Comparison of the effects of renal denervation at early or advanced stages of hypertension on cardiac, renal, and adipose tissue pathology in Dahl salt-sensitive rats.

Renal denervation (RDN) has emerged as a novel therapy for drug-resistant hypertension. We here examined the effects of RDN at early versus advanced stages of hypertension on blood pressure and organ pathology in rats with salt-sensitive hypertension. Dahl salt-sensitive (DahlS) rats fed an 8% NaCl diet from 6 weeks of age were subjected to RDN (surgical ablation and application of 10% phenol in ethanol) or sham surgery at 7 (early stage) or 9 (advanced stage) weeks and were studied at 12 weeks. RDN at early or advanced stages resulted in a moderate lowering of blood pressure. Although RDN at neither stage affected left ventricular (LV) and cardiomyocyte hypertrophy, it ameliorated LV diastolic dysfunction, fibrosis, and inflammation at both stages. Intervention at both stages also attenuated renal injury as well as downregulated the expression of angiotensinogen and angiotensin-converting enzyme (ACE) genes and angiotensin II type 1 receptor protein in the kidney. Furthermore, RDN at both stages inhibited proinflammatory gene expression in adipose tissue. The early intervention reduced both visceral fat mass and adipocyte size in association with downregulation of angiotensinogen and ACE gene expression. In contrast, the late intervention increased fat mass without affecting adipocyte size as well as attenuated angiotensinogen and ACE gene expression. Our results thus indicate that RDN at early or late stages after salt loading moderately alleviated hypertension and substantially ameliorated cardiac and renal injury and adipose tissue inflammation in DahlS rats. They also suggest that cross talk among the kidney, cardiovascular system, and adipose tissue may contribute to salt-sensitive hypertension. Supposed mechanism for the beneficial effects of RDN on hypertension and target organ damage in DahlS rats. RDN at early or late stages after salt loading moderately alleviated hypertension and substantially ameliorated renal injury in DahlS rats. Cross talk among the kidney, cardiovascular system, and adipose tissue possibly mediated by circulating RAS may contribute to salt-sensitive hypertension. LV; left ventricular, NE; norepinephrine, RAS; renin-angiotensin system, RDN; renal denervation.

Staining, magnification, and algorithmic conditions for highly accurate cell detection and cell classification by deep learning.

OBJECTIVES: Research into cytodiagnosis has seen an active exploration of cell detection and classification using deep learning models. We aimed to clarify the challenges of magnification, staining methods, and false positives in creating general purpose deep learning-based cytology models. METHODS: Using 11 types of human cancer cell lines, we prepared Papanicolaou- and May-Grünwald-Giemsa (MGG)-stained specimens. We created deep learning models with different cell types, staining, and magnifications from each cell image using the You Only Look Once, version 8 (YOLOv8) algorithm. Detection and classification rates were calculated to compare the models. RESULTS: The classification rates of all the created models were over 95.9%. The highest detection rates of the Papanicolaou and MGG models were 92.3% and 91.3%, respectively. The highest detection rates of the object detection and instance segmentation models, which were 11 cell types with Papanicolaou staining, were 94.6% and 91.7%, respectively. CONCLUSIONS: We believe that the artificial intelligence technology of YOLOv8 has sufficient performance for applications in screening and cell classification in clinical settings. Conducting research to demonstrate the efficacy of YOLOv8 artificial intelligence technology on clinical specimens is crucial for overcoming the unique challenges associated with cytology.

Relationship between a deep learning model and liquid-based cytological processing techniques.

