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The effects of immunoglobulin G (IgG) from patients with the antiphospholipid syndrome (APS) upon monocyte activation have not been fully characterized. We carried out a comprehensive proteomic analysis of human monocytes treated with IgG from patients with different manifestations of the APS. Using 2-dimensional differential gel electrophoresis (2D DiGE), 4 of the most significantly regulated proteins (vimentin [VIM], zinc finger CCH domain-containing protein 18, CAP Gly domain-containing linker protein 2, and myeloperoxidase) were differentially regulated in monocytes treated with thrombotic or obstetric APS IgG, compared with healthy control (HC) IgG. These findings were confirmed by comparing monocytes isolated from APS patients and HC. Anti-VIM antibodies (AVAs) were significantly increased in 11 of 27 patients (40.7%) with APS. VIM expression on HC monocytes was stimulated more strongly by APS IgG from patients with higher-avidity serum AVA. We further characterized the proteome of thrombotic APS IgG-treated monocytes using a label-free proteomics technique. Of 12 proteins identified with the most confidence, 2 overlapped with 2D DiGE and many possessed immune response, cytoskeletal, coagulation, and signal transduction functions which are all relevant to APS and may therefore provide potential new therapeutic targets of this disease., (© 2014 by The American Society of Hematology.)

Background: IL-17A and IL-17F are pro-inflammatory cytokines which induce the expression of several cytokines, chemokines and matrix metalloproteinases (MMPs) in target cells. IL-17 cytokines have recently attracted huge interest due to their pathogenic role in diseases such as arthritis and inflammatory bowel disease although a role for IL-17 cytokines in myocardial infarction (MI) has not previously been described., Methods: In vivo MI was performed by coronary artery occlusion in the absence or presence of a neutralizing IL-17 antibody for blocking IL-17 actions in vivo. IL-17 signaling was also assessed in isolated primary cardiomyocytes by Western blot, mRNA expression and immunostaining., Results: Expression of IL-17A, IL-17F and the IL-17 receptor (IL-17RA) were all increased following MI. Expression of several IL-17 target genes, including Cxcl1, Cxcl2, IL-1β, iNOS and IL-6 was also upregulated following MI. In addition, IL-17A promoted the expression of Cxcl1 and IL-6 in isolated cardiomyocytes in a MAPK and PI(3)K-dependent manner. IL-17A and ischaemia/reperfusion (I/R) injury were found to have an additive effect on Cxcl1 expression, suggesting that IL-17 may enhance myocardial neutrophil recruitment during MI. Moreover, protein levels of both IL-17R and IL-17A were enhanced following in vivo MI. Finally, blocking IL-17 signaling in vivo reduced the levels of apoptotic cell death markers following in vivo MI., Conclusions: These data imply that the expression of IL-17 cytokines and their receptor are elevated during myocardial I/R injury and may play a fundamental role in post infarct inflammatory and apoptotic responses., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Heat shock proteins are known to be induced during and following different forms of cardiac stress. It has previously been shown that their expression is beneficial for the heart following trauma such as ischaemia-reperfusion (I/R) injury. Heat shock protein 56 (HSP56) belongs to the family of FK506-binding immunophilin proteins and is found in steroid receptor complexes, notably the glucocorticoid receptor. We have previously shown that HSP56 and other HSPs are induced in cardiac myocytes treated with cardiotrophin-1, a cytokine with potent hypertrophic and protective properties on cardiac cells. The hypertrophic action of cardiotrophin-1 on cardiac cells is dependent on HSP56 induction and overexpression of HSP56 is sufficient for inducing hypertrophy in cardiac cells. To investigate this phenomenon in vivo, we have generated transgenic mice overexpressing HSP56 and assessed them for the development cardiac hypertrophy and resistance of their hearts to I/R-injury by Langendorff perfusion. Mice generated demonstrated stable, yet varying expression levels of HSP56. Initial characterisation identified a sex-specific phenotype where male overexpressing mice exhibited a moderate, but significant, reduced body weight compared to wild-type controls. In ex vivo stress analyses we found, unexpectedly, that significant overexpression of HSP56 does not induce myocardial hypertrophy and nor does it protect the intact heart from I/R-injury. These observations now suggest a more intricate HSP56-Sp. Cardiophenotype that requires further studies to determine if HSP56 is necessary in mediating hypertrophy induced by other myocardial stimuli., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The transcription factor STAT1 plays a role in promoting apoptotic cell death, whereas the related STAT3 transcription factor protects cardiac myocytes from ischemia/reperfusion (I/R) injury or oxidative stress. Cytokines belonging to the IL-6 family activate the JAK-STAT3 pathway, but also activate other cytoprotective pathways such as the MAPK-ERK or the PI3-AKT pathway. It is therefore unclear whether STAT3 is the only cytoprotective mediator against oxidative stress-induced cell death. Overexpression of STAT3 in primary neonatal rat ventricular myocytes (NRVM) protects against I/R-induced cell death. Moreover, a dominant negative STAT3 adenovirus (Ad ST3-DN) enhanced apoptotic cell death (81.2+/-6.9%) compared to control infected NRVM (46.0+/-3.1%) following I/R. Depletion of STAT3 sensitized cells to apoptotic cell death following oxidative stress. These results provide direct evidence for the role of STAT3 as a cytoprotective transcription factor in cells exposed to oxidative stress.

