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Articles from this Volume

Andrea Nolte, Tobias Walker, Martina Schneider, Oya Kray, Meltem Avci-Adali, Gerhard Ziemer, and Hans Peter Wendel

New drug-eluting stent (DES) methods have recently been demonstrated to improve outcomes of intravascular interventions. A novel technique is the design of gene-silencing stents that elute specific small-interfering RNAs (siRNAs) for better vascular wall regeneration. Although siRNAs used to alter gene expression have surpassed expectations in in vitro experiments, the functional and local delivery of siRNAs is still the major obstacle for the in vivo application of RNA interference. In this preliminary in vitro study we investigated a surface-immobilized siRNA delivery technique that would be readily adaptable for local intravascular applications in vivo. The transfection potency of gelatin coatings consisting of a specific siRNA complexed with polyethylenimine (PEI) was examined in primary human endothelial cells by flow cytometry and quantitative real-time polymerase chain reaction. Several media conditions, such as the presence or absence of serum during cultivation, were investigated. Furthermore, different siRNA and PEI amounts, as well as nitrogen/phosphate ratios, were tested for their transfection efficiency. Gelatin coatings consisting of PEI and siRNA against an exemplary endothelial adhesion molecule receptor achieved a significant knockdown of around 70%. The transfection efficiency of the coatings was not influenced by the presence of serum. The results of this preliminary study support the expectation that this novel coating may be favorable for local in vivo gene silencing (for example, when immobilized on stents or balloons for percutanous transluminal coronary angioplasty). However, further animal experiments are needed to confirm the translation into clinical practice. This intriguing technology leads the way to more sophisticated and individualized coatings for the post-DES era, toward silencing of genes involved in the pathway of intimal hyperplasia.

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Posted by Leah Caracappa on Nov 12, 2011 12:00 AM CST
Youping Wang and Donna H Wang

To investigate the effects of the transient receptor potential vanilloid type 1 (TRPV1) channel on renal extracellular matrix (ECM) protein expression including collagen deposition and the transforming growth factor β (TGF-β)/Smad signaling pathway during salt-dependent hypertension, wild-type (WT) and TRPV1-null (TRPV1–/–) mutant mice were uninephrectomized and given deoxycorticosterone acetate (DOCA)-salt for 4 wks. TRPV1 gene ablation exaggerated DOCA-salt-induced impairment of renal function as evidenced by increased albumin excretion (μg/24 h) compared with WT mice (83.7 ± 7.1 versus 28.3 ± 4.8, P < 0.05), but had no apparent effect on mean arterial pressure (mmHg) as determined by radiotelemetry (141 ± 4 versus 138 ± 3, P > 0.05). Morphological analysis showed that DOCA-salt-induced glomerulosclerosis, tubular injury and macrophage infiltration (cells/mm2) were increased in TRPV1–/– compared with WT mice (0.74 ± 0.08 versus 0.34 ± 0.04; 3.14 ± 0.26 versus 2.00 ± 0.31; 68 ± 5 versus 40 ± 4, P < 0.05). Immunostaining studies showed that DOCA-salt treatment decreased nephrin but increased collagen type I and IV as well as phosphorylated Smad2/3 staining in kidneys of TRPV1–/– compared with WT mice. Hydroxyproline assay and Western blot showed that DOCA-salt treatment increased collagen content (μg/mg dry tissue) and fibronectin protein expression (%β-actin arbitrary units) in the kidney of TRPV1–/– compared with WT mice (26.7 ± 2.7 versus 17.4 ± 1.8; 0.93 ± 0.07 versus 0.65 ± 0.08, P < 0.05). Acceleration of renal ECM protein deposition in DOCA-salt-treated TRPV1–/– mice was accompanied by increased TGF-β1, as well as phosphorylation of Smad2/3 protein expression (%β-actin arbitrary units) compared with DOCAsalt-treated WT mice (0.61 ± 0.07 versus 0.32 ± 0.05; 0.57 ± 0.07 versus 0.25 ± 0.05; 0.71 ± 0.08 versus 0.40 ± 0.06, P < 0.05). These
results show that exaggerated renal functional and structural injuries are accompanied by increased production of ECM protein and activation of the TGF-β/Smad2/3 signaling pathway. These data suggest that activation of TRPV1 attenuates the progression of renal fibrosis possibly via suppression of the TGF-β and its downstream regulatory signaling pathway.