OBJECTIVE: Artificial intelligence (AI)-based cytopathology studies conducted using deep learning have enabled cell detection and classification. Liquid-based cytology (LBC) has facilitated the standardisation of specimen preparation; however, cytomorphology varies according to the LBC processing technique used. In this study, we elucidated the relationship between two LBC techniques and cell detection and classification using a deep learning model. METHODS: Cytological specimens were prepared using the ThinPrep and SurePath methods. The accuracy of cell detection and cell classification was examined using the one- and five-cell models, which were trained with one and five cell types, respectively. RESULTS: When the same LBC processing techniques were used for the training and detection preparations, the cell detection and classification rates were high. The model trained on ThinPrep preparations was more accurate than that trained on SurePath. When the preparation types used for training and detection were different, the accuracy of cell detection and classification was significantly reduced (P < 0.01). The model trained on both ThinPrep and SurePath preparations exhibited slightly reduced cell detection and classification rates but was highly accurate. CONCLUSIONS: For the two LBC processing techniques, cytomorphology varied according to cell type; this difference affects the accuracy of cell detection and classification by deep learning. Therefore, for highly accurate cell detection and classification using AI, the same processing technique must be used for both training and detection. Our assessment also suggests that a deep learning model should be constructed using specimens prepared via a variety of processing techniques to construct a globally applicable AI model.

Relationship between Liquid-Based Cytology Preservative Solutions and Artificial Intelligence: Liquid-Based Cytology Specimen Cell Detection Using YOLOv5 Deep Convolutional Neural Network.

INTRODUCTION: Deep learning is a subset of machine learning that has contributed to significant changes in feature extraction and image classification and is being actively researched and developed in the field of cytopathology. Liquid-based cytology (LBC) enables standardized cytological preparation and is also applied to artificial intelligence (AI) research, but cytological features differ depending on the LBC preservative solution types. In this study, the relationship between cell detection by AI and the type of preservative solution used was examined. METHODS: The specimens were prepared from five preservative solutions of LBC and stained using the Papanicolaou method. The YOLOv5 deep convolutional neural network algorithm was used to create a deep learning model for each specimen, and a BRCPT model from five specimens was also created. Each model was compared to the specimen types used for detection. RESULTS: Among the six models, a difference in the detection rate of approximately 25% was observed depending on the detected specimen, and within specimens, a difference in the detection rate of approximately 20% was observed depending on the model. The BRCPT model had little variation in the detection rate depending on the type of the detected specimen. CONCLUSIONS: The same cells were treated with different preservative solutions, the cytologic features were different, and AI clarified the difference in cytologic features depending on the type of solution. The type of preservative solution used for training and detection had an extreme influence on cell detection using AI. Although the accuracy of the deep learning model is important, it is necessary to understand that cell morphology differs depending on the type of preservative solution, which is a factor affecting the detection rate of AI.

Pharmacological inhibition of the lipid phosphatase PTEN ameliorates heart damage and adipose tissue inflammation in stressed rats with metabolic syndrome.

Phosphatidylinositol 3-kinase (PI3K) signaling promotes the differentiation and proliferation of regulatory B (Breg) cells, and the lipid phosphatase phosphatase and tensin homolog deleted on chromosome 10 (PTEN) antagonizes the PI3K-Akt signaling pathway. We previously demonstrated that cardiac Akt activity is increased and that restraint stress exacerbates hypertension and both heart and adipose tissue (AT) inflammation in DS/obese rats, an animal model of metabolic syndrome (MetS). We here examined the effects of restraint stress and pharmacological inhibition of PTEN on heart and AT pathology in such rats. Nine-week-old animals were treated with the PTEN inhibitor bisperoxovanadium-pic [bpV(pic)] or vehicle in the absence or presence of restraint stress for 4 weeks. BpV(pic) treatment had no effect on body weight or fat mass but attenuated hypertension in DS/obese rats subjected to restraint stress. BpV(pic) ameliorated left ventricular (LV) inflammation, fibrosis, and diastolic dysfunction as well as AT inflammation in the stressed rats. Restraint stress reduced myocardial capillary density, and this effect was prevented by bpV(pic). In addition, bpV(pic) increased the proportions of Breg and B-1 cells as well as reduced those of CD8+ T and B-2 cells in AT of stressed rats. Our results indicate that inhibition of PTEN by bpV(pic) alleviated heart and AT inflammation in stressed rats with MetS. These positive effects of bpV(pic) are likely due, at least in part, to a reduction in blood pressure, an increase in myocardial capillary formation, and an altered distribution of immune cells in fat tissue that result from the activation of PI3K-Akt signaling.

Characterizing the Effect of Processing Technique and Solution Type on Cytomorphology Using Liquid-Based Cytology.