The Brn-3 family of transcription factors play a critical role in regulating expression of genes that control cell fate, including the small heat shock protein Hsp27. The aim of this study was to investigate the relationship between Brn-3a and Brn-3b and Hsp27 expression in the developing rodent heart. Brn-3a and Brn-3b were detected from embryonic days 9.5-10.5 (E9.5-E10.5) in the mouse heart, with significant increases seen later during development. Two isoforms (long and short) of each protein were detected during embryogenesis and postnatally. Brn-3a messenger RNA (mRNA) and protein were localized by E13.0 to the atrio-ventricular (AV) valve cushions and leaflets, outflow tract (OFT), epicardium and cardiac ganglia. By E14.5, Brn-3a was also localised to the septa and compact ventricular myocardium. An increase in expression of the long Brn-3a(l) isoform between E17 and adult coincided with a decrease in expression of Brn-3b(l) and a marked increase in expression of Hsp27. Hearts from Brn-3a-/- mice displayed a partially penetrant phenotype marked by thickening of the endocardial cushions and AV valve leaflets and hypoplastic ventricular myocardium. Loss of Brn-3a was correlated with a compensatory increase in Brn-3b and GATA3 mRNA but no change in Hsp27 mRNA. Reporter assays in isolated cardiomyocytes demonstrated that both Brn-3a and Brn-3b activate the hsp27 promoter via a consensus Brn-3-binding site. Therefore, Brn-3 POU factors may play an important role in the development and maintenance of critical cell types and structures within the heart, in part via developmental regulation of myocardial Hsp27 expression. Furthermore, Brn-3a may be necessary for correct valve and myocardial remodelling and maturation.

Background: We have recently reported that pretreatment of rats with endotoxin (lipopolysaccharide, LPS) and selective agonists of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) protect the kidney against ischemia/reperfusion (I/R) injury. Here we investigate the hypothesis that the renoprotective effects of LPS may be due to an enhanced formation of endogenous ligands of PPARgamma, rather than an up-regulation of PPARgamma expression., Methods: Rats were pretreated with LPS (1 mg/kg, IP, 24 hours prior to ischemia) in the absence (control) or presence of the selective PPARgamma antagonist GW9662 (1 mg/kg, IP, 24 and 12 hours prior to ischemia). Twenty-four hours after injection of LPS, rats were subjected to 60 minutes of bilateral renal ischemia, followed by 6 hours of reperfusion. Serum and urinary indicators of renal injury and dysfunction were measured, specifically serum creatinine, aspartate aminotransferase, and gamma-glutamyl-transferase, creatinine clearance, urine flow, and fractional excretion of sodium. Kidney PPARgamma1 mRNA levels were determined by reverse transcriptase-polymerase chain reaction., Results: Pretreatment with LPS significantly attenuated all markers of renal injury and dysfunction caused by I/R. Most notably, GW9662 abolished the protective effects of LPS. Additionally, I/R caused an up-regulation of kidney PPARgamma1 mRNA levels compared to sham animals, which were unchanged in rats pretreated with LPS., Conclusion: We document here for the first time that endogenous ligands of PPARgamma may contribute to the protection against renal I/R injury afforded by LPS pretreatment in the rat.