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Posted by Leah Caracappa on Nov 11, 2011 12:00 AM CST
Jennifer E Bond, Andrew Bergeron, Peter Thurlow, M Angelica Selim, Edith V Bowers, Anna Kuang, and Howard Levinson

Aberrant fibroblast migration in response to fibrogenic peptides plays a significant role in keloid pathogenesis. Angiotensin II (Ang II) is an octapeptide hormone recently implicated as a mediator of organ fibrosis and cutaneous repair. Ang II promotes cell migration but its role in keloid fibroblast phenotypic behavior has not been studied. We investigated Ang II signaling in keloid fibroblast behavior as a potential mechanism of disease. Primary human keloid fibroblasts were stimulated to migrate in the presence of Ang II and Ang II receptor 1 (AT1), Ang II receptor 2 (AT2) or nonmuscle myosin II (NMM II) antagonists. Keloid and the surrounding normal dermis were immunostained for NMM IIA, NMM IIB, AT2 and AT1 expression. Primary human keloid fibroblasts were stimulated to migrate with Ang II and the increased migration was inhibited by the AT1 antagonist EMD66684, but not the AT2 antagonist PD123319. Inhibition of the promigratory motor protein NMM II by addition of the specific NMM II antagonist blebbistatin inhibited Ang II–stimulated migration. Ang II stimulation of NMM II protein expression was prevented by AT1 blockade but not by AT2 antagonists. Immunostaining demonstrated increased NMM IIA, NMM IIB and AT1 expression in keloid fibroblasts compared with scant staining in normal surrounding dermis. AT2 immunostaining was absent in keloid and normal human dermal fibroblasts. These results indicate that Ang II mediates keloid fibroblast migration and possibly pathogenesis through AT1 activation and upregulation of NMM II.

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Posted by Leah Caracappa on Nov 10, 2011 12:00 AM CST
Fabio Ghiotto, Paolo Marcatili, Claudya Tenca, Maria Grazia Calevo, Xiao-Jie Yan, Emilia Albesiano, Davide Bagnara, Monica Colombo, Giovanna Cutrona, Charles C Chu, Fortunato Morabito, Silvia Bruno, Manlio Ferrarini, Anna Tramontano, Franco Fais, and Nicholas Chiorazzi

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hypermutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV–diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

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Posted by Leah Caracappa on Nov 9, 2011 12:00 AM CST
Yan Liu, Lihua Sun, Zhenwei Pan, Yunlong Bai, Ning Wang, Jinlong Zhao, Chaoqian Xu, Zhi Li, Baoxin Li, Zhimin Du, Yanjie Lu, Xu Gao, and Baofeng Yang

The present study was designed to investigate the cardiac benefits of M3 muscarinic receptor (M3-mAChR) overexpression and whether these effects are related to the regulation of the inward rectifying K+ channel by microRNA-1 (miR-1) in a conditional overexpression mouse model. A cardiac-specific M3-mAChR transgenic mouse model was successfully established for the first time in this study using microinjection, and the overexpression was confirmed by both reverse transcriptase–polymerase chain reaction and Western blot techniques. We demonstrated that M3-mAChR overexpression dramatically reduced the incidence of arrhythmias and decreased the mortality in a mouse model of myocardial ischemia-reperfusion (I/R). By using whole-cell patch techniques, M3-mAChR overexpression significantly shortened the action potential duration and restored the membrane repolarization by increasing the inward rectifying K+ current. By using Western blot techniques, M3-mAChR overexpression also rescued the expression of the inward rectifying K+ channel subunit Kir2.1 after myocardial I/R injury. This result was accompanied by suppression of upregulation miR-1. We conclude that M3-mAChR overexpression reduced the incidence of arrhythmias and mortality after myocardial I/R by protecting the myocardium from ischemia in mice. This effect may be mediated by increasing the inward rectifying K+ current by downregulation of arrhythmogenic miR-1 expression, which might partially be a novel strategy for antiarrhythmias, leading to sudden cardiac death.

