Platelet procoagulant activity and microvesicle formation. Its putative role in hemostasis and thrombosis.

Spontaneously hypertensive rats recieved 1 mg/kg of Adriamycin intravenously once a week for up to 12 weeks; their hearts were excised and perfused with buffer containing 4 mM [1-¹³C]glucose. Histological evidence of Adriamycin cardiotoxicity was evident after 8 and 12 weeks of treatment and was accompanied by a significant decrease in cardiac function. There were only minor changes in the ³¹P-NMR spectra in hearts following treatment; however, ¹³C-NMR spectra revealed decreased incorporation of label into the lactate, alanine and glutamate pools in hearts with severe tissue damage to hearts from untreated animals.     

Effect of dose schedule of vitamin E and hydroxethylruticide on intestinal toxicity induced by adriamycin

CDF1 mice receiving Adriamycin, 12 mg/kg IP develop a toxic GI mucositis. The mean survival in CDF1 mice after Adriamycin injection was found to be 6.5 +/- 2.0 weeks and could be increased by alcohol or acetate Vitamin E pretreatment (with 2 g/kg qDx7d) to 22.06 +/- 12.3 weeks or by treatment with Venoruton after Adriamycin (qDx7 with 1.5 g/kg) to 23.7 +/- 12.7 weeks. Other schedules were ineffective or harmful. The ability of Venoruton to enhance survival when given after Adriamycin encouraged us to proceed to tumor bearing mice. The maximum survival with CDF1 mice bearing 5 X 10(6) L1210 cells was 1 +/- 0.2 week which could be increased to 2.17 +/- 0.8 weeks with optimal dose Adriamycin (10 mg/kg). Optimum survival with Venoruton and a single dose of Adriamycin was 2.45 +/- 0.91 weeks with Venoruton, 1.5 g, qd X 14, and 12 mg/kg Adriamycin. Treatment of L1210 bearing mice with Adriamycin, 10 mg/kg on days 1 and 8, yielded a survival of 2.23 +/- 0.7 weeks. An equitoxic regimen of Adriamycin, 11 mg/kg on days 1 and 9, plus Venoruton, 1.5 g, qd X 14, increased survival 30% to 3.08 +/- 2.9 weeks. Venoruton is a promising agent to increase the therapeutic index of Adriamycin.     

Doxorubicin-induced late cardiotoxicity: delayed impairment of Ca2+-handling mechanisms in the sarcoplasmic reticulum in the rat

Doxorubicin treatment causes delayed development of cardiotoxicity. Whether the doxorubicin-induced impairment of cardiac functions reverses or progresses with time after the cessation of the treatment was examined. The rats were injected with doxorubicin (2.5 mg/kg, i.v., once a week for 3 weeks) and sacrificed at 1 (1W), 13 (13W), or 18 (18W) weeks after the final doxorubicin administration. The time to peak of twitch contraction observed at 2-Hz stimulation was not altered in left atrial or ventricular muscle preparations isolated from 1W rats, but it was prolonged in those from 13W and 18W rats. The reduction of the magnitude of postrest contraction and the alteration of force-frequency relationships in left atrial muscle preparations in 1W rats were not significant, but were intensified in the 13W and 18W groups. Alterations in the postrest contraction and the force-frequency relationships in ventricular muscle preparations isolated from doxorubicin-treated rat hearts were weaker, but the pattern of alteration was similar to that observed in left atrial muscle preparations. Caffeine-induced contraction observed in skinned fibers that were isolated from the 1W rats was not altered, but it was reduced in the 18W rats. The Ca2+ sensitivity of contractile proteins was not altered in doxorubicin-treated rat hearts in any of the groups. The K(d) values estimated from a [3H]ryanodine binding study were not altered, but the B(max) values were significantly lower in the 13W and 18W groups than those observed in control rats. These results suggest that the dysfunction of the sarcoplasmic reticulum progresses after the completion of doxorubicin treatment and contributes to the doxorubicin-induced late cardiotoxicity.     

