Absence of thrombospondin-2 causes age-related dilated cardiomyopathy

BACKGROUND: The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. METHODS AND RESULTS: TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene-mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes. CONCLUSIONS: TSP-2 expression in the heart protects against age-dependent dilated cardiomyopath

MRI Contrast-enhancement with superparamagnetic iron oxide nanoparticles amplify macrophage foam cell apoptosis in human and murine atherosclerosis

(Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidemic models of atherosclerosis.RAW264.7 foam cells, exposed in vitro to Ferumoxide (dextran-coated SPIO), Ferumoxtran (dextran-coated USPIO), or Ferumoxytol (carboxymethyl dextran-coated USPIO) (all 1 mg Fe/ml) showed increased apoptosis and ROS accumulation for Ferumoxide and Ferumoxtran, whereas Ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of Ferumoxide (0.3 mg Fe/kg) and Ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared to saline-treated controls. Again, Ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P Ferumoxide and Ferumoxtran, but not Ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis.Biopharmaceutic

Reduced Paneth cell antimicrobial protein levels correlate with activation of the unfolded protein response in the gut of obese individuals.

The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth cell alpha-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI > 35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human alpha-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth cells of obese subjects. Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth cell compromise. Copyright (c) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Prevention and reversal of hepatic steatosis with a high-protein diet in mice

The hallmark of NAFLD is steatosis of unknown etiology. We tested the high-protein (HP)(2) diet on diet-induced steatosis in male C57BL/6 mice without pre-existing fatty liver. Mice were fed all combinations of low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets lipids to ~40% of corresponding LP-containing diets, were more efficient respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP- diets showed increased mitochondrial oxidative capacity (elevated mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C0]/[C16+C18] carnitine ratio); gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma-glucose concentration without increased G6pase mRNA); reduced desaturation (decreased Scd1 expression and [C16:1n-7]/[C16:0] ratio) increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced eIF2alpha, and Fgf21 and Chop expression); and a trend toward less (lower Mcp1 and Cd11b expression and less phosphorylated NFkappaB). HP diets prevent and reverse steatosis independently of fat and intake more efficiently than a 20% reduction in energy intake. The to result from fuel-generated, highly distributed small, synergistic lipid and BCAA catabolism, and a decrease in cell stress

Level of activation of the unfolded protein response correlates with paneth cell apoptosis in human small intestine exposed to ischemia/reperfusion.

BACKGROUND & AIMS: In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. METHODS: Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-alpha and interleukin-6 levels were assessed. RESULTS: In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. CONCLUSIONS: ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammatio

Cardiotin localization in mitochondria of cardiomyocytes in vivo and in vitro and its down-regulation during dedifferentiation

BACKGROUND: Cardiotin expression is observed in adult cardiac tissue. In the present study, we provide evidence for the specific localization of cardiotin in cardiac mitochondria and for its down-regulation during adaptive remodeling (dedifferentiation) of cardiomyocytes. METHODS: Immunocytochemistry was used to study cardiotin localization in adult rabbit papillary muscle, in late-stage embryonic rabbit left ventricular tissue, and in left ventricle samples of rabbits suffering from pressure and volume overload. Western blot analysis of cardiotin was performed in purified pig heart mitochondrial fractions. Cardiotin expression was monitored in vitro in isolated adult rat and rabbit left ventricular cardiomyocytes. RESULTS: Western blot analysis revealed the presence of cardiotin in the mitochondrial fractions of pig heart. Immunoelectron microscopy confirmed the presence of cardiotin in cardiac mitochondria of normal adult rabbits both in vivo and in vitro. Quantification of the localization of immunogold particles suggests an association of cardiotin with the mitochondrial inner membrane. Cardiotin expression is initiated in late-stage embryonic rabbit heart, whereas in adult ventricular tissue cardiotin clearly stained longitudinal arrays of mitochondria. Pressure- and volume-overloaded myocardium showed a reduction in cardiotin expression in dispersed local myocardial areas. Cell cultures of adult cardiomyocytes showed a gradual loss in cardiotin expression in parallel with a sarcomeric remodeling. CONCLUSIONS: Our results demonstrate the specific localization of cardiotin in adult cardiomyocyte mitochondria and propose its use as an early marker for cardiomyocyte adaptive remodeling and dedifferentiation

Perilipin 2 improves insulin sensitivity in skeletal muscle despite eleveted intramuscular lipid levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance

Morphological and microarray analyses of human hepatocytes from xenogeneic host livers.

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body. To do so, we performed immunohistochemistry, morphometric analyses, and electron microscopic observations on chimeric mouse livers, and used microarray analyses to compare gene expression patterns in hepatocytes derived from chimeric mouse hepatocytes (c-heps) and h-heps. Morphometric analysis revealed that the ratio of hepatocytes to m-HSCs in the chimeric mouse livers were twofold higher than those in the SCID mouse livers, corresponding to twin-cell plates in the chimeric mouse liver. The h-heps in the chimeric mouse did not show hypoxia even in the twin-cell plate structure, probably because of low oxygen consumption by the h-heps relative to the mouse hepatocytes (m-heps). Immunohistochemical and electron microscopic examinations revealed that the sinusoids in the chimeric mouse livers were normally constructed with h-heps and m-HSCs. However, a number of microvilli projected into the intercellular clefts on the lateral aspects of the hepatocytes, features typical of a growth phase. Microarray profiles indicated that similar to 82% of 16 605 probes were within a twofold range difference between h-heps and c-heps. Cluster and principal component analyses showed that the gene expression patterns of c-heps were extremely similar to those of h-heps. In conclusion, the chimeric mouse livers were normally reconstructed with h-heps and m-HSCs, and expressed most human genes at levels similar to those in human livers, although the chimeric livers showed morphological characteristics typical of growth. Laboratory Investigation (2013) 93, 54-71; doi:10.1038/labinvest.2012.158; published online 12 November 201