation of fibrosis-related genes. Decreased miR-29c and increased NOX2, which induce cardiac fibrosis in other conditions, may uniquely mediate LPS-induced cardiac fibrosis.

AdipoR1/APPL1 potentiates the protective effects of globular adiponectin on angiotensin II-induced cardiac hypertrophy and fibrosis in neonatal rat atrial myocytes and fibroblasts.

Atrial hypertrophy and fibrosis are essential pathological features of atrial fibrillation. Recently, adiponectin has become a protein of interest due to its beneficial effects on cardiovascular diseases. However, the molecular mechanism of atrial structural remodeling and signaling pathways evoked by adiponectin remain unclear. In the present study, we investigated the cardioprotective effect of globular adiponectin (gAcrp) on angiotensin II-induced atrial hypertrophy and fibrosis in neonatal Sprague-Dawley rat. To further investigate the molecular mechanisms underlying the preventive effect of gAcrp, transfection of cells with siRNA was used to suppress the mRNA expression of adiponectin receptor 1 (AdipoR1) and its downstream adaptor protein APPL1. Non-silencing-Cy-3 labelled siRNA was used to determine transfection efficiency using fluorescence microscopy. The expression of atrial natriuretic peptide and procollagen type1 α-1, hypertrophy marker and fibrosis one, respectively, was detected by real-time PCR. Furthermore, the expression of adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K) and Akt was detected by western blotting. In addition, nuclear p65 translocation activity was analyzed by EMSA supershift assay. Our results showed that AdipoR1 and the adaptor protein APPL1 mediated the protective effects of gAcrp. In addition, the function of adiponectin and phosphorylation of AMPK were prominently diminished by inhibition of PI3K. Furthermore, nuclear factor-κB (NF-κB) transcription was diminished by the specific inhibition of AMPK. Taken together, AMPK pivotally interacts with NF-κB and PI3K, mediating the cardioprotective effect of adiponectin, and may serve as a therapeutic target for preventing atrial hypertrophy and fibrosis. Our present study suggests that gAcrp could ameliorate AngII-induced cardiac hypertrophy and fibrosis in rat atrial cells, which is mediated by the activation of AMPK signaling pathways. APPL1 and AdipoR1 are the key factors involved in the downstream of gAcrp approach.

Diffuse myocardial fibrosis by T1-mapping in children with subclinical anthracycline cardiotoxicity: relationship to exercise capacity, cumulative dose and remodeling.

BACKGROUND: The late cardiotoxic effects of anthracycline chemotherapy influence morbidity and mortality in the growing population of childhood cancer survivors. Even with lower anthracycline doses, evidence of adverse cardiac remodeling and reduced exercise capacity exist. We aim to examine the relationship between cardiac structure, function and cardiovascular magnetic resonance (CMR) tissue characteristics with chemotherapy dose and exercise capacity in childhood cancer survivors. METHODS: Thirty patients (15 ± 3 years), at least 2 years following anthracycline treatment, underwent CMR, echocardiography, and cardiopulmonary exercise testing (peak VO(2)). CMR measured ventricular function, mass, T(1) and T(2) values, and myocardial extracellular volume fraction, ECV, a measure of diffuse fibrosis based on changes in myocardial T1 values pre- and post-gadolinium. Cardiac function was also assessed with conventional and speckle tracking echocardiography. RESULTS: Patients had normal LVEF (59 ± 7%) but peak VO(2) was 17% lower than age-predicted normal values and were correlated with anthracycline dose (r = -0.49). Increased ECV correlated with decreased mass/volume ratio (r = -0.64), decreased LV wall thickness/height ratio (r = -0.72), lower peak VO(2)(r = -0.52), and higher cumulative dose (r = 0.40). Echocardiographic measures of systolic and diastolic function were reduced compared to normal values (p < 0.01), but had no relation to ECV, peak VO(2) or cumulative dose. CONCLUSIONS: Myocardial T1 and ECV were found to be early tissue markers of ventricular remodeling that may represent diffuse fibrosis in children with normal ejection fraction post anthracycline therapy, and are related to cumulative dose, exercise capacity and myocardial wall thinning.

CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes.

AIMS: Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX). METHODS AND RESULTS: Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure-volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1(+/+)), and these effects were markedly attenuated in CB1 knockouts (CB1(-/-)). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT-PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX +/- AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation. CONCLUSION: CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular diseases.

