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Background: Despite contemporary therapy, coronary artery disease (CAD) remains a leading cause of mortality. Genetic variants at ADAMTS7 have been associated with CAD and the loss of ADAMTS7 is protective for atherosclerosis. ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) is a secreted metalloproteinase and complex proteoglycan, yet the mechanism linking ADAMTS7 to CAD risk remains unresolved. Methods and Results: We established a new purification strategy for full-length mouse ADAMTS7 and demonstrated the loss of activity in the catalytic mutant form of ADAMTS7. To test if the enzymatic activity of ADAMTS7 mediates atherosclerosis, we generated a catalytically inactive mutant mouse allele and compared it to the Adamts7 knockout. Using two models of atherosclerosis, we found that reducing either ADAMTS7 dosage or catalytic function decreased the burden of atherosclerosis. We demonstrate impaired vascular smooth muscle migration in both Adamts7 catalytic mutant and null cells using a lateral migration wound healing assay. Expression of the WT allele rescued the migration phenotype in Adamts7 null cells while expression of the catalytic mutant protein did not. We then characterized a human ADAMTS7 coding variant rs3825807 (Ser214Pro) associated with reduced CAD risk. This variant had a hypomorphic effect on ADAMTS7 secretion and migration of vascular smooth muscle cells, findings consistent with our mouse studies. Conclusions: We demonstrated that loss of ADAMTS7 catalytic function protects against atherosclerosis via phenotype switch of VSMCs and that the atherosclerosis protective effects could be mediated by a loss-of-function coding variant associated with CAD risk. In aggregate, we provide compelling evidence that dosage of ADAMTS7 and catalytic function are responsible for the atherosclerotic phenotype, suggesting that the catalytic domain would be an attractive therapeutic target for CAD.

SUMMARYSertoli cells are highly polarized testicular supporting cells that simultaneously nurture progressively maturing germ cells. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, are vital for successful spermatogenesis. However, the mechanisms and developmental timing that underlie the establishment of polarity are poorly understood. To investigate this aspect of testicular function, we conditionally deleted Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Cdc42 deletion disrupted adult Sertoli cell maturation and localization of polarity proteins, but did not affect fetal and early postnatal testicular development, nor the onset of the first wave of spermatogenesis. By early adulthood, however, conditional knockout males exhibited a loss of spermatogenic cells, resulting in a complete lack of sperm. These findings demonstrate that Cdc42 plays an essential role in establishing adult Sertoli cell polarity and, thus, maintaining steady-state spermatogenesis and healthy sperm production.

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial–mesenchymal Notch signaling interactions. Depletion of Nestin + progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.

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

Sertoli cells are highly polarized testicular supporting cells that simultaneously nurture multiple stages of germ cells during spermatogenesis. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, is vital for spermatogenesis. However, the mechanisms and developmental timing that underlie Sertoli cell polarity are poorly understood. We investigate this aspect of testicular function by conditionally deleting Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Sertoli Cdc42 deletion leads to increased apoptosis and disrupted polarity of juvenile and adult testes but does not affect fetal and postnatal testicular development. The onset of the first wave of spermatogenesis occurs normally, but it fails to progress past round spermatid stages, and by young adulthood, conditional knockout males exhibit a complete loss of spermatogenic cells. These findings demonstrate that Cdc42 is essential for Sertoli cell polarity and for maintaining steady-state sperm production.

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.

Background Transforming growth factor beta ( TGF ‐β) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte‐specific expression of a mutant αB‐crystallin (Cry AB R 120G ), which causes human desmin‐related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so‐called myofibroblast state and TGF ‐β binding mediates an essential signaling pathway underlying this process. Here, we test the hypothesis that fibroblast‐based TGF ‐β signaling can result in significant cardiac fibrosis in a disease model of cardiac proteotoxicity that has an exclusive cardiomyocyte‐based etiology. Methods and Results Against the background of cardiomyocyte‐restricted expression of Cry AB R 120G , we have partially ablated TGF ‐β signaling in cardiac myofibroblasts to observe whether cardiac fibrosis is reduced despite the ongoing pathogenic stimulus of Cry AB R 120G production. Transgenic Cry AB R 120G mice were crossed with mice containing a floxed allele of TGF ‐β receptor 2 ( Tgfbr2 f/f ). The double transgenic animals were subsequently crossed to another transgenic line in which Cre expression was driven from the periostin locus ( Postn ) so that Tgfbr2 would be ablated with myofibroblast conversion. Structural and functional assays were then used to determine whether general fibrosis was affected and cardiac function rescued in Cry AB R 120G mice lacking Tgfbr2 in the myofibroblasts. Ablation of myofibroblast specific TGF ‐β signaling led to decreased morbidity in a proteotoxic disease resulting from cardiomyocyte autonomous expression of Cry AB R 120G . Cardiac fibrosis was decreased and hypertrophy was also significantly attenuated, with a significant improvement in survival probability over time, even though the primary proteotoxic insult continued. Conclusions Myofibroblast‐targeted knockdown of Tgfbr2 signaling resulted in reduced fibrosis and improved cardiac function, leading to improved probability of survival.