Mobilizing of haematopoietic stem cells to ischemic myocardium by plasmid mediated stromal-cell-derived factor-1alpha (SDF-1alpha) treatment

A concentration gradient of stromal-cell-derived factor-1alpha (SDF-1alpha) is the major mechanism for homing of haematopoietic stem cells (HSCs) in bone marrow. We tested the hypothesis that a gene therapy using SDF-1alpha can enhance HSCs recruiting to the heart upon myocardial infarction (MI). Adult mice with surgically induced myocardial ischemia were injected intramyocardially with either saline (n=12) or SDF-1alpha plasmid (n=12) in 50 microl volume in the ischemic border zone of the infarcted heart 2 weeks after myocardial infarction. Donor Lin-c-kit+ HSCs from isogenic BalB/c mice were harvested, sorted through magnetic cell sorting (MACS) and labeled with PKH26 Red. Three days after plasmid or saline injection, 1x10(5) labeled cells were injected intravenously (i.v.) into saline mice (n=4) and SDF-1alpha plasmid mice (n=4). The hearts and other tissue were removed for Western blot assay 2 weeks after plasmid or saline treatment. The labeled Lin-c-kit+ cells were identified with immunofluoresent staining and endogenous c-kit+ cells were identified by immunohistochemical staining. In mice killed at 1 month postinfarct, Western blot showed higher levels of SDF-1alpha expression in SDF-1alpha-treated mouse ischemic hearts compared to saline-treated hearts and other tissues. In the SDF-1alpha plasmid-treated hearts, SDF-1alpha is overexpressed in the periinfarct zone. The labeled stem cells engrafted to the SDF-1alpha positive site in the myocardium. There was also evidence for endogenous stem cell recruiting. The density of c-kit+ cells in border zone, an index of endogenous stem cell mobilization, was significantly higher in the SDF-1alpha-treated group than in the saline group (14.63+/-1.068 cells/hpf vs. 11.31+/-0.65 cells/hpf, P=0.013) at 2 weeks after SDF-1alpha or saline treatment. Following myocardial infarction, treatment with SDF-1alpha recruits stem cells to damaged heart where they may have a role in repairing and regeneration. The gene therapy with an SDF-1alpha vector offers a promising therapeutic strategy for mobilizing stem cells to the ischemic myocardium.     

Glycogen metabolism: a 13C-NMR study on the isolated perfused rat heart

Glycogen synthesis from D-[1-13C]glucose was observed in the perfused rat heart by 13C-NMR spectroscopy at 62.9 MHz. The glycogenogenesis was stimulated by pretreatment of the animals with isoprenaline. Whereas in hearts from control rats the incorporation of D-[1-13C]glucose into the glycogen remained below the detection threshold, 5 min proton-decoupled 13C-NMR spectra revealed, in hearts from treated rats, a significant labelling of the glycogen within the first minutes of the perfusion and a further linear increase of the glycogen resonance for up to 25 min. This model was used to monitor the appearance of 13C-labelled lactate during ischemia.     

HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3

Aims

HMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts.

Methods and Results

Adult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit⁺ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20–25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206.

Conclusions

HMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.     

Cardiac remodeling associated with protein increase and lipid accumulation in early-stage chronic kidney disease in rats

Chronic kidney disease (CKD) is associated with increased risks of cardiovascular morbidity and mortality. Cardiac remodeling including myocardial fibrosis and hypertrophy is frequently observed in CKD patients. In this study, we investigate the mechanism involved in cardiac hypertrophy associated with CKD using a rat model, by morphological and chemical component changes of the hypertrophic and non-hypertrophic hearts. Sprague–Dawley rats were 4/5 nephrectomized (Nx) at 11 weeks of age and assigned to no treatment and treatment with AST-120, which was reported to affect the cardiac damage, at 18 weeks of age. At 26 weeks of age, the rats were euthanized under anesthesia, and biochemical tests as well as analysis of cardiac condition were performed by histological and spectrophotometric methods. Cardiac hypertrophy and CKD were observed in 4/5 Nx rats even though vascular calcification and myocardial fibrosis were not detected. The increasing myocardial protein was confirmed in hypertrophic hearts by infrared spectroscopy. The absorption of amide I and other protein bands in hypertrophic hearts increased at the same position as in normal cardiac absorption. Infrared spectra also showed that lipid accumulation was also detected in hypertrophic heart. Conversely, the absorptions of protein were obviously reduced in the myocardium of non-hypertrophic heart with CKD compared to that of hypertrophic heart. The lipid associated absorption was also decreased in non-hypertrophic heart. Our results suggest that cardiac remodeling associated with relatively early-stage CKD may be suppressed by reducing increased myocardial protein and ameliorating cardiac lipid load.     