The UCN homologues SCP and SRP bind specifically to the CRFR2 receptor, whereas UCN binds to both CRFR1 and CRFR2. We have previously demonstrated that all three peptides are cardioprotective, and both the Akt and MAPK p42/44 pathways are essential for this effect. Here we tested the hypertrophic effects of these peptides. We examined the effects of the peptides on cell area, protein synthesis, and induction of the natriuretic peptides ANP and BNP. All three peptides were able to increase all the markers of hypertrophy examined, with SCP being the most potent of the three, followed by UCN and SRP last. In addition, we provide a mechanism of action for the three peptides and show that Akt phosphorylation is important for their hypertrophic action, whereas MAPK p42/44 is not involved in this effect.

BAG-1 is a multi-functional protein that exists as three functionally distinct and differentially localized isoforms which originate from a single mRNA and interact with a wide range of cellular targets. These include heat shock proteins, nuclear hormone receptors, signalling molecules, the anti-apoptotic BCL-2 protein and components of the ubiquitylation/proteasome machinery. Overexpression of BAG-1 isoforms has been demonstrated to regulate apoptosis, proliferation, transcription, metastasis and cell motility in a wide variety of cell systems. Since BAG-1 has a role in many biological pathways there is increasing evidence supporting the view that BAG-1 is an important molecule in disease, for example, potentially modulating both cell survival and response to nuclear hormones in breast cancer, and BAG-1 is a potential molecular target for therapeutic intervention.

We have previously shown, using Affymetrix gene chip technology, that urocortin induces the expression of several diverse genes in cardiac myocytes. An ATP sensitive inwardly rectifying potassium channel, Katp (Kir6.1), the enzyme calcium independent phospholipase A2 (iPLA2), and protein kinase C epsilon (PKCepsilon) and that these genes are involved in the cardioprotective mechanism of action of urocortin. Here we demonstrate that these gene products are localized to cardiac myocyte mitochondria and for the first time show that urocortin protects cardiac myocytes from ischaemia/reperfusion induced cell death by preventing mitochondrial damage. Using pharmacological agents to Katp channels and iPLA2 and synthetic peptide inhibitors of PKCepsilon, we go on to demonstrate that these three gene products are involved in the urocortin induced protection of cardiac myocyte mitochondria. These proteins may interact at the mitochondria to produce the protective effect.

The ability of CREB binding protein (CBP) and p300 co-activators to stimulate transcription has previously been shown to be enhanced by treatment of cardiac cells with the hypertrophic agent phenylephrine (PE). This effect is dependent on activation of the mitogen activated protein kinase pathway (p42/44 MAPK). Here, we demonstrate the first identification of potential phosphorylation sites targeted by PE within the proteins CBP and p300. We show that serine 2015 of CBP and serine 89 of p300 are necessary for PE to stimulate the transcriptional activity of these proteins. Furthermore, we have shown that PE is capable of mediating phosphorylation of endogenous p300 at serine 89. This phosphorylation mediated regulation of CBP and p300 suggests a potential signal transduction pathway for the induction of cardiac cell hypertrophy by PE.

Overexpression of heat shock protein (Hsp) 70 and Hsp27 in vivo was proclaimed as a potential tool in therapy of ischemia-reperfusion injury. However, it was so far not known whether these Hsps can beneficially act when increased in cells just at the stage of postischemic reperfusion. This issue was examined in a model of ischemia-reperfusion stress when cultures of endothelial cells (EC) from human umbilical vein were infected with virus-based vectors expressing Hsp70 or Hsp27, or Hsp56, or green fluorescent protein (GFP) and exposed to 20 hours of hypoxia followed by reoxygenation. The infection was performed either 10 hours before hypoxia or immediately after hypoxia, or at different time points of reoxygenation. Only low cell death was detected during hypoxia, but later, up to 40% of the treated cells died via caspase-dependent apoptosis between 6 and 12 hours of reoxygenation. The percentage of apoptotic cells was 1.6- to 3-fold greater in Hsp56- and GFP-infected EC than in Hsp70- or Hsp27-infected EC. The last 2 groups exhibited a lesser extent of procaspase-9 and procaspase-3 activation within 6-9 hours of reoxygenation. The cytoprotective effects of overexpressed Hsp70 and Hsp27 were observed not only in the case of infection before hypoxia but also when EC were infected at the start of reoxygenation or 1-2 hours later. An increase in the Hsp70 and Hsp27 levels in infected EC correlated well with their resistance to apoptosis under reoxygenation. These findings suggest that overexpression of Hsp70 or Hsp27, if it occurs in the involved cells at the early stage of postischemic reperfusion, can still be cytoprotective.