INTRODUCTION: Liquid-based cytology (LBC) is increasingly used for nongynecologic applications. However, the cytological preparation of LBC specimens is influenced by the processing technique and the preservative used. In this study, the influence of the processing techniques and preservatives on cell morphology was examined mathematically and statistically. METHODS: Cytological specimens were prepared using the ThinPrep (TP), SurePath (SP), and AutoSmear methods, with 5 different preservative solutions. The cytoplasmic and nuclear areas of Papanicolaou-stained specimens were measured for all samples. RESULTS: The cytoplasmic and nuclear areas were smaller in cells prepared using the 2 LBC methods, compared to that prepared using the AutoSmear method, irrespective of the preservative used. The cytoplasmic and nuclear areas of cells prepared using the SP method were smaller than those of cells prepared using the TP method, irrespective of the preservative used. There were fewer differences among the cytoplasmic areas of cells prepared with different preservative solutions using the TP method; however, the cytoplasmic areas of cells prepared using the SP method changed with the preservative solution used. CONCLUSIONS: The most significant difference affecting the cytoplasmic and nuclear areas was the processing technique. The TP method increased the cytoplasmic and nuclear areas, while the methanol-based PreservCyt solution enabled the highest enlargement of the cell. LBC is a superior preparation technique for standardization of the specimens. Our results offer a better understanding of methods suitable for specimen preparation for developing precision AI-based diagnosis in cytology.

Surgical ablation of whitened interscapular brown fat ameliorates cardiac pathology in salt-loaded metabolic syndrome rats.

Brown adipose tissue (BAT) is an endocrine organ that contributes to thermogenesis and energy consumption. We investigated the effects of salt loading and surgical removal of whitened interscapular BAT (iBAT) on cardiac and adipose tissue pathology in DahlS.Z-Leprfa /Leprfa (DS/obese) rats, an animal model of metabolic syndrome (MetS). DS/obese rats were subjected to surgical removal of iBAT or sham surgery at 8 weeks of age and were provided with drinking water containing or not containing 0.3% NaCl for 4 weeks beginning at 9 weeks of age. Removal of iBAT suppressed the salt-induced exacerbation of left ventricular inflammation, fibrosis, and diastolic dysfunction, but not that of hypertension development, in DS/obese rats. Salt loading attenuated adipocyte hypertrophy but enhanced inflammation in both visceral white adipose tissue (WAT) and iBAT. Although iBAT removal did not affect visceral WAT pathology in salt-loaded DS/obese rats, it attenuated the elevation of circulating interleukin-6 levels in these animals. Downregulation of uncoupling protein-1 expression in iBAT of DS/obese rats was not affected by salt loading. Our results suggest that the conversion of iBAT to WAT-like tissue contributes to a salt-induced elevation of circulating proinflammatory cytokine levels that leads to exacerbation of cardiac pathology in this model of MetS.

The prebiotic fiber inulin ameliorates cardiac, adipose tissue, and hepatic pathology, but exacerbates hypertriglyceridemia in rats with metabolic syndrome.

Prebiotics ameliorate dysbiosis and influence metabolism and the immune system, but their effects on cardiovascular complications in metabolic disorders remain largely unknown. We here investigated the effects of the soluble fiber inulin on cardiac, adipose tissue, and hepatic pathology as well as on metabolic disorders in DahlS.Z-Leprfa/Leprfa (DS/obese) rats, an animal model of metabolic syndrome (MetS). DS/obese rats and their homozygous lean (DahlS.Z-Lepr+/Lepr+, or DS/lean) littermate controls were fed a purified diet containing 5% or 20% inulin from 9 to 13 wk of age. The high-fiber diet ameliorated hypertension, left ventricular inflammation, fibrosis and diastolic dysfunction; attenuated adipose tissue inflammation and fibrosis; and alleviated the elevation of interleukin-6 levels, without affecting insulin resistance, in DS/obese rats. In addition, high fiber intake ameliorated lipid accumulation, inflammation, and fibrosis; attenuated the reduction in AMPK activity; upregulated sterol regulatory element-binding protein-1c gene expression; and increased the expression of microsomal triglyceride transfer protein gene in the liver of DS/obese rats. It also mitigated increases in total and non-high-density lipoprotein cholesterol levels but increased the triglyceride concentration in serum in these rats. None of these parameters were affected by high dietary fiber in DS/lean rats. The proportion of regulatory T cells in adipose tissue was influenced by dietary fiber but not by genotype. Our results indicate that inulin exacerbates hypertriglyceridemia but alleviates hypertension and cardiac injury as well as adipose tissue and hepatic pathology in MetS rats.NEW & NOTEWORTHY Prebiotics ameliorate dysbiosis and influence metabolism and the immune system, but their effects on cardiovascular complications in metabolic disorders remain largely unknown. Inulin ameliorated hypertension, cardiac injury, and diastolic dysfunction without affecting obesity or insulin resistance in a rat model of metabolic syndrome. The favorable cardiac effects of inulin may be related to inhibition of systemic inflammation associated with a reduction in circulating interleukin-6 levels. Additionally, inulin exacerbated hypertriglyceridemia but alleviates adipose tissue and hepatic pathology in these animals, as well as increased the number of regulatory T cells in adipose tissue.