Contribution of exogenous substrates to acetyl coenzyme A: measurement by 13C NMR under non-steady-state conditions

A method is presented for the rapid determination of substrate selection in a manner that is not restricted to conditions of metabolic and isotopic steady state. Competition between several substrates can be assessed directly and continuously in a single experiment, allowing the effect of interventions to be studied. It is shown that a single proton-decoupled 13C NMR spectrum of glutamate provides a direct measure of the contribution of exogenous 13C-labeled substrates to acetyl-CoA without measurement of oxygen consumption and that steady-state conditions need not apply. Two sets of experiments were performed: one in which a metabolic steady state but a non-steady-state 13C distribution was achieved and another in which both metabolism and labeling were not at steady state. In the first group, isolated rat hearts were supplied with [1,2-13C]acetate, [3-13C]lactate, and unlabeled glucose. 13C NMR spectra of extracts from hearts perfused under identical conditions for 5 or 30 min were compared. In spite of significant differences in the spectra, the measured contributions of acetate, lactate, and unlabeled sources to acetyl-CoA were the same. In the second set of experiments, the same group of labeled substrates was used in a regional ischemia model in isolated rabbit hearts to show regional differences in substrate utilization under both metabolic and isotopic non steady state. This sensitive probe of substrate selection was also demonstrated in intact hearts where excellent time resolution (3 min) of substrate selection was feasible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic studies of pyruvate- and lactate-perfused guinea pig hearts by 13C NMR. Determination of substrate preference by glutamate isotopomer distribution

Guinea pig hearts perfused in vitro with 99% enriched [3-13C]pyruvate and [3-13C]lactate have been examined by 13C NMR spectroscopy at 75.45 MHz. Resonances from the intracellular metabolites, glutamate, aspartate, alanine, citrate, malate, lactate, and acetylcarnitine are detected in the [3-13C]pyruvate-perfused hearts while glutamate is the only metabolite observed in the [3-13C]lactate-perfused hearts. Extracts obtained from individual freeze-clamped hearts run under high resolution conditions show similar distributions of metabolites indicating that the intracellular concentrations of these metabolites are indeed quite dependent upon the substrate being utilized. The excellent signal to noise in the spectra of lactate-perfused hearts allows observation of spin-spin coupling between 13C-enriched nuclei in the various glutamate isotopomers within the intact heart. It is shown that the steady-state distribution of glutamate isotopomers is determined by the amount of 12C- versus 13C-enriched acetyl-CoA entering the citric acid cycle and this provides the basis for a direct determination of substrate utilization by the heart in the presence of competing substrates. Similar information may be derived from the 13C spectrum of an extract prepared from a single [3-13C]pyruvate-perfused heart. Our results indicate that lactate is preferred over glucose by the guinea pig heart even in the presence of insulin.     

Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin

Calsequestrin (CSQ) is the major Ca2+ binding protein of the cardiac sarcoplasmic reticulum (SR). Transgenic mice overexpressing CSQ at the age of 7 weeks exhibit concentric cardiac hypertrophy, and by 13 weeks the condition progresses to dilated cardiomyopathy. The present study used a differential display analysis to identify genes whose expressions are modulated in the CSQ-overexpressing mouse hearts to provide information on the mechanism of transition from concentric cardiac hypertrophy to failure. Cardiac ankyrin repeat protein (CARP), glutathione peroxidase (Gpx1), and genes which participate in the formation of extracellular matrix including decorin, TSC-36, Magp2, Osf2, and SPARC are upregulated in CSQ mouse hearts at 7 and 13 weeks of age compared to those of non-transgenic littermates. In addition, two novel genes without sequence similarities to any known genes are upregulated in CSQ-overexpressing mouse hearts. Several genes are downregulated at 13 weeks, including SR Ca2+-ATPase (SERCA2) and adenine nucleotide translocase 1 (Ant1) genes. Further, a functionally yet unknown gene (NM_026586) previously identified in the mouse wolffian duct is dramatically downregulated in CSQ mice with dilated hearts. Thus, CARP, Gpx1, and genes encoding extracellular matrix proteins may participate in the development of cardiac hypertrophy and fibrosis, and changes in SERCA2, Ant1, and NM_026586 mRNA expression may be involved in transition from concentric to dilated cardiac hypertrophy.     

