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Biomedical subjects

B D Shames

Publications and source records attributed to B D Shames.

At least 19 recordsLinked to original sources
PubMed2001

Suppression of tumor necrosis factor alpha production by cAMP in human monocytes: dissociation with mRNA level and independent of interleukin-10.

BACKGROUND: Elevation of cellular cAMP inhibits lipopolysaccharide(LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) production and increases the expression of interleukin (IL)-10 in mononuclear cells. TNF-alpha gene expression obligates activation of the transcription factor nuclear factor kappaB (NF-kappaB). Exogenous IL-10 inhibits NF-kappaB in monocytes and thus attenuates TNF-alpha production. We examined the role of endogenous IL-10 in the regulation of NF-kappaB activation and TNF-alpha production in human monocytes by cAMP. METHODS: Human monocytes were stimulated with Escherichia coli LPS (100 ng/ml) with and without forskolin (FSK, 50 microM) or dibutyryl cyclic AMP (dbcAMP, 100 microM). Cytokine (TNF-alpha and IL-10) release was measured by immunoassay. TNF-alpha mRNA was measured by reverse transcription polymerase chain reaction, and NF-kappaB DNA binding activity was assessed by gel mobility shift assay. RESULTS: cAMP-elevating agents inhibited LPS-stimulated TNF-alpha release (0.77 +/- 0.13 ng/10(6) cells in LPS + dbcAMP and 0.68 +/- 0.19 ng/10(6) cells in LPS + FSK, both P < 0.05 vs 1.61 +/- 0.34 ng/10(6) cells in LPS alone). Conversely, cAMP enhanced LPS-stimulated IL-10 release (100 +/- 21.5 pg/10(6) cells in LPS + dbcAMP and 110 +/- 25.2 pg/10(6) cells in LPS + FSK, both P < 0.05 vs 53.3 +/- 12.8 pg/10(6) cells in LPS alone). Neither TNF-alpha mRNA expression nor NF-kappaB activation stimulated by LPS was inhibited by the cAMP-elevating agents. Neutralization of IL-10 with a specific antibody did not attenuate the effect of cAMP-elevating agents on TNF-alpha production. CONCLUSION: The results indicate that cAMP inhibits LPS-stimulated TNF-alpha production through a posttranscriptional mechanism that is independent of endogenous IL-10.

Antibodies↗
PubMed2001

Inhibition of cyclic-3',5'-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-alpha production.

BACKGROUND: Local tumor necrosis factor (TNF)-alpha production by resident macrophages (M phi) contributes to posttraumatic tissue injury. Hypoxia decreases cellular cyclic adenosine monophosphate (cAMP) levels and enhances M phi secretion of TNF-alpha following lipopolysaccharide (LPS) stimulation. Thus, tissue hypoxia associated with trauma likely synergizes with proinflammatory mediators in the induction of M phi TNF-alpha production through an influence on cAMP generation or degradation. It is unclear whether elevation of cellular cAMP inhibits LPS-stimulated TNF-alpha production by hypoxic M phi. Moreover, it is unknown whether the synergism of hypoxia with LPS can be abrogated by promotion of cAMP generation or inhibition of cAMP degradation. METHODS: Rat peritoneal M phi were stimulated with Escherichia coli LPS (20 ng/ml) in a normoxic (room air with 5% CO(2)) or hypoxic (95% N(2) with 5% CO(2)) condition. TNF-alpha levels in cell-free supernatants were measured by enzyme-linked immunoassay. The beta-adrenoceptor agonist isoproterenol (ISP; 5.0 microM) and the adenylate cyclase activator forskolin (FSK; 50 microM) were applied to promote cAMP generation. The nonselective cyclic-3',5'-nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM) and the PDE III-specific inhibitor milrinone (200 microM) were used to inhibit cAMP degradation. The nondegradable cAMP analogue dibutyryl cAMP (dbcAMP; 100 microM) was applied to further determine the role of PDE. RESULTS. Although hypoxia alone had a minimal effect on TNF-alpha production, it dramatically enhanced LPS-stimulated TNF-alpha production (4.08 +/- 0.28 ng/10(6) cells in hypoxia plus LPS vs 1.63 +/- 0.26 ng/10(6) cells in LPS, 2.5-fold, P < 0.01). Promotion of cAMP generation by either ISP or FSK reduced TNF-alpha production by hypoxic cells. However, neither of these two agents abolished the synergism of hypoxia with LPS (1.68 +/- 0.13 ng/10(6) cells in ISP plus hypoxia plus LPS vs 0.55 +/- 0.04 ng/10(6) cells in ISP plus LPS, threefold; 1.17 +/- 0.03 ng/10(6) cells in FSK plus hypoxia plus LPS vs 0.33 +/- 0.02 ng/10(6) cells in FSK plus LPS, 3.5-fold; both P < 0.01). Inhibition of cAMP degradation with IBMX reduced TNF-alpha production in hypoxic cells and abrogated the synergism (0.31 +/- 0.11 ng/10(6) cells in IBMX plus hypoxia plus LPS vs 0.27 +/- 0.04 ng/10(6) cells in IBMX plus LPS, P > 0.05), and the PDE III inhibitor milrinone had a comparable effect. Moreover, dbcAMP also attenuated TNF-alpha production with abrogation of the synergistic effect of hypoxia (0.56 +/- 0.08 ng/10(6) cells in dbcAMP plus hypoxia plus LPS vs 0.46 +/- 0.04 ng/10(6) cells in dbcAMP plus LPS, P > 0.05). CONCLUSIONS: The results show that elevation of cellular cAMP, either by promotion of generation or by inhibition of degradation, suppresses LPS-stimulated TNF-alpha production in hypoxic M phi. It appears that hypoxia synergizes with LPS in the induction of M phi TNF-alpha production through PDE-mediated cAMP degradation. Inhibition of PDE may be a therapeutic approach for suppression of synergistic induction of M phi TNF-alpha production by hypoxia and LPS in posttraumatic tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Differential cellular immunolocalization of renal tumour necrosis factor-alpha production during ischaemia versus endotoxaemia.

