Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of ß2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis.

OBJECTIVE: IgG aPL against domain I of ß2-glycoprotein I (ß2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS. METHODS: Two polyclonal IgG fractions were isolated from serum of a patient with APS, both with high aPL activity but differing in aDI activity (aDI-rich and aDI-poor). These IgG fractions were tested for their pathogenic ability in an in vivo mouse model of thrombosis. Male CD1 mice were injected intraperitoneally with either aDI-rich or aDI-poor IgG; as a control, IgG isolated from healthy serum was used. A pinch injury was applied to the right femoral vein and thrombus dynamics and tissue factor activity in isolated tissue were evaluated. RESULTS: Both aDI-rich and aDI-poor IgG retained aCL and anti-ß2GPI activity, while only aDI-rich IgG displayed high aDI activity, as defined by our in-house cut-offs for positivity in each assay. aDI-rich IgG induced significantly larger thrombi in vivo compared with aDI-poor IgG (P < 0.0001). Similarly, aDI-rich IgG significantly enhanced the procoagulant activity of carotid artery endothelium and peritoneal macrophages isolated from experimental animals (P < 0.01). CONCLUSION: These data directly demonstrate that the ability to cause thrombosis in vivo is concentrated in the aDI fraction of aPL.

Changes in regulation of human monocyte proteins in response to IgG from patients with antiphospholipid syndrome.

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.

Regulation of myocardial interleukin-6 expression by p53 and STAT1.

Cardiovascular diseases are a major cause of morbidity and mortality worldwide. The interferon inducible transcriptional activator signal transducer and activator of transcription-1 (STAT1) and p53 are two critical transcriptional factors that have pivotal roles in cardiac biology and pathology. Here we describe a novel interplay between these two key players that critically regulate the levels of the pleiotropic interleukin 6 (IL6) in the heart. We provide in vivo evidence to demonstrate that, in cardiac tissues, STAT1 is a positive regulator of IL6 expression and it competes with the suppressive effect of p53 to sustain basal IL6 levels. Induction of IL6 expression in response to interferon gamma (IFNγ), a well-characterized activator of STAT1, parallels that of STAT1 phosphorylation and induction of STAT1 target genes, such as the interferon regulatory factor-1 (IRF-1), major histocompatibility complex class II transactivator (C2ta), and ß2-microglobulin (B2m). Furthermore, hearts from STAT1 knockout mice fail to induce IL6 expression in response to IFNγ. More importantly, we showed that this regulatory system is not functional in mouse embryonic fibroblasts, suggesting that activation of IL6 expression by STAT1 may be tissue specific. IL6 is a major effector of inflammation and cardiac hypertrophy, two major processes involved in heart failure, and therefore, understanding the molecular mechanisms regulating IL6 expression will enable better therapies and treatments for cardiovascular disease patients.

Signal transducer and activator of transcription-1 localizes to the mitochondria and modulates mitophagy.

The signal transducer and activator of transcription (STAT) proteins are latent transcription factors that have been shown to be involved in cell proliferation, development, apoptosis, and autophagy. STAT proteins undergo activation by phosphorylation at tyrosine 701 and serine 727 where they translocate to the nucleus to regulate gene expression. STAT1 has been shown to be involved in promoting apoptotic cell death in response to cardiac ischemia/reperfusion and has recently been shown by our laboratory to be involved in negatively regulating autophagy. These processes are thought to promote cell death and restrict cell survival leading to the generation of an infarct. Here we present data that shows STAT1 localizes to the mitochondria and co-immunoprecipitates with LC3. Furthermore, electron microscopy studies also reveal mitochondria from ex vivo I/R treated hearts of STAT1KO mice contained within a double membrane autophagosome indicating that STAT1 may be involved in negatively regulating mitophagy. This is the first description of STAT1 being localized to the mitochondria and also having a role in mitophagy.

Endothelial microparticle release is stimulated in vitro by purified IgG from patients with the antiphospholipid syndrome.