Introduction: Impairment of protein quality control leads to the accumulation of intracellular misfolded protein aggregates, contributing to cardiac disease and heart failure. Essential protein quality control pathways, including proteasomal degradation and autophagy, are involved in maintaining cellular homeostasis and cardiac function during proteotoxic insult. In a prior study, we undertook an unbiased, total genomewide screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes. Forkhead box F1 (Foxf1) gene was one of the hits from our high throughput screening. In this study, we have performed further validation of Foxf1, a transcription factor and previously unknown player in proteotoxic processes. Aims and Methods: The objective of this study was to validate Foxf1 as a contributor to protein quality control and understand its role in contributing to protein aggregate formation during proteotoxic-induced cardiac disease. We knocked down Foxf1 expression using siRNA mediated transfection in neonatal rat ventricular cardiomyocytes that expressed the protein aggregate inducing mutation of the chaperone α B crystallin (CryABR120G). Proteasomal function and relative activity in cells was analyzed using a degron-destablized green fluorescent protein (GFPu) based reporter protein. Similarly, autophagy activity was determined by autophagic flux assay using bafilomycin treatment. Results: CryABR120G expression leads to aggregate formation and decreased proteasomal function in cardiomyocytes. Knockdown of Foxf1 gene expression in CryABR120G-transfected cardiomyocytes reduced the proteotoxic sequelae, decreasing aggregate concentration. Knockdown of Foxf1 significantly increased proteasomal function in the model without altering autophagy activity. Conclusion: Decreased Foxf1 expression reduced CryABR120G induced aggregate protein accumulation and decreased proteotoxic stress in cardiomyocytes, likely through the maintenance of clearance of the misfolded protein via the proteasomal pathway.

Background: Mutations in cardiac myosin binding protein C ( MYBPC3 ) account for more than one third of identified hypertrophic cardiomyopathy (HCM). Data from both human patients and mouse models suggest that cardiac fibrosis preceeds hypertrophy and cardiac dysfunction, implying that fibrosis related signaling may be one of the initial signs of developing cardiac disease. We wished to determine if cardiomyocyte-autonomous TGFβ signaling is sufficient to initiate pathogenic fibrosis in the heart in the context of a cMyBP-C-induced disease. Methods and Results: Neonatal rat ventricular cardiomyocytes (NRVMs) expressing either wild-type or Mybpc3 mutations were mediated by adenovirus for 48 hours. Both cells and culture medium (conditional medium) were harvested. The fibrotic effects of the conditional medium on neonatal rat ventricular fibroblasts (NRVFs) were determined using “fibrotic” gene promoters driving luciferase (luc) expression ( αSMA-luc , postn-luc and col1a1-luc ). TGFβ levels in the conditional medium were determined using ELISA assays. Expression of cMybpc3 mutations activated TGFβ promoter activity ( Tgfb1-luc , Tgfb2-luc and Tgfb3-luc ) in a TGFβ receptor(s) (TβRs) dependent manner. Upregulation of TGFβ was also confirmed in transgenic mice expressing a fragment of cMyBP-C (cMyBP-C 40kDa ) whose expression results in significant fibrosis and cardiac disease. To establish causality, we genetically ablated TGFβ receptors ( Tgfbr1 and Tgfbr2 ) in the cardiomyocytes of the cMybpc3 40kDa mice. TGFβ receptors ablation blocked the induction of TGFβ expression, alleviating cardiac fibrosis and decreasing cardiac hypertrophy. Cardiac function was significantly improved and survival of the cMyBP-C 40kDa mice was increased. Conclusions: Cardiomyocytes are a critical source of TGFβ in cMyBP-C 40kDa hearts during the initial stages of pathogenesis. Thus, TβRs ablation at the early stage completely prevent the progression of the pathological process in vivo .