The novel hydroxylamine derivative, bimoclomol, has been shown previously to act as a co-inducer of several heat shock proteins (Hsp-s), enhancing the amount of these proteins produced following a heat shock compared to heat shock alone. Here we show that the co-inducing effect of bimoclomol on Hsp expression is mediated via the prolonged activation of the heat shock transcription factor (HSF-1). Bimoclomol effects are abolished in cells from mice lacking HSF-1. Moreover, bimoclomol binds to HSF-1 and induces a prolonged binding of HSF-1 to the respective DNA elements. Since HSF-1 does not bind to DNA in the absence of stress, the bimoclomol-induced extension of HSF-1/DNA interaction may contribute to the chaperone co-induction of bimoclomol observed previously. These findings indicate that bimoclomol may be of value in targeting HSF-1 so as to induce up-regulation of protective Hsp-s in a non-stressful manner and for therapeutic benefit.

Peripheral blood mononuclear cells (PBMCs) represent a significant target for gene delivery both for therapeutic and experimental purposes. Thus far however, it has proved difficult to develop vectors capable of high efficient gene delivery to unstimulated PBMCs. We have tested a range of different vectors derived from herpes simplex virus (HSV) which differ in their degree of disablement in terms of their gene delivery efficiency to unstimulated human PBMCs and ability to deliver a reporter gene. None of the viruses had any significant toxic effect in PBMCs. However, optimal gene delivery to unstimulated PBMCs was obtained with a semidisabled virus lacking functional genes encoding ICP34.5 and Vmw65 which was more efficient than either nondisabled or more extremely disabled viruses. Expression of green fluorescent protein (GFP) with this virus was observed in up to 50% of PBMCs 1 day after infection, and reporter gene expression was detectable by Western blotting and immunofluorescence at undiminished levels at the longest time points tested, up to 5 days after infection. This optimised HSV vector may thus represent an effective tool for gene delivery to unstimulated PBMCs in culture.

Urocortin (Ucn) is a 40 amino acid peptide which is closely related to corticotrophin-releasing factor (CRF). It is expressed in specific regions of the brain but is also detectable in other organs notably the heart. Although some of the effects of Ucn in the nervous system such as enhanced anxiety and activity mimic those of CRF, Ucn is a much more potent suppressor of appetite/feeding behaviour. Moreover, Ucn has much more potent effects on the cardiovascular system than CRF, including enhanced cardiac contractility/heart rate and enhanced resistance of cardiac cells to injury induced, for example, by ischaemia/reperfusion. This suggests Ucn may play a key role in the response of the cardiovascular system to stress. In addition, Ucn represents a novel cardioprotective agent which may be of therapeutic use in treating the damaging effects of cardiac ischaemia and subsequent reperfusion.

The Brn-3a POU domain transcription factor is able to regulate the transcription of promoters containing a Brn-3 response element via its POU domain. In addition, the POU domain of Brn-3a has been shown to functionally interact with the estrogen receptor and regulate transcription from estrogen responsive promoters. The steroid receptor coactivator, Src-1, enhances transcription with a variety of steroid receptors. Here we describe a functional interaction between Brn-3a and Src-1. In glutathione S-transferase pull-down assays Src-1 was shown to specifically interact with Brn-3 proteins. Moreover, Src-1 co-immunoprecipitated from intact cells with Brn-3a. The transactivation potential of the Brn-3a/Src-1 complex was tested on both the Brn-3 responsive SNAP-25 promoter and the estrogen responsive vitellogenin promoter, in each of two different cell lines, the neuronal ND7 cell line, and the kidney BHK21 cell line. Src-1 consistently and strongly potentiated the activation of Brn-3a on the SNAP promoter construct in both the ND7 and BHK21 cell lines. The vitellogenin promoter construct, however, was only weakly activated by the Brn-3/Src-1 complex in the ND7 cells and there was even less effect on this promoter in the BHK21 cells. These results suggest a functional role for Src-1 in enhancing Brn-3a mediated transactivation, seemingly independent of nuclear hormone receptors, thus broadening the transcriptional repertoire of both Brn-3a and Src-1., ((c) 2002 Elsevier Science (USA).)