Alleviation of salt-induced exacerbation of cardiac, renal, and visceral fat pathology in rats with metabolic syndrome by surgical removal of subcutaneous fat.

OBJECTIVES: Evidence suggests that visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) should be considered as distinct types of white fat. Although VAT plays a key role in metabolic syndrome (MetS), the role of subcutaneous adipose tissue (SAT) has been unclear. DahlS.Z-Leprfa/Leprfa (DS/obese) rats, an animal model of MetS, develop adipocyte hypertrophy and inflammation to similar extents in SAT and VAT. We have now investigated the effects of salt loading and SAT removal on cardiac, renal, and VAT pathology in DS/obese rats. METHODS: DS/obese rats were subjected to surgical removal of inguinal SAT or sham surgery at 8 weeks of age. They were provided with a 0.3% NaCl solution as drinking water or water alone for 4 weeks from 9 weeks of age. RESULTS: Salt loading exacerbated hypertension, insulin resistance, as well as left ventricular (LV) hypertrophy, inflammation, fibrosis, and diastolic dysfunction in DS/obese rats. It also reduced both SAT and VAT mass but aggravated inflammation only in VAT. Although SAT removal did not affect LV hypertrophy in salt-loaded DS/obese rats, it attenuated hypertension, insulin resistance, and LV injury as well as restored fat mass and alleviated inflammation and the downregulation of adiponectin gene expression in VAT. In addition, whereas salt loading worsened renal injury as well as upregulated the expression of renin-angiotensin-aldosterone system-related genes in the kidney, these effects were suppressed by removal of SAT. CONCLUSIONS: SAT removal attenuated salt-induced exacerbation of MetS and LV and renal pathology in DS/obese rats. These beneficial effects of SAT removal are likely attributable, at least in part, to inhibition of both VAT and systemic inflammation.

Anti-inflammatory effects of heat-killed Lactobacillus plantarum L-137 on cardiac and adipose tissue in rats with metabolic syndrome.

The effects of heat-killed Lactobacillus plantarum L-137 (HK L-137) on chronic inflammation associated with metabolic disorders have remained unknown. We examined the effects of HK L-137 on cardiac and adipose tissue pathophysiology in DahlS.Z-Lepr fa /Lepr fa (DS/obese) rats as a model of metabolic syndrome. DS/obese rats were treated orally with HK L-137 (2 or 75 mg kg-1 day-1) from 9 to 13 weeks of age. HK L-137 attenuated left ventricular (LV) inflammation and fibrosis as well as adipocyte hypertrophy, inflammation, and up-regulation of sterol regulatory element-binding protein-1c (SREBP-1c) gene expression in visceral and subcutaneous adipose tissue, without affecting body weight gain or hypertension. The low dose of HK L-137 also ameliorated LV diastolic dysfunction, the increase in subcutaneous fat mass, and insulin resistance as well as attenuated the down-regulation of Akt phosphorylation in visceral and subcutaneous adipose tissue, and the elevation of the circulating interleukin-6 concentration. Furthermore, the proportion of regulatory T (Treg) cells among CD4+ T cells in the spleen was increased by HK L-137. These results suggest that the anti-inflammatory effects of HK L-137 on the heart and adipose tissue are related, at least partly, to suppression of systemic inflammation associated with an increase in splenic Treg cell.