IgG antiphospholipid antibodies (aPL) exert direct effects on various cell types, contributing to the pathogenesis of thrombosis and pregnancy morbidity in patients with the antiphospholipid syndrome (APS). Some IgG samples from these patients activate endothelial cells (EC) in vitro as judged by surface expression of adhesion molecules such as E-selectin, which can promote thrombosis. Endothelial microparticles (EMP), which themselves are potentially prothrombotic, are released by activated EC. Though elevated circulating EMP levels have been reported in patients with APS, it is not known whether these EMP are released due to a direct effect of aPL on the cells. We tested the effect of purified polyclonal IgG from patients with APS (APS-IgG) and healthy controls (HC-IgG) upon cultured human umbilical vein EC (HUVEC). HUVEC exposed to APS-IgG produced significantly more EMP than those exposed to HC-IgG (p=0.0036) and a greater proportion of these EMP carried surface E-selectin (6.2% ± 4.0 for APS-IgG vs. 3.4% ± 2.0 for HC IgG, p=0.0172). This study therefore demonstrates that purified polyclonal APS-IgG can drive EMP release. We propose that EMP generation may be a useful measure of aPL-mediated pathogenic effects upon EC.

Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury.

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-1ß, 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.

Autophagy in the stress-induced myocardium.

Cardiovascular disease is a leading cause of death worldwide, particularly in Western societies. During an ischaemic insult, ventricular pressure from the heart is diminished as a result of cardiac myocyte death by necrosis and apoptosis. Autophagy is a process whereby cells catabolise intracellular proteins in order to generate ATP in times of stress such as nutrient starvation and hypoxia. Emerging evidence suggests that autophagy plays a positive role in cardiac myocyte survival during periods of cellular stress performing an important damage limitation function. By promoting cell survival, cardiac myocyte loss is reduced thereby minimising the potential of heart failure. In contrast, it has been reported that autophagy can also be a form of cell death. By considering the various animal models of autophagy, we examine the role of the Signal Transducers and Activator of Transcription (STAT) proteins in the autophagic response. Additionally we review the role of the tumour suppressor, p53 and its family member p73 and their potential role in the autophagic response.

The role of minocycline in ischemia-reperfusion injury: a comprehensive review of an old drug with new implications.

Minocycline is a semi-synthetic tetracycline that inhibits bacterial protein synthesis and hence is used for the treatment of many infectious diseases. Over the years, many other interesting properties of minocycline have been identified and been used to make patents which include anti-inflammatory, anti-apoptotic, matrix metalloproteinase inhibitor and free oxygen radical scavenger activity. Ischemia-reperfusion injury is a concern for almost every clinical specialty and minocycline seems to be an attractive cytoprotective agent that can ameliorate the damage due to these properties. Ischemia-reperfusion injury is a complex process and involves various pathways that lead to cell death. This review focuses on the body of evidence describing various proposed mechanisms of action of minocycline and its current experimental use in various animal models of ischemia-reperfusion injury.

Transcriptional modulation of heat-shock protein gene expression.

Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

Transgenic overexpression of HSP56 does not result in cardiac hypertrophy nor protect from ischaemia/reperfusion injury.

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.

STAT1 interacts directly with cyclin D1/Cdk4 and mediates cell cycle arrest.

In response to IFN-γ, the latent cytoplasmic STAT1 protein is tyrosine phosphorylated and translocates to the nucleus where it transactivates STAT1-responsive genes. We now present data that shows that STAT1 has additional non-transcriptional functions. We first demonstrate that STAT1 can interact directly with the G1 cell cycle regulatory cyclin D1 and CDK4 proteins, suggesting a role for STAT1 in G1 cell cycle regulation. Acute IFN-γ treatment dramatically reduced cyclin D1 protein expression and the interaction of STAT1 with cyclin D1. The IFN-γ-induced reduction in cyclin D1 was dependent on the proteasome pathway. Interestingly, the STAT1 serine 727 phosphorylation site and not the STAT1 tyrosine 701 site is required for cyclin D1-dependent proteosomal degradation. Furthermore, IFN-γ-STAT1 cyclin D1 reduction correlated with decreased amount of p-Rb Ser-795, cyclin E and increased amounts of the cell cycle inhibitors p27(Kip1) and p21(Cip1). Finally, STAT1 deficient cells not only proliferate at a greater rate, but have enhanced phosphorylated pRb-(S795), cyclin E and reduced p27(Kip1) and p21(Cip1). Our results suggest that there is a time-dependent hierarchy of events following IFN-γ-STAT1 which begins with the rapid reduction of cyclin D1 levels that is dependent on STAT1 directly interacting with the cyclin D1/Cdk4 complex. This is then followed by a later sustained up-regulation of p27(Kip1) and p21(Cip1) that may be dependent on STAT1 transcriptional activity. Thus, these results highlight a dual role of STAT1 that may require both its non-transcriptional as well as it known transcriptional function.

