Hot off the presses! May 01 J Mol Cell Cardiol

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Latest Articles Include:

• Editorial Board
  - J Mol Cell Cardiol 46(5):i (2009)
  
• Multiple protective pathways against reperfusion injury: A SAFE path without Aktion?
  - J Mol Cell Cardiol 46(5):607-609 (2009)
  
• Regulation of myofibroblast differentiation by convergence of the Wnt and TGF-β1/Smad signaling pathways
  - J Mol Cell Cardiol 46(5):610-611 (2009)
  
• CB2 cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion
  - J Mol Cell Cardiol 46(5):612-620 (2009)
  Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB2 cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB2 selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27% ± 1.91) as co! mpared to vehicle-treated mice (31.77% ± 2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-α, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-α induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils.
• Transcriptional regulation of SM22α by Wnt3a: Convergence with TGFβ1/Smad signaling at a novel regulatory element
  - J Mol Cell Cardiol 46(5):621-635 (2009)
  The role of canonical Wnt signaling in myofibroblast biology has not been fully investigated. The C3H10T1/2 mesenchymal cell line recapitulates myofibroblast differentiation in vitro and in vivo, including SM22α expression. Using this model, we find that Wnt3a upregulates SM22α in concert with TGFβ1. Wnt1, Wnt5a and BMP2 could not replace Wnt3a and TGFβ1 signals. Chromatin immunoprecipitation identified that Wnt3a enhances both genomic SM22α histone H3 acetylation and β-catenin association, hallmarks of transcriptional activation. By analyzing a series of SM22α promoter-luciferase (LUC) reporter constructs, we mapped Wnt3a-regulated DNA transcriptional activation to nucleotides − 213 to − 192 relative to the transcription initiation site. In gel shift assays, DNA–protein complexes assembled on this element were disrupted with antibodies to β-catenin, Smad2/3, and TCF7, confirming the participation of known Wnt3a and TGFβ transcriptional mediators. Mutati! on of a CAGAG motif within this region abrogated recognition by these DNA binding proteins. Wnt3a treatment increased Smad2/3 binding to this element. Mutation of the cognate within the context of the native 0.44 kb SM22α promoter resulted in a 70% decrease in transcription, and reduced Wnt3a + TGFβ1 induction. A concatamer of SM22α [− 213 to − 192] conveyed Wnt3a + TGFβ1 activation to the unresponsive RSV promoter. Dominant negative TCF inhibited SM22α [− 213 to − 192]x6 RSVLUC activation. Moreover, ICAT (inhibitor of β-catenin and TCF) decreased while TCF7L2 and β-catenin enhanced 0.44 kb SM22α promoter induction by Wnt3a + TGFβ1. RNAi "knockdown" of β-catenin inhibited Wnt3a induction of SM22α. Thus, Wnt/β-catenin signaling interacts with TGFβ/Smad pathways to control SM22α gene transcription.
• In situ co-distribution and functional interactions of SAP97 with sinoatrial isoforms of HCN channels
  - J Mol Cell Cardiol 46(5):636-643 (2009)
  The sinoatrial node is a region of specialized cardiomyocytes that is responsible for the repetitive activity of the adult heart. The sinoatrial node is heavily innervated compared to the other regions of the heart, and the specialized cardiomyocytes of this region receive neural and hormonal input from the autonomic nervous system, which leads to changes in heart rate. A key regulator of sinoatrial beating frequency in response to autonomic input is the hyperpolarization-activated cyclic nucleotide gated (HCN) channel, a mixed cationic channel whose activity is increased by the binding of cAMP to its cytoplasmic side. HCN channels localize to distinct regions or "hot spots" on the cell surface of sinoatrial myocytes, but how these regions are formed, whether they correspond to specific signaling domains and the specific HCN isoforms and other proteins therein are not known. In this paper, we show that both HCN2 and HCN4 isoforms co-distribute with the adapter prot! ein SAP97, an important component of distinct punctae in the sinoatrial node of the rabbit heart. HCN4, but not HCN2, also co-distributes with the post-synaptic marker β-catenin, thus identifying diverse organized domains within this tissue. Furthermore, we show, using heterologous expression systems, whole-cell patch clamp electrophysiology and imaging, that SAP97 interacts functionally with HCN in a manner that depends upon the PDZ compatible binding motif of the C-terminus, but that its effects on If behaviour are HCN isoform and context dependent. Together, the data suggest that SAP97 contributes to isoform specific organization of HCN channels within specific domains in the sinoatrial node of the rabbit.
