James T. Colston

Regulation of CCAAT/Enhancer Binding Protein, Interleukin-6, Interleukin-6 Receptor, and gp130 Expression During Myocardial Ischemia/Reperfusion

BACKGROUND Interleukin (IL)-6 is elevated in myocardium after ischemia and reperfusion. The IL-6 promoter/enhancer region contains response elements for nuclear factor-kappaB, AP-1, and CCAAT/enhancer binding protein (C/EBP). Expression and regulation of C/EBP in rat myocardium after ischemia and reperfusion has not been defined, nor has the behavior of the specific IL-6 receptor (IL-6R) or the signal transducer gp130. METHODS AND RESULTS C/EBP DNA binding activity was not detected in shams or in previously ischemic tissue at 15 minutes of reperfusion; it was detected at 30 minutes of reperfusion, increased at 1 hour of reperfusion, and declined by 6 hours of reperfusion. A supershift was observed with anti-C/EBP-beta but not with anti-alpha or anti-delta antibodies. mRNA and protein levels of IL-6 and gp130 were detected at low levels in controls, increased at 1 hour of reperfusion, and remained high until 6 hours of reperfusion. IL-6R mRNA and protein were not detected in controls, but its mRNA was induced at 1 hour of reperfusion and its protein at 2 hours of reperfusion. Although effects of reperfusion were rapid, in ischemic tissue not reperfused, low levels of C/EBP were detected at 4 hours, and at 24 hours the levels were slightly elevated. Significant upregulation in IL-6, IL-6R, and gp130 occurred only at 24 hours of sustained ischemia. CONCLUSIONS Reperfusion after a brief period of ischemia caused induction of myocardial C/EBP (beta-subunit). The rapid and sustained production of IL-6 with concomitant expression of IL-6 receptor and gp130 suggest that these factors may participate in a local inflammatory cascade after myocardial ischemia and reperfusion.

Inhibition of nuclear factor κB attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium

We have previously reported that induction of nuclear factor-kappa B (NF-kappa B) occurs in a biphasic manner in postischemic myocardium. Because interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-alpha), and inducible nitric-oxide synthase (iNOS) contain kappa B-response elements, and since transforming growth factor-beta 1 (TGF-beta 1) down-modulates both cytokine and iNOS expression, we studied their temporal expression during myocardial ischemia/reperfusion (I/R). Northern and Western analyses showed low levels of IL-6 and no signal for IL-1 beta, TNF-alpha and iNOS under basal conditions. Their expression rose significantly over sham-operated controls by 1 h reperfusion, and persisted high for various periods. Under basal conditions, low levels of TGF-beta 1 were detected, which rose significantly at 3 h reperfusion, and remained high until 24 h reperfusion. Administration of diethyldithiocarbamate (DDC) inhibited induction of NF-kappa B and concomitantly the expression of IL-1 beta, IL-6, TNF-alpha as well as iNOS. However, expression of TGF-beta was not altered. Our results indicate that ischemia/reperfusion induces NF-kappa B, and upregulates kappa B-response genes. Administration of DDC inhibits NF-kappa B levels, and attenuates expression of inflammatory cytokines and iNOS.

TNF-α and H2O2 induce IL-18 and IL-18Rβ expression in cardiomyocytes via NF-κB activation

Abstract Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-α induced rapid and sustained activation of NF-κB whereas H 2 O 2 induced delayed and transient activation. Both TNF-α and H 2 O 2 induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-α led to sustained expression. Expression of IL-18Rβ, but not α, was induced by both agonists. TNF-α and H 2 O 2 induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-α and H 2 O 2 induced IL-18 and IL-18Rβ, but not basal expression of IL-18Rα. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-κB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.