STAT3 modulates the DNA damage response pathway.

The STAT3 transcription factor is well known to function as an anti-apoptotic factor, especially in numerous malignancies. Recently we showed that STAT3 is cytoprotective and that cells lacking STAT3 are more sensitive to oxidative stress. A key feature of oxidative stress involves activation of the DNA damage pathway. However, a role for STAT3 or its contribution in response to DNA damage has not been described. In the present study we show that cells lacking STAT3 are less efficient in repairing damaged DNA. Moreover, STAT3 deficient cells show reduced activity of the ATM-Chk2 and ATR-Chk1 pathways, both important pathways in sensing DNA damage. Finally we show that MDC1, a regulator of the ATM-Chk2 pathway and facilitator of the DNA damage response, is modulated by STAT3 at the transcriptional level. These findings demonstrate that STAT3 is necessary for efficient repair of damaged DNA, partly by modulating the ATM-Chk2 and ATR-Chk1 pathways.

Cell-specific regulation of the pro-survival Brn-3b transcription factor by microRNAs.

We have previously shown that the Brn-3b transcription factor is subjected to post-transcriptional gene regulation by specific microRNAs (mir-23 and mir-214) in the ND7 and SHSY-5Y neuronal cell lines (Calissano et al., 2007). As Brn-3b plays an essential role in the survival of retinal ganglion cells in the rat (Erkman et al., 1996; Gan et al., 1996; Gan et al., 1999; Erkman et al., 2000), we wanted to investigate whether mir-23 and mir-214 are expressed and target Brn-3b mRNA in a retinal ganglion cell line (RGC-5) thus potentially killing the cells expressing it. Here we show that, possibly due to its pro-survival role, Brn-3b is protected from degradation by microRNAs in RGC-5 cells in contrast to its fate in other cell types. This seems to be accomplished by i) the lack of expression of one of the two microRNAs targeting its 3'UTR and by ii) the requirement of at least two distinct microRNAs to mediate its down-regulation in retinal ganglion cells. We speculate that this mechanism could have a widespread role in the regulation of mRNAs encoding for essential proteins.

Effects of polyclonal IgG derived from patients with different clinical types of the antiphospholipid syndrome on monocyte signaling pathways.

A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs, p38 MAPK, and NF-kappaB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 microg/ml IgG for 6 h, and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-kappaB. To further investigate intracellular signaling pathways induced by these IgGs, specific inhibitors of p38 MAPK, NF-kappaB, TLR4, and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM-) caused phosphorylation of NF-kappaBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT-/PM+), anti-phospholipid Ab-positive patients without APS, or healthy controls. TF upregulation caused by the VT+/PM- samples was reduced by inhibitors of p38 MAPK, NF-kappaB, and TLR4. The effects of VT+/PM- IgG on signaling and TF upregulation were concentrated in the fraction that bound beta-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-kappaB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4.

New targets of urocortin-mediated cardioprotection.