Effect of estrogen replacement treatment on ischemic preconditioning in isolated rat hearts

In the present study, we tested the effects of long-term estrogen replacement treatment on myocardial ischemia-reperfusion injury and on the cardioprotection of ischemic preconditioning in isolated hearts from ovariectomized rats. Ovariectomized rats were treated with 17beta-estradiol (30 micro g/kg/d, s.c.) for 12 weeks. Isolated rat hearts were perfused in the Langendorff mode. Heart rate, coronary flow, left ventricular pressure and its first derivative (+/-LVdp/dtmax) were recorded. Fifteen-min global ischemia and 30-min reperfusion caused a significant decrease of cardiac mechanical function, which were not affected by ovariectomy or estrogen replacement treatment. The isolated hearts in all groups could be preconditioned, and the cardioprotection afforded by preconditioning in the sham-operated rats was greater compared with ovariectomized rats with or without estrogen treatment. These results suggest that long-term estrogen replacement treatment exerts no effect on the inhibition of mechanical function after ischemia-reperfusion, and this study also suggests that estrogen does not affect ischemic preconditioning in isolated hearts of ovariectomized rats.     

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts.

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.     

Alteration of antioxidants during the progression of heart disease in streptozotocin-induced diabetic rats

Involvement of oxidative stress is implicated in the progression of complication of diabetes mellitus. With respect to heart diseases, we have studied role of oxidative stress/antioxidants using rats treated with streptozotocin to induce diabetes (DM). Hemodynamic and echocardiographic measurements showed thickening of the wall and an increase in the internal dimension of the left ventricle (LV) in DM rats at 8th week. Decrease in diastolic posterior wall velocity and rate of LV pressure change, and increase in LV end diastolic pressures also proved cardiac dysfunction. These changes were further developed in DM rats after 12 weeks. Utilizing rat hearts at 8th and 12th weeks, the following estimations were performed. There was a decrease in the activity of Mn-superoxide dismutase (SOD), suggesting abnormal mitochondrial metabolism of reactive oxygen species. The level of glutathione (GSH) decreased concomitant with a decrease in the expression of γ-glutamylcysteine synthetase (γ-GCS). The expression of transforming growth factor-β1 (TGF-β1), known as a growth factor and a suppressor of GSH synthesis, elevated in DM rat hearts. Immunohistochemical estimation showed an increase in type IV collagen in DM hearts. Collectively, it was suggested a linkage between mitochondrial damage to generate reactive oxygen species and inactivation of Mn-SOD and elevation of the expression of TGF-β1 to lead suppression of GSH synthesis and induction of fibrous change for the consequent cardiac dysfunction in DM.     

Modification of subcellular organelles in pressure-overloaded heart by etomoxir, a carnitine palmitoyltransferase I inhibitor.

To examine the signals regulating cardiac growth and molecular structure of subcellular organelles, cardiac hypertrophy was induced in rats by constriction of the abdominal aorta for 12-13 wk or by treatment with a carnitine palmitoyltransferase I inhibitor, etomoxir (12-15 mg/kg body wt) for 12-13 wk. In contrast to pressure overload, etomoxir redistributed the myosin isozyme population from V3 to V1 and increased the sarcoplasmic reticulum (SR) Ca(2+)-stimulated ATPase activity. When rats with pressure-overloaded hearts were treated with etomoxir, the cardiac hypertrophy was increased whereas the shift in myosin isozymes from V1 to V3 was prevented and the depression in SR Ca(2+)-stimulated ATPase activity was reversed. Plasma thyroid hormone and insulin concentrations were not altered but triglyceride concentrations were reduced in etomoxir-treated rats with pressure overload. The data demonstrate a dissociation between cardiac muscle growth and changes in subcellular organelles and indicate that a shift in myocardial substrate utilization may represent an important signal for molecular remodeling of the heart.     