Overexpression of collagen type III in injured myocardium prevents cardiac systolic dysfunction by changing the balance of collagen distribution.

OBJECTIVE: Left ventricular (LV) remodeling alters the contractile and relaxation properties and induces myocardial stiffness. As LV remodeling progresses, the amount of collagen type III (Col3) is gradually decreased, being replaced by collagen type I (Col1). We evaluated whether Col3 overexpression improved cardiac function and remodeling in a rat with ischemic cardiomyopathy (ICM). We also investigated the functional motif and mechanism of thrombin-cleaved N-terminal osteopontin (N-OPN) on cardiac remodeling. METHODS: The rats with ICM were divided into 3 groups: ligation only (Control) group and groups transplanted with nontransfected fibroblast sheets (normal Fb group) or with Col3-secretory fibroblast sheets (Col3 Fb group). A gelatin hydrogel containing the N-terminal fragment (N-OPN), N-OPN lacking the SVVYGLR sequence (⊿SV), the Arg-Gly-Asp (RGD) sequence (⊿RGD), RGD and SVVYGLR sequences (⊿RGD-SV), SVVYGLR alone (SV), or a random SV peptide was implanted into an ICM model rat. RESULTS: The Col3 Fb group exhibited significantly attenuated LV systolic dysfunction. LV dilatation, myocyte hypertrophy, and LV fibrosis at the infarcted area were also attenuated by Col3 Fb implantation. Furthermore, N-OPN, ⊿RGD, and SV peptide suppressed the depression of cardiac function, LV dilatation, and myocyte hypertrophy, and also induced increased Col3 expression and reduction in the ratio of Col1 to Col3 in the infarcted and border areas. CONCLUSIONS: Overexpression of Col3 improved cardiac function by changing the balance of collagen distribution in LV remodeling. The SVVYGLR motif of the thrombin-cleaved N-OPN and SV peptide attenuated cardiac dysfunction by increasing Col3 and changing the pattern of collagen balance in the impaired area.

Effects of ramelteon on cardiac injury and adipose tissue pathology in rats with metabolic syndrome.

Melatonin regulates circadian rhythms but also has antioxidative and anti-inflammatory effects that ameliorate metabolic disorders. We investigated the effects of the selective melatonin agonist ramelteon on cardiac and adipose tissue pathology in the DahlS.Z-Leprfa /Leprfa (DS/obese) rat, a model of metabolic syndrome (MetS). Rats were treated with a low (0.3 mg/kg per day) or high (8 mg/kg per day) dose of ramelteon from 9 to 13 weeks of age. Ramelteon treatment at either dose attenuated body weight gain, left ventricular fibrosis, and diastolic dysfunction, as well as cardiac oxidative stress and inflammation, without affecting hypertension or insulin resistance. Although ramelteon did not affect visceral white adipose tissue (WAT) mass, it attenuated inflammation and downregulated insulin signaling in this tissue. In contrast, ramelteon reduced fat mass, adipocyte hypertrophy, and inflammation, and ameliorated impaired insulin signaling in subcutaneous WAT. In addition, ramelteon attenuated adipocyte hypertrophy, downregulated mitochondrial uncoupling protein 1, and upregulated 11ß-hydroxysteroid dehydrogenase type 1 expression in interscapular brown adipose tissue (BAT). In summary, ramelteon treatment attenuated obesity and cardiac injury, improved insulin signaling in visceral and subcutaneous WAT, and inhibited the whitening of BAT in rats with MetS.

Effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism in rats with metabolic syndrome.