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Posted by Leah Caracappa on Nov 8, 2011 12:00 AM CST
Ling Li, Zongyu Miao, Rui Liu, Mengliu Yang, Hua Liu, and Gangyi Yang

Liraglutide is a glucagonlike peptide (GLP)-1 analog that reduces blood glucose levels, increases insulin secretion and improves insulin sensitivity through mechanisms that are not completely understood. Therefore, we aimed to evaluate the metabolic impact and underlying mechanisms of liraglutide in a hypoadiponectinemia and high-fat diet (HFD)-induced insulin resistance (IR) model. Adiponectin gene targeting was achieved using adenovirus-transduced RNAi and was used to lower plasma adiponectin levels. Liraglutide (1 mg/kg) was given twice daily for 8 wks to HFD-fed apolipoprotein (Apo)E–/– mice. Insulin sensitivity was examined by a hyperinsulinemic-euglycemic clamp. Gene mRNA and protein expressions were measured by quantitative real-time polymerase chain reaction (PCR) and Western blot, respectively. Administration of liraglutide prevented hypoadiponectinemia-induced increases in plasma insulin, free fatty acids, triglycerides and total cholesterol. Liraglutide also attenuated hypoadiponectinemia-induced deterioration in peripheral and hepatic insulin sensitivity and alterations in key regulatory factors implicated in glucose and lipid metabolism. These findings demonstrated for the first time that liraglutide could be used to rescue IR induced by hypoadiponectinemia and HFD via regulating gene and protein expression involved in glucose and lipid metabolism.

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Posted by Leah Caracappa on Nov 7, 2011 12:00 AM CST
Victoria Catalán, Javier Gómez-Ambrosi, Amaia Rodríguez, Beatriz Ramírez, Fernando Rotellar, Victor Valentí, Camilo Silva, María J Gil, José Manuel Fernández-Real, Javier Salvador, and Gema Frühbeck

Calprotectin has been recently described as a novel marker of obesity. The aim of this study was to determine the circulating concentrations and expression levels of calprotectin subunits (S100A8 and S100A9) in visceral adipose tissue (VAT), exploring its impact on insulin resistance and inflammation and the effect of weight loss. We included 53 subjects in the study. Gene expression levels of the S100A8/A9 complex were analyzed in VAT as well as in both adipocytes and stromovascular fraction cells (SVFCs). In addition, circulating calprotectin and soluble receptor for the advanced glycation end product (sRAGE) concentrations were measured before and after weight loss achieved by Roux-en-Y gastric bypass (RYGB) (n = 26). Circulating concentrations and VAT expression of S100A8/A9 complex were increased in normoglycemic and type 2 diabetic obese patients (P < 0.01) and associated with markers of inflammation (P < 0.01). Oppositely, concentrations of sRAGE were significantly lower (P < 0.001) in both obese groups compared to lean volunteers. Elevated calprotectin levels in obese patients decreased (P < 0.00001) after RYGB, whereas sRAGE concentrations tended to increase. Calprotectin was mainly expressed by SVFCs, and its expression was significantly correlated (P < 0.01) with mRNA levels of the monocyte-macrophage–related molecules macrophage-specific antigen CD68 (CD68), monocyte chemotactic protein 1 (MCP1), integrin α-M (CD11B), and NADPH oxidase 2 (NOX2). Tumor necrosis factor-α treatment significantly enhanced (P < 0.05) the mRNA levels of S100 calcium-binding protein A8 (S100A8) of human visceral adipocytes. The increased levels of calprotectin in obesity and obesity-associated type 2 diabetes, its positive association with inflammation as well as the higher expression levels in the SVFCs in VAT suggests a potential role of this protein as a chemotactic factor in the recruitment of macrophages to VAT, increasing inflammation and the development of obesity-associated comorbidities.