Alteration of antioxidants during the progression of heart disease in streptozotocin-induced diabetic rats

Involvement of oxidative stress is implicated in the progression of complication of diabetes mellitus. With respect to heart diseases, we have studied role of oxidative stress/antioxidants using rats treated with streptozotocin to induce diabetes (DM). Hemodynamic and echocardiographic measurements showed thickening of the wall and an increase in the internal dimension of the left ventricle (LV) in DM rats at 8th week. Decrease in diastolic posterior wall velocity and rate of LV pressure change, and increase in LV end diastolic pressures also proved cardiac dysfunction. These changes were further developed in DM rats after 12 weeks. Utilizing rat hearts at 8th and 12th weeks, the following estimations were performed. There was a decrease in the activity of Mn-superoxide dismutase (SOD), suggesting abnormal mitochondrial metabolism of reactive oxygen species. The level of glutathione (GSH) decreased concomitant with a decrease in the expression of γ-glutamylcysteine synthetase (γ-GCS). The expression of transforming growth factor-β1 (TGF-β1), known as a growth factor and a suppressor of GSH synthesis, elevated in DM rat hearts. Immunohistochemical estimation showed an increase in type IV collagen in DM hearts. Collectively, it was suggested a linkage between mitochondrial damage to generate reactive oxygen species and inactivation of Mn-SOD and elevation of the expression of TGF-β1 to lead suppression of GSH synthesis and induction of fibrous change for the consequent cardiac dysfunction in DM.     

A decrease in cyclin B1 levels leads to polyploidization in DNA damage‐induced senescence

Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration‐dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin‐induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated β‐galactosidase activity. In DNA damage‐induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage‐induced senescence.     

Distribution of Adriamycin in mice bearing mammary adenocarcinoma 16/C

The response of solid mammary adenocarcinoma 16/C to treatment with Adriamycin is highly variable and ranges from growth under treatment to complete regression. Tumour and host factors were evaluated to determine the influence of each on the response. We determined that the concentration of Adriamycin in plasma and tumour was a function of tumour size and treatment history in mice bearing mammary adenocarcinoma 16/C. The plasma concentrations following a single dose of Adriamycin (10 mg/kg) increased in proportion to tumour mass without a concurrent increase in tumour concentration. When mice bearing large tumours (greater than 1.0 g) were treated with a multidose protocol, the plasma concentrations were higher and the tumour concentrations lower following the initial dose than following subsequent doses; in tumour-free mice, prior treatment with Adriamycin did not affect the plasma level achieved after a second dose. The magnitude of the decrease in plasma and increase in tumour concentrations was a function of the initial tumour size and the treatment schedule. The increase in tumour levels represented the sum of residual Adriamycin and drug bound as a result of the dose immediately prior to analysis. At the time of the initial treatment, the Adriamycin was distributed within each tumour in proportion to vascular perfusion. The percent of the tumour mass that was well-perfused appeared to increase with repeated treatments. The results indicate that the plasma concentration of Adriamycin did not necessarily reflect the tumour exposure in the mammary adenocarcinoma 16/C model. In hosts bearing mammary adenocarcinoma 16/C--or, possibly, other tumours that produce similar effects on the host--a low initial dose of Adriamycin might modify the distribution, possibly reduce the toxicity and allow escalation of subsequent doses with increased exposure of the tumour.     

Pulmonary fibrosis in L-NAME-treated mice is dependent on an activated endothelin system

Activation of the endothelin (ET) system promotes vasoconstriction, inflammation, and fibrosis in various tissues, including the lung. Therefore, ET-1 transgenic mice overexpressing ET-1 develop pulmonary fibrosis in a slow, age-dependent manner. In vivo, NO is the most important counterregulatory mediator of the ET system and decreases ET-1 promoter activity. The aim of our study was to elucidate the impact on pulmonary inflammation and fibrosis of the interaction between NO and the ET system in young ET-1 transgenic mice before the onset of pulmonary fibrosis. Male ET-1 transgenic mice and wild-type littermates at the age of 8 weeks were randomly allocated to the following 6 groups: WT (n = 11), wild-type animals without treatment; WT + l-NAME (n = 14), wild-type animals receiving l-NAME, an inhibitor of NO synthase; WT + l-NAME + LU (n = 13), wild-type animals receiving l-NAME and LU 302872, a dual ETA/ETB-receptor antagonist; ET1tg (n = 10), ET-1 transgenic mice; ET1tg + l-NAME (n = 13); and ET1tg + l-NAME + LU (n = 13). After 6 weeks, animals were euthanized, and hearts and lungs were harvested for histology and immunohistochemistry. No differences in pulmonary inflammation, as indicated by macrophage infiltration, or in interstitial fibrosis were observed between WT and ET1tg mice at baseline; however, inflammation and interstitial fibrosis were significantly enhanced in ET1tg mice, but not in WT groups, after l-NAME treatment. The combined ETA/ETB-receptor antagonist LU 302872 abolished inflammation and interstitial fibrosis in l-NAME-treated ET1tg mice. Perivascular fibrosis and media/lumen ratio of pulmonary bronchi and arteries did not differ between all study groups. In our study l-NAME induced pulmonary fibrosis and inflammation only in young ET1tg mice. Additional treatment with LU 302872 abolished these effects. We thus conclude that an imbalance between an activated ET system and a suppressed NO system contributes to pulmonary inflammation and fibrosis.     