• Normal impulse propagation in the atrioventricular conduction system of Cx30.2/Cx40 double deficient mice
  - J Mol Cell Cardiol 46(5):644-652 (2009)
  Connexin (Cx) 30.2, Cx40 and Cx45 containing gap junctional channels contribute to electrical impulse propagation through the mouse atrioventricular node (AV-node). The cross talk in between these Cxs may be of great importance for AV-nodal conduction. We generated Cx30.2/Cx40 double deficient mice (Cx30.2LacZ/LacZCx40−/−) and analyzed the relative impact of Cx30.2 and Cx40 on cardiac conductive properties in vivo by use of electrophysiological examination. Cx30.2LacZ/LacZCx40−/− mice exhibited neither obvious cardiac malformations nor impaired contractile function. In surface-ECG analyses, Cx30.2LacZ/LacZCx40−/− and Cx40 deficient animals (Cx40−/−) showed significantly longer P-wave durations, PQ-intervals and prolonged QRS-complexes relative to wildtype littermates (WT). Cx30.2-deficient mice (Cx30.2LacZ/LacZ) developed shorter PQ-intervals as compared to WT, Cx40−/− or Cx30.2/Cx40 double deficient mice. Intracardiac evaluation of the atria-His (A! H) and His-ventricle (HV) intervals representing supra and infra-Hisian conduction yielded significant acceleration of supra-Hisian conductivity in Cx30.2LacZ/LacZ (AH: 28.2 ± 4.3 ms) and prolongation of infra-Hisian conduction in Cx40−/− mice (HV: 13.7 ± 2.6 ms). These parameters were unchanged in the Cx30.2LacZ/LacZCx40−/− mice (AH: 37.3 ± 5.5 ms, HV: 11.7 ± 2.6 ms), which exhibited AV-nodal and ventricular conduction times similar to WT animals (AH: 35.9 ± 4.4 ms, HV: 10.5 ± 1.9 ms). We conclude that the remaining Cx45 gap junctional channels are sufficient to maintain electrical coupling and cardiac impulse propagation in the AV-node and proximal ventricular conduction system in mice. We suggest that Cx30.2 and Cx40 act as counterparts in the AV-node and His-bundle, decreasing or increasing, respectively, electrical coupling and conduction velocity in these areas.
• Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes
  - J Mol Cell Cardiol 46(5):653-662 (2009)
  Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as dem! onstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.