The mammalian target of rapamycin (mTOR) is a regulator of metabolism and is implicated in pathological conditions such as obesity and diabetes. We aimed to investigate the role of mTOR in obesity. A new animal model of metabolic syndrome (MetS), named DahlS.Z-Leprfa /Leprfa (DS/obese) rats was established previously in our laboratory. In this study, we used this model to evaluate the effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism. DS/obese rats were treated with the mTOR inhibitor, everolimus, (0.83 mg/kg per day, per os) for 4 weeks at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr+ /Lepr+ or DS/lean) littermates of DS/obese rats were used as controls. Treatment with everolimus ameliorated hypertension, left ventricular (LV) hypertrophy and fibrosis, and LV diastolic dysfunction, and attenuated cardiac oxidative stress and inflammation in DS/obese rats, but had no effect on these parameters in DS/lean rats. Treatment with everolimus reduced Akt Thr308 phosphorylation in the heart of DS/obese rats. It also alleviated obesity, hyperphagia, adipocyte hypertrophy, and adipose tissue inflammation in DS/obese rats. Everolimus treatment exacerbated glucose intolerance, but did not affect Akt phosphorylation levels in the fat or liver in these rats. Pancreatic ß-cell mass was increased in DS/obese rats compared with that in DS/lean rats and this effect was attenuated by everolimus. Activation of mTOR signaling contributes to the pathophysiology of MetS and its associated complications. And mTOR inhibition with everolimus ameliorated obesity as well as cardiac and adipose tissue pathology, but exacerbated glucose metabolism in rats with MetS.

Evaluation of dermal wound healing activity of synthetic peptide SVVYGLR.

SVVYGLR peptide (SV peptide) is a 7-amino-acid sequence with angiogenic properties that is derived from osteopontin in the extracellular matrix and promotes differentiation of fibroblasts to myofibroblast-like cells and the production of collagen type Ⅲ by cardiac fibroblasts. However, the effects of SV peptide on dermal cells and tissue are unknown. In this study, we evaluated the effects of this peptide in a rat model of dermal wound healing. The synthetic SV peptide was added to dermal fibroblasts or keratinocytes, and their cellular motility was evaluated. In an in vivo wound healing exeriment, male rats aged 8 weeks were randomly assigned to the SV peptide treatment, non-treated control, or phosphate-buffered saline (PBS) groups. Wound healing was assessed by its repair rate and histological features. Scratch assay and cell migration assays using the Chemotaxicell method showed that SV peptide significantly promoted the cell migration in both fibroblasts and keratinocytes. In contrast the proliferation potency of these cells was not affected by SV peptide. In the rat model, wound healing progressed faster in the SV peptide-treated group than in the control and PBS groups. The histopathological analyses showed that the SV peptide treatment stimulated the migration of fibroblasts to the wound area and increased the number of myofibroblasts. Immunohistochemical staining showed a marked increase of von Willebland factor-positive neomicrovessels in the SV peptide-treated group. In conclusion, SV peptide has a beneficial function to promote wound healing by stimulating granulation via stimulating angiogenesis, cell migration, and the myofibroblastic differentiation of fibroblasts.

Atorvastatin reduces cardiac and adipose tissue inflammation in rats with metabolic syndrome.

BACKGROUND: Statins are strong inhibitors of cholesterol biosynthesis and help to prevent cardiovascular disease. They also exert additional pleiotropic effects that include an anti-inflammatory action and are independent of cholesterol, but the molecular mechanisms underlying these additional effects have remained unclear. We have now examined the effects of atorvastatin on cardiac and adipose tissue inflammation in DahlS.Z-Leprfa/Leprfa (DS/obese) rats, which we previously established as a model of metabolic syndrome (MetS). METHODS AND RESULTS: DS/obese rats were treated with atorvastatin (6 or 20mgkg-1day-1) from 9 to 13weeks of age. Atorvastatin ameliorated cardiac fibrosis, diastolic dysfunction, oxidative stress, and inflammation as well as adipose tissue inflammation in these animals at both doses. The high dose of atorvastatin reduced adipocyte hypertrophy to a greater extent than did the low dose. Atorvastatin inhibited the up-regulation of peroxisome proliferator-activated receptor γ gene expression in adipose tissue as well as decreased the serum adiponectin concentration in DS/obese rats. It also activated AMP-activated protein kinase (AMPK) as well as inactivated nuclear factor-κB (NF-κB) in the heart of these animals. The down-regulation of AMPK and NF-κB activities in adipose tissue of DS/obese rats was attenuated and further enhanced, respectively, by atorvastatin treatment. CONCLUSIONS: The present results suggest that the anti-inflammatory effects of atorvastatin on the heart and adipose tissue are attributable at least partly to increased AMPK activity and decreased NF-κB activity in this rat model of MetS.

