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Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease (CAD). ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating CAD; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EMEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease related biological substrates and facilitate activity-based ADAMTS7 biomarker development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/469331v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1520ad4org.highwire.dtl.DTLVardef@144f148org.highwire.dtl.DTLVardef@13cdbc1org.highwire.dtl.DTLVardef@7f7b4f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Rationale: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc3 40kDa ). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy—one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. Objective: TGFβ (transforming growth factor-β) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFβ signaling during chronic Mybpc3 40kDa expression. Methods and Results: To specifically block TGFβ signaling only in the activated myofibroblasts in Mybpc3 40kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFβ receptor II (TβRII) alleles ( Tgfbr2 ) were ablated using the periostin ( Postn ) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm ) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc3 40kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc3 40kDa mouse survival but failed to reverse cardiac hypertrophy. Conclusions: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc3 40kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFβ signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.

Purpose: Hypertrophic cardiomyopathy (HCM) is considered one of the most common genetic heart disorders with a prevalence of about 1 in 500 people, with 35% of those affected being attributed to mutations within the gene encoding cardiac myosin-binding protein C (cMyBP-C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes ( Mybp3 40kDa ) leads to HCM, fibrosis and heart failure, mimicking human disease progression. The transforming growth factor-β (TGFβ) signaling pathway has been implicated in a variety of fibrotic processes. The goal of this study is to define the role of TGFβ signaling in distinct cell populations, the cardiomyocyte and fibroblast, in the cMyBP-C HCM/HF model. Methods and results: Masson’s Trichrome staining, PCR arrays, immunohistochemistry and western blots were performed to characterize the fibrotic progression in Mybp3 40kDa transgenic mice. Cardiac fibrosis was initially detected 4 weeks after transgene expression. Extensive interstitial fibrosis and severe atrial fibrosis were detected at 16 weeks. Both canonical and non-canonical TGFβ pathways were active during fibrotic progression. To specifically block TGFβ signaling in Mybp3 40kDa transgenic mice, compound mutant mice were generated, in which the tgfbr1 or tgfbr2 alleles were ablated, either in cardiomyocytes or in activated fibroblasts (myofibroblasts) by αMHC-Cre or Periostin-MerCreMer-Cre respectively. Blockage of TGFβ signaling in either cardiomyocytes or myofibroblasts alleviated cardiac fibrosis. Furthermore, treatment with the non-canonical TGFβ signaling inhibitor MMI-0100 also alleviated cardiac fibrosis and increased the life span of the Mybp3 40kDa transgenic mice. Conclusions: TGFβ signaling is activated in the Mybp3 40kD HCM/HF model. Genetic or pharmaceutical inhibition of TGFβ signaling inhibited fibrosis and increased the life span in this model.

The endoplasmic reticulum (ER) is the major site of calcium storage and protein folding. It has a unique oxidizing-folding environment due to the predominant disulfide bond formation during the process of protein folding. Alterations in the oxidative environment of the ER and also intra-ER Ca2+ cause the production of ER stress-induced reactive oxygen species (ROS). Protein disulfide isomerases, endoplasmic reticulum oxidoreductin-1, reduced glutathione and mitochondrial electron transport chain proteins also play crucial roles in ER stress-induced production of ROS. In this article, we discuss ER stress-associated ROS and related diseases, and the current understanding of the signaling transduction involved in ER stress.

Hyperlipidemia is a major contributor for atherosclerosis and hypolipidemic drugs such as statin are highly prescribed to treat elevated lipid level in plasma. Rubus coreanus, which is widely cultivated in south eastern Asia, have been reported to show significant cholesterol lowering action in hyperlipidemic subjects. Our objective was to determine the cellular effect of Rubus coreanus extract (RCE) on cholesterol biosynthesis in human hepatic cells (HepG2) and to elucidate the molecular mechanism by which it causes change in cholesterol metabolism. RCE treatment lowered cholesterol biosynthesis as well as secretion from HepG2 cells. This effect was associated with lowering the release of apolipoproteins from hepatic cells. RCE treatment also showed an increase in phosphorylation of foxhead box protein 01 (FoXo-1) and 5-adenosine monophosphate-activated protein kinase (AMPK), thus lowering expression of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, which might be a major pathway for cholesterol biosynthesis inhibition. Apart from this; RCE also lowered sterol regulatory element-binding protein-1 (SREBP-1) expression in HepG2 cells, showing a long term regulation of cholesterol biosynthesis activity. These results indicate that one of the anti-hyperlipidemic actions of RCE is due to inhibition of cholesterol biosynthesis in hepatic cells and provides first documentation of a hypolipidemic bio-molecular action of Rubus coreanus.

Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3Kδ inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-κB (NF-κB) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1α (IRE1α)-dependent degradation (RIDD) of IRE1α was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3Kδ may induce severe airway inflammation and hyperresponsiveness by activating NF-κB signaling through ER-associated ROS and RIDD-RIG-I activation. The PI3Kδ inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.

Endoplasmic reticulum (ER) stress and associated protein aggregation are closely associated with human diseases, including alterations in hepatic lipid metabolism. Inhibition of ER stress can have a significant effect on the prevention of hepatic dyslipidemia. Here, we studied the role of 4-phenylbutyric acid (4-PBA), a chemical chaperone, on ER stress-induced hepatic lipid accumulation. We studied ER stress induction following CCl4 exposure and delineated mechanisms of the CCl4-induced ER stress response in liver tissue from mice. CCl4 affected the formation of disulfide bonds through excessive hyper-oxidation of protein disulfide isomerase (PDI). Increased complex formation between PDI and its client proteins persisted in CCl4-exposed samples. Conversely, 4-PBA inhibited ER stress via secretion of apolipoprotein B and prevention of hepatic lipid accumulation. We also studied the mechanism-based pharmacokinetic and pharmacodynamic profiles and identified the ER stress-related proteins GRP78 and CHOP, along with plasma apolipoprotein B and triglyceride levels, as novel biomarkers of ER stress-induced hepatic lipid accumulation. ER stress and its clinical relevance for therapeutic approaches were well correlated with the activity of the ER stress regulator 4-PBA, which may be a promising drug candidate for the treatment of hepatic lipid accumulation, such as hepatic steatosis.

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

IC87114 is a selective PI3Kδ inhibitor. A simple, sensitive and reliable LC–MS/MS method with rapid sample preparation was developed and validated for the determination of IC87114 in mouse plasma, bronchoalveolar lavage, and lung. Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C 18 column (150 mm × 2.1 mm internal diameter, 3.5 μm particle size). Mass spectrometric detection was conducted by electrospray ionization in positive ion multiple reaction monitoring modes. The calibration curve was linear over a concentration range of 0.01–1000 ng/mL for plasma/BAL and 0.1–250 ng/mL for lung tissue. Recoveries were as high as 97.29%, 102.81% and 89.70% for plasma, BAL fluid and lung sample, respectively. The lower limit of quantification was 0.01 ng/mL. Intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. Finally, the method was successfully used in a pharmacokinetic study that measured IC87114 in mouse plasma, BAL fluid and lung tissue after administration of a single 1 mg/kg intratracheal dose of IC87114. The percentage change for incurred sample reanalysis (ISR) was within ±15.0% and met the acceptance criteria for ISR.

Background Salvia miltiorrhiza (SM) has long been used as a traditional oriental medicine for cardiovascular disease. Accumulating evidence also indicates that SM has anti-osteoporotic effects. This study was conducted to examine the SM-induced anti-osteoporotic effect and its possible mechanisms with various doses of SM. Methods We studied Sprague-Dawley female rats aged 12 weeks, divided into six groups: sham-operated control (SHAM), OVX rats supplemented with SM (1, 3, 10 and 30 mg/kg) orally for 8 weeks. At the end of the experiment, blood samples were collected and biochemistry analysis was performed. Specimens from both tibia and liver were processed for light microscopic examination. DEXA and μ-CT analyses of the tibia were also performed. Results SM treatment significantly ameliorated the decrease in BMD and trabecular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by μ-CT scanning. In serum biochemical analysis, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX. Conclusions The preventive effect of SM was presumably due to its anti-oxidative stress partly via modulation of osteoclast maturation and number. In current study, SM appears to be a promising osteoporosis therapeutic natural product.