• Endothelium-targeted overexpression of constitutively active FGF receptor induces cardioprotection in mice myocardial infarction
  - J Mol Cell Cardiol 46(5):663-673 (2009)
  Fibroblast growth factor receptor (FGFR) is expressed in a variety of cells and is involved in their proliferation/migration/survival. To elucidate FGFR-mediated specific action of vascular endothelial cells (ECs) on myocardial ischemia, we generated endothelium-targeted transgenic mice overexpressing constitutively active FGFR2 using Tie2 promoter (FGFR2-Tg). Infarct size, vessel formation and blood perfusion were significantly improved 28 days after myocardial infarction (MI) in FGFR2-Tg, compared with wild-type mice. Aortic ECs isolated from FGFR-Tg showed a marked increase in migratory capacity and tube formation. These in vitro angiogenic activities were blocked by PI3-kinase inhibitor. Whereas, parameters obtained from echocardiography were already improved at three days after MI. Cardiomyocyte apoptosis at the ischemic border zone was decreased in FGFR2-Tg (32.1%, p < 0.05) and cardiac mRNA expression of FGF2 (basic FGF) was also up-regulated (142%, p < 0.05) at! 3 days after MI. 1% oxygen-mediated apoptosis was significantly inhibited in FGFR2-Tg-ECs and this inhibition was abolished by PI3-kinase inhibitor. FGFR2-Tg-ECs exposed to 1% oxygen exhibited enhanced phosphorylation of 416-Tyr-Src, 473-Ser-Akt, and HIF1α accumulation. The production of FGF2 was enhanced 2.1-fold in FGFR-Tg-ECs under 1% oxygen via the Src/Akt/HIF1α pathway, which induced the peri-vessel migration of vascular smooth muscle cells (VSMCs) and anti-apoptotic effects on VSMCs and cardiomyocytes. FGF receptor signaling in ECs promoted migration, survival and autocrine production of FGF2, leading to reduced infarct size, which is associated with anti-apoptotic action in the early stage and with enhanced angiogenesis in the late stage after MI.
• The A-kinase anchor protein AKAP121 is a negative regulator of cardiomyocyte hypertrophy
  - J Mol Cell Cardiol 46(5):674-681 (2009)
  Pathologic cardiac hypertrophy imposes a significant clinical burden on patients, yet the precise intracellular mechanisms responsible for its induction are only partially understood. We examined a potential role for AKAP121 to regulate cardiomyocyte hypertrophy, since recent reports have implicated other AKAPs in this process. We report here that knockdown of AKAP121 expression in isolated neonatal rat cardiomyocytes results in pronounced cellular hypertrophy. Loss of AKAP121 expression is associated with dephosphorylation and nuclear localization of NFATc3, a downstream effector of the hypertrophic phosphatase calcineurin. We also demonstrate that over-expression of AKAP121 in cardiac myocytes reduces basal cell size, and blocks hypertrophy induced by isoproterenol, indicating that AKAP121 negatively regulates the hypertrophic process. Co-immunoprecipitation data indicates that AKAP121 and calcineurin directly interact. Our findings are consistent with a model in whi! ch loss of AKAP121 expression leads to the release of an active pool of calcineurin, in turn causing nuclear translocation of NFATc3 and activation of the hypertrophic gene program. These results are the first to identify AKAP121 as a negative regulator of cardiomyocyte hypertrophy, and highlight AKAP121 as a potential target for therapeutic exploitation.
• Novel signaling pathways promote a paracrine wave of prostacyclin-induced vascular smooth muscle differentiation
  - J Mol Cell Cardiol 46(5):682-694 (2009)
  The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the pr! ostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.
• Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome
  - J Mol Cell Cardiol 46(5):695-703 (2009)
  Recent studies have demonstrated an association between mutations in CACNA1c or CACNB2b and Brugada syndrome (BrS). Previously described mutations all caused a loss of function secondary to a reduction of peak calcium current (ICa). We describe a novel CACNB2b mutation associated with BrS in which loss of function is caused by accelerated inactivation of ICa. The proband, a 32 year old male, displayed a Type I ST segment elevation in two right precordial ECG leads following a procainamide challenge. EP study was positive with induction of polymorphic VT/VF. Interrogation of implanted ICD revealed brief episodes of very rapid ventricular tachycardia. He was also diagnosed with vasovagal syncope. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 15 ion channel genes were amplified and sequenced. The only mutation uncovered was a missense mutation (T11I) in CACNB2b. We expressed WT or T11I CACNB2b in TSA201 cells co-transfected with WT CACNA1c and! CACNA2d. Patch clamp analysis showed no significant difference between WT and T11I in peak ICa density, steady-state inactivation or recovery from inactivation. However, both fast and slow decays of ICa were significantly faster in mutant channels between 0 and + 20 mV. Action potential voltage clamp experiments showed that total charge was reduced by almost half compared to WT. We report the first BrS mutation in CaCNB2b resulting in accelerated inactivation of L-type calcium channel current. Our results suggest that the faster current decay results in a loss-of-function responsible for the Brugada phenotype.