Induced bone marrow mesenchymal stem cells improve cardiac performance of infarcted rat hearts

We investigated whether transplantation of bone marrow mesenchymal stem cells (BMSC) with induced BMSC (iBMSC) or uninduced BMSC (uBMSC) into the myocardium could improve the performance of post-infarcted rat hearts. BMSCs were specified by flowcytometry. IBMSCs were cocultured with rat cardiomyocyte before transplantation. Cells were injected into borders of cardiac scar tissue 1?week after experimental infarction. Cardiac performance was evaluated by echocardiography at 1, 2, and 4?weeks after cellular or PBS injection. Langendorff working-heart and histological studies were performed 4?weeks after treatment. Myogenesis was detected by quantitative PCR and immunofluorescence. Echocardiography showed a nearly normal ejection fraction (EF) in iBMSC-treated rats and all sham control rats but a lower EF in all PBS-treated animals. The iBMSC-treated heart, assessed by echocardiography, improved fractional shortening compared with PBS-treated hearts. The coronary flow (CF) was decreased obviously in PBS and uBMSC-treated groups, but recovered in iBMSC-treated heart at 4?weeks (P?<?0.01). Immunofluorescent microscopy revealed co-localization of Superparamagnetic iron oxide (SPIO)-labeled transplanted cells with cardiac markers for cardiomyocytes, indicating regeneration of damaged myocardium. These data provide strong evidence that iBMSC implantation is of more potential to improve infarcted cardiac performance than uBMSC treatment. It will open new promising therapeutic opportunities for patients with post-infarction heart failure.     

The influence of lipoic acid on adriamycin induced nephrotoxicity in rats

Adriamycin, which is widely used in the treatment of various neoplastic conditions, exerts toxic effects in several organs. Adriamycin nephrotoxicity has been recently documented in a variety of animal species. The present study was designed to investigate the effect of lipoic acid on the nephrotoxic potential of adriamycin. The study was carried out with adult male albino rats of Wistar strain. Test animals were divided into four groups of six rats each as follows: Group I (control) received only normal saline throughout the course of the experiment. Group II (ADR) received intravenous injections of adriamycin through the tail vein (1 mg kg^–1 body wt day^–1) once a week for a period of 12 weeks. Group III (LA) received lipoic acid (35 mg kg^–1 body wt day^–1) intraperitoneally once a week for a period of 12 weeks. Group IV (ADR + LA) received a single injection of lipoic acid intraperitoneally 24 h prior to the administration of adriamycin through the tail vein once a week for a period of 12 weeks. Intravenous injections of adriamycin resulted in decreased activities of the glycolytic enzymes; hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase in the rat renal tissue. The gluconeogenic enzymes; glucose-6-phosphatase and fructose-1,6-diphosphatase, showed a decline in their activities on adriamycin administration. The transmembrane enzymes namely the Na⁺,K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brush-border enzyme alkaline phosphatase also showed a decrease in their activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush-border membrane damage. Decreased activities of the TCA cycle enzymes isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, suggest a loss in mitochondrial function and integrity. Nephrotoxicity was evident from the increased excretions of N-acetyl--D-glucosaminidase and -glutamyl transferase in the urine of adriamycin administered rats. These biochemical disturbances were effectively counteracted on pretreatment with lipoic acid, which brought about an increase in the activities of glycolytic enzymes, ATPases and the TCA cycle enzymes. On the other hand, the gluconeogenic enzymes showed a further decrease in their activities on lipoic acid pretreatment. LA pretreatment also restored the activities of the urinary enzymes to normal. These observations shed light on the nephroprotective action of lipoic acid rendered against experimental aminoglycoside toxicity.