Background Cardiac stress can trigger production of a 40‐kDa peptide fragment derived from the amino terminus of the cardiac myosin‐binding protein C. Cardiac stress, as well as cMyBP ‐C mutations, can trigger production of 1 such truncated protein fragment, a 40‐kDa peptide fragment derived from the amino terminus of cMyBP ‐C. Genetic expression of this 40‐kDa fragment in mouse cardiomyocytes ( cMyBP ‐C 40k ) leads to cardiac disease, fibrosis, and death within the first year. Fibrosis can occur in many cardiovascular diseases, and mitogen‐activated protein kinase––activated protein kinase‐2 signaling has been implicated in a variety of fibrotic processes. Recent studies demonstrated that mitogen‐activated protein kinase––activated protein kinase‐2 inhibition using the cell‐permeant peptide inhibitor MMI ‐0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI ‐0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP ‐C 40k cardiomyocyte expression. Methods and Results Nontransgenic and cMyBP ‐C 40k inducible transgenic mice were given MMI ‐0100 or PBS daily for 30 weeks. In control groups, long‐term MMI ‐0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy, or probability of survival. In the inducible transgenic group, MMI ‐0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy, and prolonged survival. Conclusions Pharmaceutical inhibition of mitogen‐activated protein kinase––activated protein kinase‐2 signaling via MMI ‐0100 treatment is beneficial in the context of fibrotic cMyBPC 40k disease.

Introduction: Transforming Growth Factor beta (TGFβ) is an important cytokine in mediating cardiac fibrosis. Cardiomyocyte-specific expression of a mutant αB-crystallin (CryAB R120G ) that is responsible for human Desmin Related Myopathy results in significant cardiac fibrosis and cardiac remodeling leading to heart failure. Onset of fibrosis is initiated by the activation of a quiescent fibroblast population to an active, “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that myofibroblast-based TGFβ signaling can result in significant cardiac fibrosis. Here, we have partially ablated TGFβ signaling in cardiac myofibroblasts to observe if cardiac fibrosis is altered. Objective: To understand the contributions of myofibroblast-based TGFβ signaling to the development of cardiac fibrosis. Methods and Results: To test the hypothesis we partially ablated myofibroblast specific TGFβ signaling by crossing CryAB R120G mice with mice containing a floxed allele of TGFβ’s receptor 1 (TGFβr1). The double transgenic animals were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter so that TGFβr1 would be ablated subsequent to myofibroblast conversion as the periostin promoter became active. Echocardiography, Masson’s Trichome staining, the hydroxyproline assay, PCR arrays, immunohistochemistry and western blots were used to characterize fibrosis and cardiac function in mice lacking TGFβr1 in the myofibroblasts were used to characterize the resultant animals. Conclusion: Myofibroblast-targeted knockdown of Tgfβr1 signaling resulted in reduced fibrosis and improved cardiac function.

Background: Hypertrophic cardiomyopathy is considered one of the most common genetic heart disorders, occurring with a frequency of about 1 in 200, with approximately 30% of those affected showing mutations within the cardiac myosin-binding protein C (cMyBP-C) gene ( MYBPC3 ). 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 (MyBP-C 40k ) leads to hypertrophic cardiomyopathy), fibrosis and heart failure. Fibrosis can occur in many cardiovascular diseases and the p38-MK2 signaling pathway has been implicated in a variety of fibrotic processes. Recent studies demonstrated that MK2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C 40k cardiomyocyte expression. Methods and Results: Non-transgenic and MyBP-C 40k transgenic (Dtg) mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy or probability of survival. In the Dtg group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy and prolonged survival. Conclusions: Pharmaceutical inhibition of p38-MK2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic MyBPC 40k disease.

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.