• PKC activation and PIP2 depletion underlie biphasic regulation of IKs by Gq-coupled receptors
  - J Mol Cell Cardiol 46(5):704-712 (2009)
  KCNQ1 is co-assembled with KCNE1 subunits in the heart to form the cardiac delayed rectifier K+ current (IKs), which is one of the main currents responsible for myocyte repolarization. The most commonly inherited form of cardiac arrhythmias, long-QT syndrome type 1 (LQT1), is due to mutations on KCNQ1. Gq-coupled receptors (GqPCRs) are known to mediate positive inotropism in human ventricular myocardium. The mechanism of IKs current modulation by GqPCRs remains incompletely understood. Here we studied the molecular mechanisms underlying Gq regulation of the IKs channel. Heterologously expressed IKs (human KCNQ1/KCNE1 subunits) was measured in Xenopus oocytes, expressed together with GqPCRs. Our data from several GqPCRs shows that IKs is regulated in a biphasic manner, showing both an activation and an inhibition phase. Receptor-mediated inhibition phase was irreversible when recycling of agonist-sensitive pools of phosphatidylinositol-4,5-bisphosphate (PIP2) was blocke! d by the lipid kinase inhibitor wortmannin. In addition, stimulation of PIP2 production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PIP5-kinase), decreased receptor-mediated inhibition. The receptor-mediated activation phase was inhibited by the PKC inhibitor calphostin C and by a mutation in a putative PKC phosphorylation site in the KCNE1 subunit. Our results indicate that the depletion of membrane PIP2 underlies receptor-mediated inhibition of IKs and that phosphorylation by PKC of the KCNE1 subunit underlies the GqPCR-mediated channel activation.
• Cardioprotection against myocardial infarction with PTD-BIR3/RING, a XIAP mimicking protein
  - J Mol Cell Cardiol 46(5):713-718 (2009)
  The purpose of the present study was to investigate the potential cardioprotective effects of an original approach based on the properties of the X chromosome-linked Inhibitor of Apoptosis (XIAP), the most effective endogenous inhibitor of apoptosis. For this purpose, the C-terminal part of XIAP (BIR3 and RING domains) was fused to the protein transduction domain (PTD) of the HIV1 transactivator of transcription, which confers to fused protein the ability to cross cell membranes. This protein, so-called PTD-BIR3/RING, was administered intravenously in C57BL/6J mice subjected to 30 min coronary artery occlusion and 24 h of reperfusion. Administration of PTD-BIR3/RING at 5 min before and 30 min after the onset of reperfusion reduced infarct size vs control (23 ± 2% vs 41 ± 4% and 27 ± 4% vs 41 ± 3%, respectively, p < 0.05). Similar reduction in infarct size was observed when PTD-BIR3/RING was administered prior to ischemia (28 ± 1% vs 44 ± 3%). In addition to inhib! ition of caspase-3 and -9 activities, PTD-BIR3/RING induced an inhibition of caspase-8 and several other actors of the apoptotic pathways. In conclusion, this study demonstrates that the administration of PTD-BIR3/RING reduces myocardial infarct size even when injected during reperfusion through interruption of caspase activation by pharmacologically mimicking endogenous XIAP.