H2O2 activates Nox4 through PLA2-dependent arachidonic acid production in adult cardiac fibroblasts

Stimulated production of reactive oxygen species (ROS) by plasma membrane‐associated nicotinamide adenine dinucleotide phosphate oxidases (Nox) in non‐phagocytic cells regulates a number of biological processes including growth, vessel tone, and oxygen sensing. The purpose of this study was to investigate H2O2‐stimulated ROS production in primary adult cardiac fibroblasts (CF). Results demonstrate that CF express an H2O2‐inducible oxidant generating system that is inhibitable by diphenylene iodonium (DPI) and sensitive to antioxidants. In addition to H2O2, generation of ROS was stimulated potently by 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and arachidonic acid (AA) in a protein kinase C‐independent manner. Pretreatment with arachidonyl trifluoromethyl ketone was nearly as effective as DPI at reducing H2O2‐ and OAG‐stimulated oxidant generation indicating a central role for phospholipase A2 (PLA2) in this signaling pathway. Co‐stimulation with H2O2 and OAG did not increase ROS generation as compared to OAG alone suggesting both agonists signal through a shared, rate‐limited enzymatic pathway involving PLA2. Co‐stimulation with H2O2 and AA had additive effects indicating these two agonists stimulate oxidant production through a parallel activation pathway. Reverse transcriptase‐coupled polymerase chain reaction and Western blotting demonstrate primary cardiac fibroblasts express transcripts and protein for Nox4, p22, p47, and p67 phox. Transfections with Nox4 small inhibitory ribonucleic acid oligonucleotides or p22 phox antisense oligonucleotides significantly downregulated stimulated Nox activity. Inhibitors of nitric oxide synthases were without effect. We conclude adult CF express Nox4/p22 phox‐containing oxidant generating complex activated by H2O2, OAG, and AA through a pathway that requires activation of PLA2.

Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis

OBJECTIVE The extent to which apoptosis contributes to myocyte cell loss during acute carditic viral infection is unknown. To assess whether apoptosis occurs in acute viral myocarditis, and how it is modulated, we studied mice inoculated with coxsackievirus B3 (CVB3). METHODS Five CD1 and C3H.HeJ (C3H) mice/group were sacrificed as saline vehicle-injected controls, and at 1, 2, and 3 weeks post-inoculation (p.i.) with 5 x 10(6) pfu CVB3. Histopathological status and terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assays quantified inflammation, necrosis and apoptosis in myocardium. Apoptosis-related protein immunoreactivity defined presence and location of Bax, Fas, Fas Ligand (FasL), Bcl-2, interleukin-1 beta converting enzyme (ICE), inducible nitric oxide synthase (iNOS) and the proto-oncogene p53. RESULTS Both strains exhibited significant histopathology at all time points. Saline-injected control animals showed no signs of inflammation and no significant difference in apoptosis-related protein immunoreactivity was observed between strains. Myocardial TUNEL-positive cells were exceedingly rare though apoptosis was present in thymic medulla and spleen follicles. Pro-apoptotic proteins Bax, Fas, and FasL were present in all groups though no clear correlation with histopathology was apparent. By contrast, the anti-apoptotic protein Bcl-2 showed mild immunoreactivity in controls, which increased following infection and correlated well with histopathological scores in both strains. Myocardial iNOS immunoreactivity displayed a similar though weaker staining pattern to Bcl-2 over the 3 week study period in both strains. Neither ICE nor p53 immunoreactivity could be demonstrated in myocardium. CONCLUSION Thus, despite marked inflammatory activity, myocyte apoptosis is rare in acute CVB3 myocarditis in CD1 and C3H.HeJ mice.