The urocortin (UCN) hormones UCN1 and UCN2 have been shown previously to confer significant protection against myocardial ischaemia/reperfusion (I/R) injury; however, the molecular mechanisms underlying their action are poorly understood. To further define the transcriptional effect of UCNs that underpins their cardioprotective activity, a microarray analysis was carried out using an in vivo rat coronary occlusion model of I/R injury. Infusion of UCN1 or UCN2 before the onset of reperfusion resulted in the differential regulation of 66 and 141 genes respectively, the majority of which have not been described previously. Functional analysis demonstrated that UCN-regulated genes are involved in a wide range of biological responses, including cell death (e.g. X-linked inhibitor of apoptosis protein), oxidative stress (e.g. nuclear factor erythroid derived 2-related factor 1/nuclear factor erythroid derived 2-like 1) and metabolism (e.g. Prkaa2/AMPK). In addition, both UCN1 and UCN2 were found to modulate the expression of a host of genes involved in G-protein-coupled receptor (GPCR) signalling including Rac2, Gnb1, Dab2ip (AIP1), Ralgds, Rnd3, Rap1a and PKA, thereby revealing previously unrecognised signalling intermediates downstream of CRH receptors. Moreover, several of these GPCR-related genes have been shown previously to be involved in mitogen-activated protein kinase (MAPK) activation, suggesting a link between CRH receptors and induction of MAPKs. In addition, we have shown that both UCN1 and UCN2 significantly reduce free radical damage following myocardial infarction, and comparison of the UCN gene signatures with that of the anti-oxidant tempol revealed a significant overlap. These data uncover novel gene expression changes induced by UCNs, which will serve as a platform to further understand their mechanism of action in normal physiology and cardioprotection.

Estrogen and non-genomic upregulation of voltage-gated Na(+) channel activity in MDA-MB-231 human breast cancer cells: role in adhesion.

External (but not internal) application of beta-estradiol (E2) increased the current amplitude of voltage-gated Na(+) channels (VGSCs) in MDA-MB-231 human breast cancer (BCa) cells. The G-protein activator GTP-gamma-S, by itself, also increased the VGSC current whilst the G-protein inhibitor GDP-beta-S decreased the effect of E2. Expression of GPR30 (a G-protein-coupled estrogen receptor) in MDA-MB-231 cells was confirmed by PCR, Western blot and immunocytochemistry. Importantly, G-1, a specific agonist for GPR30, also increased the VGSC current amplitude in a dose-dependent manner. Transfection and siRNA-silencing of GPR30 expression resulted in corresponding changes in GPR30 protein expression but only internally, and the response to E2 was not affected. The protein kinase A inhibitor, PKI, abolished the effect of E2, whilst forskolin, an adenylate cyclase activator, by itself, increased VGSC activity. On the other hand, pre-incubation of the MDA-MB-231 cells with brefeldin A (a trans-Golgi protein trafficking inhibitor) had no effect on the E2-induced increase in VGSC amplitude, indicating that such trafficking ('externalisation') of VGSC was not involved. Finally, acute application of E2 decreased cell adhesion whilst the specific VGSC blocker tetrodotoxin increased it. Co-application of E2 and tetrodotoxin inhibited the effect of E2 on cell adhesion, suggesting that the effect of E2 was mainly through VGSC activity. Pre-treatment of the cells with PKI abolished the effect of E2 on adhesion, consistent with the proposed role of PKA. Potential implications of the E2-induced non-genomic upregulation of VGSC activity for BCa progression are discussed.

Green tea polyphenol epigallocatechin gallate inhibits cell signaling by inducing SOCS1 gene expression.

Therapeutic effects of green tea involve an inhibitory function of its constituent polyphenol epigallocatechin gallate (EGCG) on cell signaling. The specificity and mechanism(s) by which EGCG inhibits cell signaling have remained unclear. Here, we demonstrate that green tea and EGCG induce suppressor of cytokine signaling 1 (SOCS1) gene expression, a negative regulator of specific cell signaling pathways. In mouse immune cells, EGCG induces SOCS1 expression via an oxidative (superoxide) pathway and activation of the signal transducer and activator of transcription 5 transcription factor. EGCG inhibited SOCS1-regulated cell signaling, but this inhibitory effect was abrogated in cells deficient in SOCS1. These findings identify a mechanism by which EGCG inhibits cell signaling with specificity, mediated by induction of the negative regulator SOCS1.