Laminin α2-secreting fibroblasts enhance the therapeutic effect of skeletal myoblast sheets.

Objectives: Skeletal myoblast sheet (SMB) transplantation, a method used for treating failing hearts, results in the secretion of cytokines that improve heart function. Enhancing the survival rate of implanted myoblasts should yield more continuous and effective therapies. We hypothesized that laminin-211 (merosin), a major component of skeletal muscle extracellular matrix (ECM), which mediates cell-to-ECM adhesion by binding to α -dystroglycan ( α DG) on muscle cells, could inhibit detachment of implanted myoblasts from host myocardia. Methods: Multilayered sheets composed of fibroblasts expressing laminin G-module (LG)4-5 of α 2 and skeletal myoblasts were transplanted into ischemic cardiomyopathy model rats. Animals were divided into four groups: the ligation only (Control) group, and those transplanted with SMB alone, with both myoblasts and control fibroblast sheets (SMB + normal Fb), or with myoblasts and laminin α 2 LG4-5-expressing fibroblast sheets (SMB + laminin Fb). Results: Quantitative estimation of nebulin mRNA levels indicated that the transplanted myoblasts in SMB + laminin Fb group exhibited significantly higher survival rates than those in the other groups. Consistent with these findings, the myoblasts in SMB + laminin Fb group exhibited elevated expression of growth factors, while SMB + laminin Fb rats also showed significant improvements in percent fractional shortening (%FS) and left ventricular remodelling, compared to the other groups. Conclusions: Laminin secreted by implanted fibroblasts inhibited the detachment of implanted myoblasts from grafted myocardia, resulting in more permanent therapeutic effects upon myoblast sheet transplantation.

Effects of various types of anesthesia on hemodynamics, cardiac function, and glucose and lipid metabolism in rats.

Anesthesia can affect respiratory, circulatory, and endocrine systems but is necessary for certain experimental procedures such as echocardiography and blood sampling in small animals. We have now investigated the effects of four types of anesthesia [pentobarbital sodium (PENT), ketamine-xylazine (K/X), and low- or high-dose isoflurane (ISO)] on hemodynamics, cardiac function, and glucose and lipid metabolism in Sprague-Dawley rats. Aortic pressure, heart rate, and echocardiographic parameters were measured at various time points up to 45 min after the induction of anesthesia, and blood was then collected for measurement of parameters of glucose and lipid metabolism. Systolic aortic pressure remained constant in the PENT group, whereas it showed a biphasic pattern in the K/X group and a gradual decline in the ISO groups. Marked bradycardia was observed in the K/X group. The serum glucose concentration was increased and the plasma insulin level was reduced in the K/X and ISO groups compared with the PENT group. The concentrations of free fatty acids and norepinephrine in plasma were increased in the K/X group. Despite the metabolic effects of K/X and ISO, our results suggest that the marked bradycardic effect of K-X renders this combination appropriate for measurement of Doppler-derived indexes of left ventricular diastolic function, whereas the relative ease with which the depth of anesthesia can be controlled with ISO makes it suitable for manipulations or data collection over long time periods. On the other hand, PENT may be best suited for experiments that focus on measurement of cardiac function by M-mode echocardiography and metabolic parameters.

Blockade of glucocorticoid receptors with RU486 attenuates cardiac damage and adipose tissue inflammation in a rat model of metabolic syndrome.