Contractile depression and expression of proinflammatory cytokines and iNOS in viral myocarditis

We assessed two strains of mice [CD-1 and C3H.HeJ (C3H)] with different responses to coxsackievirus B3 (CVB3) infection at 7, 14, and 21 days after inoculation with 105 pfu of CVB3. CD-1 mice developed inflammatory lesions at 7 days that nearly recovered by 21 days; C3H mice demonstrated persistence of infiltrates. Although there were differences in the baseline fractional shortening, it was further reduced at 7 and 14 days in both strains. It recovered in CD-1 mice but remained depressed at 21 days in C3H mice. Interleukin-6 and tumor necrosis factor-α transcripts were increased in both strains at 7 days. Levels dropped to near control in CD-1 mice at 21 days but remained elevated in C3H mice. Interleukin-1β was minimally elevated in CD-1 mice but increased progressively in C3H mice. mRNA for the inducible form of NO synthase (iNOS) was increased at 7 days in the CD-1 mice, returning to baseline by 14 days; it rose progressively in C3H mice, with a fivefold increase at 21 days. We conclude that mice infected with CVB3 show increased expression of proinflammatory cytokines as well as iNOS associated with reduced contractile performance. In more susceptible mice, contractile depression and cytokine and iNOS expression are more pronounced.

Induction of proinflammatory cytokine and antioxidant enzyme gene expression following brief myocardial ischaemia

The purpose of this study was to determine if proinflammatory cytokines are up‐regulated during reperfusion following sublethal ischaemia, and whether concurrent up‐regulation of antioxidant enzymes occurs. Open‐chest rats were subjected to 15 min of ischaemia followed by 1 or 3 h reperfusion (R). Myocardium from the ischaemic zone showed a significantly higher (P < 0.01) generation of thiobarbituric acid‐reactive substances at 1 h and 3 h R. Northern blots showed a weak signal in controls for IL‐6 mRNA (0.13 ± 0.01); this was elevated to 0.68 ± 0.12 at 1 h and 0.69 ± 0.10 at 3 h R. Neither IL‐1β nor tumour necrosis factor‐alpha (TNF‐α) were detectable in controls. IL‐1β rose to 0.78 ± 0.07 at 1 h and 0.51 ± 0.08 at 3 h R, and TNF‐α rose to 0.69 ± 0.10 at 1 h and 0.38 ± 0.15 at 3 h R. Western blotting showed no signals in the control, but readily detectable signals at 1 h R; these remained high (IL‐6) or decreased (IL‐1β and TNF‐α) at 3 h R. mRNA analysis for antioxidant enzymes revealed a weak signal in controls for catalase (CAT; 0.16 ± 0.08), glutathione peroxidase (GSH‐Px; 0.15 ± 0.06) and superoxide dismutase (SOD; 0.21 ± 0.05). After 1 h R, levels increased significantly for CAT (0.46 ± 0.10; P < 0.025) and GSH‐Px (0.51 ± 0.13; P < 0.01), but remained similar to controls for SOD (0.26 ± 0.15). At 3 h R the mRNA levels were significantly elevated for the three enzymes (CAT 0.48 ± 0.13; GSH‐Px 0.47 ± 0.10; SOD 0.54 ± 0.08). We conclude that mRNA for proinflammatory cytokines is expressed early in reperfusion, and that the proteins are present in heart tissue. Also, reperfusion is associated with rapid expression of genes for antioxidant enzymes, which may enhance reactive oxygen intermediate (ROI) scavenging.

Carbon monoxide donors or heme oxygenase-1 (HO-1) overexpression blocks interleukin-18-mediated NF-κB–PTEN-dependent human cardiac endothelial cell death