O bloqueio da síntese do óxido nítrico promove aumento da hipertrofia e da fibrose cardíaca em ratos submetidos a treinamento aeróbio / Nitric oxide synthesis blockade increases hypertrophy and cardiac fibrosis in rats submitted to aerobic training

OBJETIVO: O presente estudo avaliou as adaptações teciduais cardíacas em ratos submetidos a treinamento aeróbio, após o bloqueio da síntese de óxido nítrico (NO). MÉTODOS: Os animais (n = 48) foram divididos em quatro grupos: sedentários (grupo CONTROLE), hipertensos após administração de Ng-nitro-L-arginina metil éster durante sete dias (grupo L-NAME), treinados por meio de natação durante oito semanas (grupo TREINADO) e treinados e tratados com L-NAME na última semana (grupo TREINADO L-NAME). Em todos os animais foi registrada a pressão arterial (PA) e realizada a avaliação morfométrica cardíaca. RESULTADOS: Os grupos L-NAME e TREINADO L-NAME apresentaram-se hipertensos em relação aos demais (p < 0,05), porém a elevação da PA no grupo TREINADO L-NAME foi significativamente menor em relação ao L-NAME (p < 0,05). Os grupos TREINADO e TREINADO L-NAME apresentaram índice de peso cardíaco maior que os grupos CONTROLE e L-NAME (p < 0,05). Também apresentaram maiores índices de área cardíaca macroscópica e de fibrose cardíaca em relação aos demais (p < 0,05) e, quando comparados, o grupo TREINADO L-NAME mostrou-se significativamente superior (p < 0,05). CONCLUSÃO: O bloqueio a curto prazo da síntese de NO, em animais sedentários, induziu hipertensão, sem no entanto causar hipertrofia cardíaca. Nos animais treinados, a inibição da síntese de NO atenuou a hipertensão e promoveu hipertrofia cardíaca com aumento expressivo da fibrose miocárdica, sugerindo importante papel do NO nas adaptações teciduais cardíacas induzidas pelo treinamento físico aeróbio.

OBJECTIVE: The objective of the present study was to evaluate cardiac tissue adaptations in rats submitted to aerobic training after nitric oxide (NO) synthesis blockade. METHODS: The animals (n=48) were divided into four groups: sedentary (CONTROL group); hypertensive after administration of NG-nitro-L-arginine methyl ester for 7 days (L-NAME Group); trained for 8 weeks through swimming exercises (TRAINED Group);trained and treated with L-NAME during the last week (L-NAME TRAINED Group). All the animals were submitted to the experiment procedures for blood pressure (BP) readings and cardiac morphometric evaluation. RESULTS: In comparison to the other groups, the L-NAME and L-NAME TRAINED groups were hypertensive (p<0.05); however, BP elevation in the L-NAME TRAINED group was significantly lower than the L-NAME group (p<0.05). The heart weight indexes for the TRAINED and L-NAME TRAINED groups were higher than the CONTROL and L-NAME groups (p<0.05). Also they had presented higher rates of macroscopic cardiac area and cardiac fibrosis in relation to the rest (p<0.05); comparisons revealed that the values for the L-NAME TRAINED group were significantly higher (p<0.05) than the others. CONCLUSION: Short term NO synthesis blockade in sedentary animals induced hypertension but did not cause cardiac hypertrophy. In the trained animals, the inhibition of NO synthesis attenuated hypertension, induced cardiac hypertrophy and significantly increased myocardial fibrosis, indicating that NO plays an important role in cardiac tissue adaptations caused by aerobic exercise.

[Angiotensin converting enzyme inhibitor enalapril in the treatment of endomyocardial fibrosis in patients with lymphogranulomatosis subjected to radio- and chemotherapy]. / Ingibitor angiotenzinprevrashchaiushchego fermenta énalapril v lechenii postluchevogo i lekarstvenno-indutsirovannogo éndomiokardial'nogo fibroza pri limfogranulematoze.

Echocardiography was used in the study of 40 patients with stage IIA-IVB lymphogranulomatosis. In 2-6 years before the study all patients had been treated with combination radio- (36-44 Gy) and chemotherapy. Echocardiography revealed endomyocardial fibrosis and signs of cardiac remodeling (reduced left ventricular dimensions and volumes, decreased myocardial mass and impaired diastolic function). The patients were divided into 2 groups. Patients of group 1 were given enalapril (5-10 mg/day), of group 2 - potassium and magnesium aspartate and inosine. In 2 months 68% of patients in group 1 demonstrated improvement of structural and functional state of the heart, no such changes occurred in group 2.