Introduction: Transforming Growth Factor Beta (TGFβ) is an important cytokine in mediating the fibrogenic response and, in particular, cardiac fibrosis. Extensive fibrosis accompanies the cardiac remodeling that occurs during development of the protein conformation-based disease caused by cardiomyocyte-specific expression of a mutant, small, heat shock-like protein and chaperone, aB crystallin (CryABR120G). During the onset of fibrosis, fibroblasts are activated to the so-called “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that TGFβ signaling processes that result in significant cardiac fibrosis in a mouse model of proteotoxic heart disease are mediated by cardiac fibroblasts, rather than cardiomyocytes. Here, we have partially ablated TGFβ signaling only in cardiac myofibroblasts to observe if cardiac fibrosis is reduced. Aims and Methods: The objective of this study was to understand the contributions of fibroblast-derived TGFβ signaling to the development of cardiac fibrosis in a proteotoxic mouse model that results in significant cardiac fibrosis. To test the hypothesis we partially deleted the myofibroblast specific canonical and non-canonical signaling by crossing CryAB R120G mice with Tgfbr1 or Tgfbr2 floxed mice. The double transgene containing mice were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter. Echocardiography, Masson’s Trichome staining, PCR arrays, IHC and western blots were performed to characterize the fibrotic progression in CryAB R120G transgenic mice. Results: We observed that myofibroblast-targeted partial knockdown of Tgf βr1 signaling prolonged survival, modestly reducing fibrosis and improving cardiac function . Similarly, Tgf βr2 partial knockdown prolonged survival, modestly reducing fibrosis without improving cardiac function during fibrosis development in CryAB R120G mice. Conclusion: These findings suggest that, in a model of proteotoxic heart disease, myofibroblast based TGFβ signaling in the heart may contribute to cardiac hypertrophy/dysfunction but cannot account entirely for the fibrotic response.

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.

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.

Mitochondria, also known as "Power House of cell," are crucial organelles, regulating energy metabolism. Recently, an involvement of mitochondria in cancer occurrence and metastasis has been proposed. The roles of mitochondria in cancer progression/metastasis include alteration of glycolysis, regulation of ROS and suppression of intrinsic apoptosis. This mini-review explains the specific mitochondrial characteristics during cancer metastasis with past and recent findings. It may contribute to understanding mitochondria-related mechanisms of cancer metastasis.

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.

Oxidized low-density lipoprotein (oxLDL) contributes to atherosclerosis in part by being taken up into macrophages via scavenger receptors and leading to foam cell formation. Herbal compounds that have been used to treat blood stasis (a counterpart of atherosclerosis) for centuries include extracts of medicinal plants in the Rosaceae and Leguminosae families. In this study, we investigated the effect of the unripe Rubus coreanus (Korean black raspberry) fruit extract on oxLDL uptake by murine macrophage cells. In the presence of Rubus coreanus extract (RCE), Dil-labeled oxLDL uptake was inhibited in a dose-dependent manner. SP600125, a specific JNK inhibitor, inhibited the uptake of Dil-oxLDL into macrophages. RCE also inhibited JNK phosphorylation in a time- and dose-dependent manner in macrophages treated with oxLDL. These results indicate that among the mitogen-activated protein kinases, JNK phosphorylation is inhibited by RCE, which is likely the mechanism underlying the RCE-induced inhibition of oxLDL uptake by macrophages.

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.

Cardiovascular science has undergone a revolution in the past 25 years with the application of genetic engineering to the heart and creation of relevant animal models in worms, flies, mice, rats and pigs [1]. A series of evermore-powerful tools have catalyzed the creation of hearts that can express, over-express or not express targeted genes and in some cases, control the developmental timing of the manipulation. Using animal models has allowed the cardiovascular community to establish causality, confirm proof-of-principal for therapeutic approaches, and helped us to understand the relevant pathways and their most important intersections for normal and abnormal cardiac function [1]. The mouse has been the model of choice for the largest number of investigators using these tools as the advantages of dealing with a mammalian, four chambered heart in terms of the data's application to human cardiovascular function and disease remain compelling. The definition of reagents such as the α- and β-myosin heavy chain promoters (MyHC) [2,3] that allowed investigators to drive cardiomyocyte-specific expression at various developmental times and in a chamber-specific manner, cemented the mouse as an important, genetically amenable model for cardiovascular disease. The community's ability to rigorously characterize the resultant phenotype and cardiac physiology using techniques that were first developed for larger animal models, followed quickly [4,5]. The promoters were used to drive expression of a large number of normal and mutated proteins in the heart with the use of the α-MyHC promoter predominating, as it drives cardiomyocyte-specific expression in the atria during fetal development and in all four chambers starting a couple of days before birth, as thyroid hormone production begins and activates transcription from the myc6 locus [3].

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.

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.

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.