Glucocorticoids are stress hormones that modulate metabolic, inflammatory and cardiovascular processes. We recently characterized DahlS.Z-Lepr(fa)/Lepr(fa) (DS/obese) rats, derived from a cross between Dahl salt-sensitive (DS) and Zucker rats, as a new animal model of metabolic syndrome (MetS). We have now investigated the effects of glucocorticoid receptor (GR) blockade on cardiac and adipose tissue pathology and gene expression, as well as on glucose metabolism in this model. DS/obese rats were treated with the GR blocker RU486 (2 mg kg(-1) per day, subcutaneous) for 4 weeks beginning at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr(+)/Lepr(+), or DS/lean) littermates of DS/obese rats served as controls. Treatment of DS/obese rats with RU486 attenuated left ventricular (LV) fibrosis and diastolic dysfunction, as well as cardiac oxidative stress and inflammation, without affecting hypertension or LV hypertrophy. Administration of RU486 to DS/obese rats also inhibited the upregulation of GR and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) expression at the mRNA and protein levels in the heart; it attenuated adiposity and adipose tissue inflammation, as well as the upregulation of GR and 11ß-HSD1 mRNA and protein expression in adipose tissue; it ameliorated fasting hyperinsulinemia as well as insulin resistance and glucose intolerance. Our results thus implicate the glucocorticoid-GR axis in the pathophysiology of MetS, and they suggest that GR blockade has therapeutic potential for the treatment of this condition.

Roles of oxidative stress and the mineralocorticoid receptor in cardiac pathology in a rat model of metabolic syndrome.

Oxidative stress and the mineralocorticoid receptor (MR) are implicated in the pathogenesis of salt-induced left ventricular (LV) diastolic dysfunction associated with metabolic syndrome (MetS). We recently characterized DahlS.Z-Lepr(fa) /Lepr(fa) (DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats, as a new animal model of MetS. We investigated the pathophysiological roles of increased oxidative stress and MR activation in cardiac injury with this model. DS/obese rats were treated with the antioxidant tempol (1 mmol/L in drinking water) or the selective MR antagonist eplerenone (15 mg/kg per day, per os) for 5 weeks beginning at 10 weeks of age. The increased systolic blood pressure and LV hypertrophy that develop in untreated DS/obese rats were substantially ameliorated by eplerenone but not by tempol. Eplerenone also attenuated LV fibrosis and diastolic dysfunction more effectively than did tempol in DS/obese rats, whereas cardiac oxidative stress and inflammation were reduced similarly by both drugs. Both the ratio of plasma aldosterone concentration to plasma renin activity and cardiac expression of the MR and serum/glucocorticoid-regulated kinase 1 genes were decreased to a greater extent by eplerenone than by tempol. Our results indicate that both increased oxidative stress and MR activation in the heart may contribute to the development of LV remodeling and diastolic dysfunction in DS/obese rats. The superior cardioprotective action of eplerenone is likely attributable to its greater antihypertensive effect, which is likely related to its greater inhibition of aldosterone-MR activity in the cardiovascular system.

Restraint stress exacerbates cardiac and adipose tissue pathology via ß-adrenergic signaling in rats with metabolic syndrome.

Restraint stress stimulates sympathetic nerve activity and can affect adiposity and metabolism. However, the effects of restraint stress on cardiovascular and metabolic disorders in metabolic syndrome (MetS) have remained unclear. We investigated the effects of chronic restraint stress and ß-adrenergic receptor (ß-AR) blockade on cardiac and adipose tissue pathology and metabolic disorders in a rat model of MetS. DahlS.Z-Lepr(fa)/Lepr(fa) (DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats. Rats were exposed to restraint stress (restraint cage, 2 h/day) for 4 wk from 9 wk of age with or without daily subcutaneous administration of the ß-AR blocker propranolol (2 mg/kg). Age-matched homozygous lean littermates of DS/obese rats (DahlS.Z-Lepr(+)/Lepr(+) rats) served as control animals. Chronic restraint stress exacerbated hypertension as well as left ventricular hypertrophy, fibrosis, diastolic dysfunction, and oxidative stress in a manner sensitive to propranolol treatment. Restraint stress attenuated body weight gain in DS/obese rats, and this effect tended to be reversed by propranolol (P = 0.0682). Restraint stress or propranolol did not affect visceral or subcutaneous fat mass. However, restraint stress potentiated cardiac and visceral adipose tissue inflammation in DS/obese rats, and these effects were ameliorated by propranolol. Restraint stress also exacerbated glucose intolerance, insulin resistance, and abnormal lipid metabolism in a manner sensitive to propranolol. In addition, restraint stress increased urinary norepinephrine excretion, and propranolol attenuated this effect. Our results thus implicate ß-ARs in the exacerbation of cardiac and adipose tissue pathology and abnormal glucose and lipid metabolism induced by restraint stress in this model of MetS.