• Attenuation of experimental autoimmune myocarditis by blocking T cell activation through 4-1BB pathway
  - J Mol Cell Cardiol 46(5):719-727 (2009)
  4-1BB, a member of the tumor necrosis factor receptor (TNFR) family, binds the 4-1BB ligand (4-1BBL), works as a costimulatory molecule, and regulates T cell-mediated immune responses. Although inflammation is an essential pathological feature of myocarditis, the role of 4-1BB in experimental autoimmune myocarditis (EAM) remains unclear. Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish EAM. 4-1BB-immunoglobulin (4-1BBIg) was administered intraperitoneally (n = 6) a total of 9 times (3 times per week). Rats were killed on day 21 to study effects of 4-1BB/4-1BBL pathway blockade. For controls, isotype-matched human IgG was administered in other EAM rats (n = 6). Histologic and echocardiographic examination showed development of EAM attenuated by 4-1BBIg. Suppression of mRNA expression for IL-1α, IL-1β, IL-4, IL-6, and TNF-α was noted in the heart tissue treated with 4-1BBIg. Treatment with 4-1BBIg reduced production of Th1-type cyto! kines, and inhibited T cell proliferation in vitro. In the 4-1BB signaling pathway in splenocytes, 4-1BBIg suppressed JNK, p38, and IκB activity but not that of ERK1/2. Blockade of T cell activation through the 4-1BB/4-1BBL pathway regulates development of EAM; therefore, 4-1BB may be an effective target for treating myocarditis.
• Thymosin β4 mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo
  - J Mol Cell Cardiol 46(5):728-738 (2009)
  Hypoxic heart disease is a predominant cause of disability and death worldwide. Since adult mammalian hearts are incapable of regeneration after hypoxia, attempts to modify this deficiency are critical. As demonstrated in zebrafish, recall of the embryonic developmental program may be the key to success. Because thymosin β4 (TB4) is beneficial for myocardial cell survival and essential for coronary development in embryos, we hypothesized that it reactivates the embryonic developmental program and initiates epicardial progenitor mobilization in adult mammals. We found that TB4 stimulates capillary-like tube formation of adult coronary endothelial cells and increases embryonic endothelial cell migration and proliferation in vitro. The increase of blood vessel/epicardial substance (Bves) expressing cells accompanied by elevated VEGF, Flk-1, TGF-β, Fgfr-2, Fgfr-4, Fgf-17 and β-Catenin expression and increase of Tbx-18 and Wt-1 positive myocardial progenitors suggested o! rgan-wide recall of the embryonic program in the adult epicardium. TB4 also positively regulated the expression and phosphorylation of myristoylated alanine-rich C-kinase substrate (Marcks), a direct substrate and indicator of protein kinase C (PKC) activity in vitro and in vivo. PKC inhibition significantly reduced TB4 initiated epicardial thickening, capillary growth and the number of myocardial progenitors. Our results demonstrate that TB4 is the first known molecule capable of organ-wide activation of the embryonic coronary developmental program in the adult mammalian heart after systemic administration and that PKC plays a significant role in the process.
• The role of Akt/GSK-3β signaling in familial hypertrophic cardiomyopathy
  - J Mol Cell Cardiol 46(5):739-747 (2009)
  Mutations in cardiac troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). Transgenic mice expressing a missense mutation (R92Q) or a splice site donor mutation (Trunc) in the cardiac TnT gene have mutation-specific phenotypes but mice of both models have smaller hearts compared to wild type and exhibit hemodynamic dysfunction. Because growth-related signaling pathways in the hearts of mice expressing TnT mutations are not known, we evaluated the impact of increased Akt or glycogen synthase kinase-3β (GSK-3β) activity in both mutant TnT mice; molecules that increase heart size via physiologic pathways and block pathologic growth, respectively. Expression of activated Akt dramatically augments heart size in both R92Q and Trunc mice; however, this increase in heart size is not beneficial, since Akt also increases fibrosis in both TnT mutants and causes some pathologic gene expression shifts in the R92Q mice. Activated GSK-3β results in further de! creases in left ventricular size in both R92Q and Trunc hearts, but this decrease is associated with significant mutation-specific phenotypes. Among many pathologic consequences, activating GSK-3β in R92Q hearts decreases phosphorylation of troponin I and results in early mortality. In contrast, increased GSK-3β activity in Trunc hearts does not significantly impact cardiac phenotypes. These findings demonstrate that increased Akt and its downstream target, GSK-3β can impact both cardiac size and phenotype in a mutation-specific manner. Moreover, increased activity of these molecules implicated in beneficial cardiac phenotypes exacerbates the progression of disease in the R92Q TnT mutant.