Reactive oxygen species exert toxic effects during ischemia-reperfusion (I/R) injury of various organs. This study was designed to evaluate the preventive effects of various isoflavonoids such as biochanin A, daidzein, genistein, rutin and quercetin. These compounds are wellknown naturally occurring compounds with beneficial health effects and antioxidant activity. Free radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and superoxide dismutase (SOD) assay. Among the isoflavonoids tested, biochanine A, quercetin and rutin showed significant DPPH free radical scavenging activity. Similarly, treatment of biochanine A, genistein and rutin significantly increased SOD activity in neonant rat heart myocyte primary cells as well as in H9C2 cells. For ex vivo study, hearts from Sprague-Dawley rats were perfused in Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O(2) and 5% CO(2). Hearts were subjected to 20 min of pre-ischemia followed by 20 min of global ischemia, and then 50 min of reperfusion at 37°C. The test compounds were perfused 10 min before ischemia and during the entire reperfusion period. Among the isoflavonoids tested, only rutin significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/- dP/dtmax). In left ventricular end diastolic pressure (LVEDP) analysis, rutin, daidzein and biochanin A were effective. Among the isoflavonoids, rutin had consistent protective effects in I/R injury by affecting cardiac dynamic factors as well as by enhancing SOD and DPPH activity.

The role of ER stress on hepatic steatosis was investigated in a rat model. We injected CCl(4) into rats and found that CCl(4) could induce hepatic lipid accumulation, confirmed by Oil Red O staining and by measurement of triglyceride and cholesterol. The expression of ApoB, an apolipoprotein, was decreased in plasma and increased in the liver of CCl(4)-treated animals. The ER stress response was also significantly increased by CCl(4). P450 2E1 expression and activity were increased through interactions of P450 2E1 with NADPH-dependent P450 reductase (NPR) under CCl(4)-treated conditions. In HepG2 cells, intracellular lipid accumulation and its signaling were comparable to in vivo results. In order to elucidate the effect of the ER stress response itself, tunicamycin, an N-acetyl-glycosylation inhibitor, was injected into rats, followed by Oil Red O staining, lipid/triglyceride/cholesterol accumulation analysis, and examination of ApoB expression. Additionally, the ER stress response and upregulation of P450 2E1 were also confirmed in the tunicamycin-treated rats. All of the responses were similar to those seen with CCl(4). The P450 2E1 inhibitor diallyl sulphide (DAS), N-acetylcysteine (NAC), and reduced glutathione (GSH) antioxidants also regulated processes, including ApoB expression and lipid accumulation in CCl(4)-treated animals. In the presence of tunicamycin, DAS or NAC/GSH regulated all of the pathological phenomena with the exception of the ER stress response. In summary, CCl(4) induces liver steatosis, a process involving ER stress-induced P450 2E1 activation and ROS production.

Rubus coreanus fruits have been employed as a traditional medicine for centuries in the Asia-Pacific region. Its pharmacological action differs according to the different extraction methods utilized and the degree of fruit ripening. In this study, we determined the cellular effect of different ethanol extracts of mature and immature Rubus coreanus fruits in human hepatic cell line, HepG2 cells. The antioxidant activity, effect on superoxide dismutase activity and cholesterol biosynthesis efficiency was also evaluated. Immature Rubus coreanus extract showed higher antioxidant capability, compared with that of its mature fractions. Cellular antioxidant proteins including HO-1, Cu/Zn-superoxide dismutase and catalase were highly expressed in the presence of Rubus coreanus. Cholesterol levels in HepG2 cells treated with the water fraction of immature Rubus coreanus were significantly reduced. This antihyperlipidaemic action of Rubus coreanus is a consequence of cholesterol biosynthesis and extracellular secretion in HepG2 cells. These results indicate that among different ethanol fraction of mature and immature Rubus coreanus fruit extracts, water extract of immature fruit extract shows higher antioxidant as well as higher antihyperlipidaemic action.