Cardiac consequences of the systemic lupus erythematosus therapy with corticosteroids morphological study.

It is presented the case of a fifty years old women, diagnosed 3 years ago with systemic lupus erythematosus, under therapy with prednisone and cyclophosphamid therapy. She was admitted in our hospital for right decompensated heart disease and the presence of an apical right ventricular mass occluding part of the right ventricular cavity. The endomyocardial biopsy was made to clearify the nature of this mass. After processing the specimen, the histological study evidenciated an organizing apical thrombotic mass formed in a large right ventricular cavity in conditions of pulmonary hypertention. There are presented data concerning the adverse effects of the systemic lupus erythematosus drug therapy, as well. In these circumstances, we demonstrated histologically, that both conditions could alter the heart morphology.

Myocardial necrosis and cocaine. A quantitative morphologic study in 26 cocaine-associated deaths.

A quantification of different forms of acute myocardial necrosis, myocardial leukocytic infiltrates and myocardial fibrosis was accomplished in 26 chronic cocaine abusers who died of cocaine intoxication and compared to 45 normal subjects who died from head trauma and 38 who died of acquired immunodeficiency syndrome. The findings were: absence of infarct necrosis, a similar frequency and extent of coagulative myocytolysis (contraction band necrosis) and leukocytic infiltrates in cocaine abusers and normal controls, and an absence of myocardial fibrosis in cocaine abusers. These findings question both the acute and chronic cardiotoxicity of cocaine. The infarct-like pattern in some predisposed subjects may be due to an excess of catecholamine release induced by the drug resulting in coagulative myocytolysis and platelet thrombi.

Serious cardiovascular side effects of large doses of anabolic steroids in weight lifters.

Pathological cardiovascular manifestations are reported in four male patients, who had taken massive amounts of anabolic steroids while undergoing many years of strength training. One patient was referred because of ventricular fibrillation during exercise, one because of clinically manifest heart failure, and one because of arterial thrombus in his lower left leg. The fourth patient was persuaded to attend for a check-up because of a long history of massive use of anabolic steroids. All four patients had cardiac hypertrophy. Two of the patients had symptoms and signs of heart failure, and one of these two had a massive thrombosis in both right and left ventricles of his heart. After cessation of the use of anabolic steroids in the other patient with heart failure, left ventricular wall thickness reduced quickly from 12 to 10.5 mm, and fractional shortening increased from 14% to 27%. Endomyocardial biopsy revealed increased fibrosis in the myocardium in two of the three cases. HDL-cholesterol was 0.58 mmol.l-1 and 0.35 mmol.l-1 in the two patients still using multiple anabolic steroids at the time of investigation. The cardiovascular findings described in the present paper should warn all physicians and athletes about the possible serious acute and long-term side effects of the massive use of anabolic steroids.

Chemotherapy induced myocardial fibrosis.

Alkylating agents have a well established role in the treatment of malignant disease, with well documented side effects. This case history, however, illustrates the susceptibility to these agents shown by some patients. It is suggested that, when drug induced pneumonitis is noted, careful screening of left ventricular function should be performed in order to diagnose myocardial fibrosis and subsequent cardiac failure.

Cyclosporine A has no direct effect on collagen metabolism by cardiac fibroblasts in vitro.

BACKGROUND: Cyclosporine A has been implicated in the pathogenesis of myocardial interstitial fibrosis observed in heart transplant recipients. However, other confounding variables such as posttransplantation hypertension and rejection episodes may also be responsible for interstitial fibrosis development and associated abnormalities in ventricular diastolic function. Therefore, we examined whether cyclosporine A directly or indirectly affects fibrillar collagen metabolism by cardiac fibroblasts in vitro. METHODS AND RESULTS: Rat cardiac fibroblasts were isolated by collagenase digestion. Subconfluent cultures were then maintained (24 hours) in serum-containing or serum-free medium before addition of cyclosporine A (50-1,000 ng/mL). After an additional 24 hours, total procollagen synthesis, accumulation, and degradation were analyzed by measuring hydroxyproline content in the cell monolayer and in the ethanol-soluble and ethanol-precipitable fractions of the culture medium. mRNA levels for alpha 1(I) and alpha 1(III) procollagen polypeptides were assessed 2, 6, 12, and 24 hours after cyclosporine A treatment using Northern blot analysis. The results were compared with control cultures maintained in the absence of cyclosporine A. There were no differences in procollagen gene expression, total procollagen synthesis, accumulation, or degradation in cardiac fibroblasts treated directly with cyclosporine A, in concentrations up to 1,000 ng/mL, compared with untreated cells. In additional experiments, we examined whether cyclosporine A might stimulate the production of collagen regulatory substances by cardiac myocytes in culture. However, addition of conditioned media from neonatal myocytes maintained in the presence and absence of cyclosporine A (1,000 ng/mL) also had no effect on collagen deposition by cardiac fibroblasts. CONCLUSIONS: We conclude that cyclosporine A has no direct effect on collagen metabolism by cultured cardiac fibroblasts in vitro. In addition, we have excluded a paracrine effect of ventricular myocytes on collagen production in the presence of cyclosporine A. These results suggest that factors other than cyclosporine A are responsible for the interstitial fibrosis observed in cardiac allografts.