The objective of this study was to determine whether heme oxygenase-1 (HO-1) or heme metabolites exert cytoprotective effects on interleukin-18-mediated endothelial cell (EC) death. Treatment with interleukin (IL)-18 increased NF-kappaB activation and PTEN induction, suppressed Akt activation, and stimulated EC death. While ectopic expression of p65 enhanced PTEN transcription, adenoviral transduction of dnIkappaB-alpha, dnp65, or dnIKKbeta was inhibitory. Furthermore, IL-18 suppressed HO-1 mRNA expression via enhanced mRNA degradation. Overexpression of HO-1, treatment with HO-1 inducer hemin, or the CO donor cobalt (III) protoporphyrin IX all reversed IL-18-mediated NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Furthermore, hemin induced HO-1 expression, and HO-1 knockdown, HO-1 inhibition, or CO scavengers all reversed the prosurvival effects of hemin. In addition, the CO donors CORM-1 and CORM-3 and the heme metabolites biliverdin and bilirubin attenuated IL-18-induced EC death via a similar signaling pathway. IL-18 induced p38alpha MAPK activation, and suppressed p38beta isoform expression. While p38alpha knockdown attenuated, p38beta knockdown potentiated IL-18-mediated EC death. Hemin and HO-1 reversed IL-18-mediated p38alpha induction and restored p38beta levels. These results demonstrate that IL-18 suppresses HO-1 expression and induces EC death. HO-1 overexpression, HO-1 induction, or treatment with heme metabolites all reverse IL-18-mediated p38alpha MAPK and NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Thus, HO-1 inducers and CO donors may have the therapeutic potential to effectively block IL-18 signaling and reduce IL-18-dependent vascular injury and inflammation.

A Novel Peroxide-induced Calcium Transient Regulates Interleukin-6 Expression in Cardiac-derived Fibroblasts

Reperfusion of ischemic myocardium leads to a local burst of free radicals, increased [Ca2+] i , and the release of proinflammatory cytokines. The purpose of this study was to determine whether brief exposure of cardiac fibroblasts to H2O2 is associated with transient changes in [Ca2+] i levels and whether this stimulus is sufficient to induce interleukin-6 (IL-6) expression. Cardiac derived fibroblasts were isolated from adult male rats and cultured under standard conditions. Individual coverslip-attached fibroblasts were loaded with the calcium probe Fura-2/AM and exposed to a single 3-min pulse of 100 μmH2O2. In addition, low passage cultures were exposed to a pulse of H2O2 and assayed for IL-6 expression. A brief exposure of H2O2 led to a large intracellular Ca2+ transient with a mean transient magnitude of 318 ± 28 nm (mean ± S.D.,n = 12). Stimulation in the absence of [Ca2+] o led to a 59% reduction in mean transient magnitude (129 ± 23 nm, n = 10,p < 0.001), whereas pretreatment with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C resulted in a 37% reduction (199 ± 25 nm, n = 10, p < 0.01). Cells treated with xestospongin C and stimulated in the absence of [Ca2+] o did not exhibit a Ca2+ transient. Time-dependent IL-6 release was significantly elevated by 4 h (368 ± 64 pg/mg protein, p < 0.01) and increased further by 24 h (1030 ± 76 pg/mg protein). The depletion of cellular Ca2+ by pretreatment with thapsigargin in the absence of [Ca2+] o attenuated H2O2-induced IL-6 mRNA expression while blocking protein release. These data show that the exposure of cardiac fibroblasts to a brief pulse of physiological levels of H2O2 resulted in a large Ca2+transient with intracellular and extracellular Ca2+contributions. Furthermore, brief H2O2 exposure led to calcium-dependent IL-6 expression.

Induction of nuclear factor κB but not κB-responsive cytokine expression during myocardial reperfusion injury after neutropenia

Abstract Neutrophils may contribute to myocardial ischemia/reperfusion (I/R) injury by generating reactive oxygen intermediates (ROIs). ROIs activate nuclear factor (NF)-κB, which regulates genes for cytokines with negative inotropic effects (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α). We investigated the impact of neutrophil depletion on NF-κB DNA binding activity, and expression of these cytokines during myocardial I/R injury. Male WKY rats (n = 28) received a single dose of antineutrophil antiserum (i/v). Twenty two hours later, when the peripheral blood neutrophil counts were profoundly decreased (94% reduction), the animals underwent 15 min of left anterior descending coronary artery ligation followed by reperfusion for 0.25, 0.5, 1, 2, 3, and 6 h (n = 4/group). Saline-treated animals underwent a similar protocol, and served as controls (n = 28, 4/group). Neutrophil accumulation, defined by myeloperoxidase activity, was present in controls, but not in anti-PMN antisera-treated animals (at least p