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Posted by Leah Caracappa on Nov 6, 2011 12:00 AM CDT
James L Wynn, Natalie Z Cvijanovich, Geoffrey L Allen, Neal J Thomas, Robert J Freishtat, Nick Anas, Keith Meyer, Paul A Checchia, Richard Lin, Thomas P Shanley, Michael T Bigham, Sharon Banschbach, Eileen Beckman, and Hector R Wong

Septic shock is a frequent and costly problem among patients in the pediatric intensive care unit (PICU) and is associated with high mortality and devastating survivor morbidity. Genome-wide expression patterns can provide molecular granularity of the host response and offer insight into why large variations in outcomes exist. We derived whole-blood genome-wide expression patterns within 24 h of PICU admission from children with septic shock. We compared the transcriptome between septic shock developmental-age groups defined as neonates (≤28 d, n = 17), infants (1 month to 1 year, n = 62), toddlers (2–5 years, n = 54) and schoolage (≥6 years, n = 47) and age-matched controls. Direct intergroup comparisons demonstrated profound changes in neonates, relative to older children. Neonates with septic shock demonstrated reduced expression of genes representing key pathways of innate and adaptive immunity. In contrast to the largely upregulated transcriptome in all other groups, neonates exhibited a predominantly downregulated transcriptome when compared with controls. Neonates and school-age subjects had the most uniquely regulated genes relative to controls. Age-specific studies of the host response are necessary to identify developmentally relevant translational opportunities that may lead to improved sepsis outcomes.

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Supplementary Data
Posted by Leah Caracappa on Nov 5, 2011 12:00 AM CDT
Mu-Yan Cai, Zhu-Ting Tong, Wei Zhu, Zhu-Zhi Wen, Hui-Lan Rao, Ling-Ling Kong, Xin-Yuan Guan, Hsiang-Fu Kung, Yi-Xin Zeng, and Dan Xie

Trimethylation of lysine 27 on histone H3 (H3K27me3) is an epigenetic change which plays a critical role in tumor development and/or progression. However, the molecular status of H3K27me3 and its clinicopathologic/prognostic significance in nasopharyngeal carcinoma (NPC) have not been elucidated. In this study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to examine the expression of H3K27me3 protein in NPC tissues and nonneoplastic nasopharyngeal epithelial tissues. Receiver operating characteristic (ROC) curve analysis was used to determine the cutpoint for H3K27me3 high expression. High expression of H3K27me3 could be observed in 127/209 (60.8%) of NPCs and in 8/50 (16.0%) normal nasopharyngeal epithelial tissues (P < 0.001). Further correlation analysis demonstrated that high expression of H3K27me3 was positively associated with tumor later T classification, tumor metastasis, advanced clinical stage and chemoradioresistance (P < 0.05). Moreover, high expression of H3K27me3 was closely associated with NPC patient shortened survival time as evidenced by univariate and multivariate analysis (P < 0.05). Consequently, a new clinicopathologic prognostic model with three poor prognostic factors (H3K27me3 expression, distant metastasis and treatment regimen) was constructed. The model could stratify risk significantly (low, intermediate and high) for overall survival and progression-free survival (P < 0.0001). These findings provide evidence that H3K27me3 expression, as examined by IHC, has the potential to be used as an immunomarker to predict NPC chemoradiotherapy response and patient prognosis. The combined clinicopathologic prognostic model may become a useful tool for identifying NPC patients with different clinical outcomes.

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Posted by Leah Caracappa on Nov 4, 2011 12:00 AM CDT

Michalis V Karamouzis and Athanasios G Papavassiliou

It has long been shown that many of the presently used anticancer drugs exert their effects partly through modulating the activity of vital transcription factors. The intricacy of transcriptional regulation still represents the main obstacle for the design of transcription factor–directed agents. Systematic mapping of tumor-specific transcriptional networks and application of new molecular tools have reinforced research interest and efforts in this venue. The case of breast cancer is discussed as a representative example.

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Posted by Leah Caracappa on Nov 3, 2011 12:00 AM CDT
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