Long-term hemodynamic follow-up of cardiac transplant patients treated with cyclosporine and prednisone.

To evaluate the long-term hemodynamic results in cardiac transplant patients treated with cyclosporine and prednisone, 19 patients were studied by cardiac catheterization and endomyocardial biopsy 13 +/- 3 months after transplantation. Immunosuppression consisted of 6 +/- 4 mg/kg/day cyclosporine and 20 +/- 8 mg/day prednisone. Eighteen patients were asymptomatic but had developed postoperative systemic hypertension (17 on antihypertensive therapy). These patients were compared with a normotensive control group of 18 patients without cardiovascular disease. Significant differences were found in heart rate; right atrial, pulmonary arterial, pulmonary arterial wedge, systemic arterial, and left ventricular end-diastolic pressures; cardiac index and stroke volume index; systemic and pulmonary vascular resistance; and end-diastolic volume index and left ventricular ejection fraction. The most frequent hemodynamic abnormalities included an elevated arterial pressure in 10 patients (56%), an elevated left ventricular end-diastolic pressure in six patients (33%), and a reduced ejection fraction in five patients (28%). Hemodynamic abnormalities tended to resolve or improve in the five patients restudied 2 years after transplantation. There was no significant relationship between fibrosis or inflammation on endomyocardial biopsy and hemodynamic abnormalities. We conclude that mild-to-moderate hemodynamic abnormalities are common in asymptomatic cardiac transplant patients receiving cyclosporine and prednisone.

Cyclosporine induced myocardial fibrosis: a unique controlled case report.

Many patients treated with cyclosporine in the Stanford Heart Transplant Program have exhibited perimyocytic fibrosis in the donor heart, seen on endomyocardial biopsy specimen. Although restrictive hemodynamic effects have not been observed up to three years after transplantation, there is a possibility that such changes could become a problem. With the increasing use of cyclosporine in non-cardiac organ transplantation, it is of interest to examine the effect of this drug upon the non transplanted heart. This was possible in a man who underwent a heterotopic heart transplantation 3 1/2 years ago, and who has been followed with serial endomyocardial biopsies from both hearts. Although there were no episodes of rejection the donor heart developed significant perimyocytic fibrosis which was not seen in the native heart. This study suggests that cyclosporine induced fibrosis only occurs in the transplanted heart.

Simulation of acute myopericarditis by constrictive pericardial disease with endomyocardial fibrosis due to methysergide therapy.

Methysergide ( Sansert ) has been associated with numerous fibrotic disorders. In particular, multiple cardiac lesions have been described in cases where methysergide was thought to have played a causative role. A patient is described who presented with cardiac findings suggestive of acute myopericarditis . An inflammatory myocarditis was subsequently excluded by endomyocardial biopsy. Hemodynamic findings suggested the presence of constrictive pericarditis or restrictive cardiomyopathy, or both. Radiographic evidence of constrictive pericarditis and biopsy evidence of endocardial fibrosis were documented in this patient with a long history of interrupted methysergide therapy.

Endomyocardial fibrosis in rats treated with N-nitrosomorpholine.

Endomyocardial fibrosis was observed after long lag periods in male Sprague-Dawley rats treated for 1-14 weeks with the carcinogen N-nitrosomorpholine. The fibrosis developed predominantly in the left ventricle. It occurred during 29-78 weeks after withdrawal of the carcinogen in 5% and 79-108 weeks after withdrawal in 20% of the experimental animals, but was never observed in controls of the same age. We suggest that endomyocardial fibrosis was induced by a direct effect of the carcinogen on the fibroblasts of the endomyocardium.