Christopher J. Carroll

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-κB 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 μg/ml IgG for 6 h, and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-κB. To further investigate intracellular signaling pathways induced by these IgGs, specific inhibitors of p38 MAPK, NF-κB, 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-κBand 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-κB, and TLR4. The effects of VT+/PM− IgG on signaling and TF upregulation were concentrated in the fraction that bound β-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-κB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4.

Cardioprotection mediated by urocortin is dependent on PKCepsilon activation.

Urocortin (Ucn) is an endogenous cardioprotective agent that protects against the damaging effects of ischemia and reperfusion injury in vitro and in vivo. We have found that the mechanism of action of Ucn involves both acute activation of specific target molecules, and using Affymetrix (Santa Clara, CA) gene chip technology, altered gene expression of different end effector molecules. Here, from our gene chip data, we show that after a 24 h exposure to Ucn, there was a specific increase in mRNA and protein levels of the protein kinase C epsilon (PKCε) isozyme in primary rat cardiomyocytes compared with untreated cells and in the Langendorff perfused ex vivo heart. Furthermore, a short 10 min exposure of these cells to Ucn caused a specific translocation/activation of PKCε in vitro and in the Langendorff perfused ex vivo heart. The importance of the PKCε isozyme in cardioprotection and its relationship to cardioprotection produced by Ucn was assessed using PKCε‐specific inhibitor peptides. The inhibitor peptide, when introduced into cardiomyocytes, caused an increase in apoptotic cell death compared with control peptide after ischemia and reperfusion. When the inhibitor peptide was present with Ucn, the cardioprotective effect of Ucn was lost. This loss of cardioprotection by Ucn was also seen in whole hearts from PKCε knockout mice. These findings indicate that the cardioprotective effect of Ucn is dependent upon PKCε.

Urocortin protects cardiac myocytes from ischemia/reperfusion injury by attenuating calcium-insensitive phospholipase A2 gene expression.

We have used Affymetrix gene chip technology to look for changes in gene expression caused by a 24 h exposure of rat primary neonatal cardiac myocytes to the cardioprotective agent urocortin. We observed a 2.5‐fold down‐regulation at both the mRNA and protein levels of a specific calcium‐insensitive phospholipase A2 enzyme. Levels of lysophosphatidylcholine, a toxic metabolite of phospholipase A2, were lowered by 30% in myocytes treated with urocortin for 24 h and by 50% with the irreversible iPLA2 inhibitor bromoenol lactone compared with controls. Both 4 h ischemia and ischemia followed by 24 h reperfusion caused a significant increase in lysophosphatidylcholine concentration compared with controls. When these myocytes were pretreated with urocortin, the ischemia‐induced increase in lysophosphatidylcholine concentration was significantly lowered. Moreover, co‐incubation of cardiac myocytes with urocortin, or the specific phospholipase A2 inhibitor bromoenol lactone, reduces the cytotoxicity produced by lysophosphatidylcholine or ischemia/reperfusion. Similarly, in the intact heart ex vivo we found that cardiac damage measured by infarct size was significantly increased when lysophoshatidylcholine was applied during ischemia, compared with ischemia alone, and that pre‐treatment with both urocortin and bromoenol lactone reversed the increase in infarct size. This, to our knowledge, is the first study linking the cardioprotective effect of urocortin to a decrease in a specific enzyme protein and a subsequent decrease in the concentration of its cardiotoxic metabolite.

The transcriptional coactivator p300 plays a critical role in the hypertrophic and protective pathways induced by phenylephrine in cardiac cells but is specific to the hypertrophic effect of urocortin

Anacardic acid is an alkylsalicylic acid obtained from cashew‐nut‐shell liquid, and is a potent inhibitor of p300 histone acetyl‐transferase (HAT) activity. We have used anacardic acid to prevent the induction of hypertrophy in isolated neonatal rat cardiomyocytes. Hypertrophy was detected as an increase in cell size, the rearrangement of sarcomeres into a striated pattern, and the induction of embryonic genes β‐MHC and ANF. p300 inhibition was equally effective at preventing hypertrophy whether it was induced by treatment with the α1‐adrenergic agonist, phenylephrine, or by treatment with urocortin, a member of the corticotrophin‐releasing‐factor family, which stimulates specific G protein‐coupled receptors. Spiruchostatin A is a natural‐product inhibitor of histone deacetylases (HDAC) similar to the depsipeptide FK228 molecule. We have recently synthesized spiruchostatin A and now show that, although HDACs act in opposition to HATs, spiruchostatin A has the same effect as anacardic acid, that is, it prevents the induction of hypertrophy in response to phenylephrine or urocortin. Pretreatment with either phenylephrine or urocortin reduced the extent of death observed after the exposure of isolated cardiomyocytes to simulated ischaemia and reoxygenation. Inhibition of p300 or HDAC activity eliminated the protection conferred by phenylephrine; however, it did not affect the protection conferred by urocortin. Therefore, it might eventually be possible to use chemical inhibitors such as these in a therapeutic setting to dissociate the protective effect and hypertrophic effect of urocortin, enhancing the survival of cardiomyocytes exposed to transient ischemia, while inhibiting the hypertrophic pathway that would otherwise be induced concurrently.

STAT1 regulates p73-mediated Bax gene expression

Although signal transducer and activator of transcription 1 (STAT1) mediated regulation of p53 transcription and apoptosis has been previously reported, modulation of other members of the p53 family of transcription factors remains poorly understood. In this study, we found that STAT1 and TA‐p73 can interact directly and that p73‐mediated Bax promoter activity was observed to be reduced by STAT1 expression in a p53‐independent manner for which STAT1 Tyrosine‐701 and Serine‐727 are key residues. This study presents the first report physically linking STAT1 and TA‐p73 signalling and highlights the modulation of the Bax promoter in the context of IFN‐γ stimulation.

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 β2-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.

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.