• PPARγ enhances IFNγ-mediated transcription and rescues the TGFβ antagonism by stimulating CIITA in vascular smooth muscle cells
  - J Mol Cell Cardiol 46(5):748-757 (2009)
  Chronic inflammatory response and active vascular remodeling are two featured pathophysiological events during atherogenesis. Gamma interferon (IFN-γ) modulates these two processes through transcriptional control of major histocompatibility complex II (MHC II) and collagen type I (COL1A2) genes, mediated by class II transactivator (CIITA). Transforming growth factor (TGF-β) antagonizes the effect of IFN-γ in part by dampening the expression of CIITA. Here we report that peroxisome proliferator activated receptor γ (PPARγ) enhanced MHC II activation and COL1A2 repression by IFN-γ while rescuing the antagonism by TGF-β in a CIITA-dependent manner in human aortic smooth muscle cells judged by quantitative PCR and luciferase reporter assays. PPARγ exerted its effect by augmenting the levels of CIITA and stimulating CIITA recruitment to target promoters as evidenced by chromatin immunoprecipitation assays. The up-regulation of CIITA levels was the result of PPARγ-m! ediated transcriptional activation of CIITA through promoter IV, and increased CIITA protein stability. Thus, our data suggest that PPARγ could be a key factor in fine-tuning inflammation as well as restructuring of vessel walls during atherogenesis by acting as a "balance tipper" of the differential effects exerted by cytokines.
• The divergent roles of protein kinase C epsilon and delta in simulated ischaemia–reperfusion injury in human myocardium
  - J Mol Cell Cardiol 46(5):758-764 (2009)
  Experimental studies suggest that cardioprotection can be achieved through either the activation of PKC-var epsilon prior to the index ischaemic episode or the inhibition of PKC-δ at the onset of reperfusion. However, whether these PKC isoforms exert such divergent roles in human myocardium, subjected to simulated ischaemia–reperfusion injury, is unclear. Human atrial trabeculae were isolated from right atrial appendages harvested from patients undergoing elective cardiac surgery. These were subjected to 90 min of hypoxia followed by 120 min of reoxygenation, at the end of which the recovery of baseline contractile function was determined. Atrial trabeculae were randomised to receive various treatment protocols comprising a peptide activator of PKC-var epsilon, a peptide inhibitor of PKC-δ and their respective inactive control peptides. Administering the PKC-δ peptide inhibitor at reoxygenation improved the recovery of function at all the concentrations tested (39.3 ± 1.4% at 5 nM, 52.4 ± 2.9% at 50 nM and 46.8 ± 2.9% at 500 nM versus the control group, 27.5 ± 1.4%: N ≥ 6/group: P < 0.02). Preconditioning with the PKC-var epsilon peptide activator improved the recovery of function (40.0 ± 0.8% at 50 nM and 49.7 ± 3.1% at 500 nM versus the control group 27.5 ± 1.4%: N ≥ 6/group: P < 0.02). This cardioprotective effect was comparable to that achieved by a standard hypoxic preconditioning protocol (52.3 ± 3.2%). Interestingly, administering the PKC-var epsilon activator (500 nM) at the onset of reperfusion also improved the recovery of contractile function (40.7 ± 2.1% versus 27.5 ± 1.5%: N ≥ 6/group: P < 0.05). In human myocardium, cardioprotection can be achieved by either inhibiting PKC-δ or activating PKC-var epsilon at the onset of reperfusion. In addition, PKC-var epsilon activation offers cardioprotection when administered as a preconditioning strategy.