Both renal ischaemia and endotoxaemia provoke renal dysfunction and cellular injury. Although the clinical manifestation of each insult is similar (global renal dysfunction), ischaemia and endotoxaemia induce different patterns of cellular injury. Tumour necrosis factor-alpha (TNF-alpha) has been implicated in both types of renal injury; however, it remains unknown whether differential cellular TNF-alpha expression accounts for these changes. We hypothesized that renal glomerular cells and tubular cells differentially express TNF-alpha in response to ischaemia compared with endotoxaemia. To investigate this hypothesis, male Sprague-Dawley rats were anaesthetized and exposed to various time-periods of renal ischaemia, with or without reperfusion (sham operation=negative control), or lipopolysaccharide (LPS) 0.5 mg/kg intraperitoneally (i.p.). The kidneys were harvested following renal injury, and rat TNF-alpha protein expression was determined (by enzyme-linked immunosorbent assay), as were TNF-alpha bioactivity (by WEHI-164 cell clone cytotoxicity assay) and TNF-alpha cellular localization (by immunohistochemistry). TNF-alpha protein expression and TNF-alpha bioactivity peaked following 1 hr of ischaemia and 2 hr of reperfusion (48 +/- 11 pg/mg of protein, P < 0.05, and 12 +/- 0.5 x 10-3 units/mg of protein, P < 0.05, respectively). The concentration of TNF-alpha increased to a similar extent following exposure to LPS; however, while TNF-alpha production following ischaemia-reperfusion injury localized predominantly to renal tubular epithelial cells, animals exposed to LPS demonstrated a primarily glomerular distribution of TNF-alpha production. Hence, the cellular localization of renal TNF-alpha production appears to be injury specific, i.e. renal tubular cells are the primary source of TNF-alpha following an ischaemic insult, whereas LPS induces glomerular TNF-alpha production. The cellular source of TNF-alpha following different insults may have therapeutic implications for targeted inhibition of TNF-alpha production.

Animals↗
PubMed2001

L-arginine attenuates lipopolysaccharide-induced lung chemokine production.

Chemokines stimulate the influx of leukocytes into tissues. Their production is regulated by nuclear factor-kappaB (NF-kappaB), an inducible transcription factor under the control of inhibitory factor kappaB-alpha (IkappaB-alpha). We have previously demonstrated that L-arginine (L-Arg) attenuates neutrophil accumulation and pulmonary vascular injury after administration of lipopolysaccharide (LPS). We hypothesized that L-Arg would attenuate the production of lung chemokines by stabilizing IkappaB-alpha and preventing NF-kappaB DNA binding. We examined the effect of L-Arg on chemokine production, IkappaB-alpha degradation, and NF-kappaB DNA binding in the lung after systemic LPS. To block nitric oxide (NO) production, a NO synthase inhibitor was given before L-Arg. LPS induced the production of chemokine protein and mRNA. L-Arg attenuated the production of chemokine protein and mRNA, prevented the decrease in IkappaB-alpha levels, and inhibited NF-kappaB DNA binding. NO synthase inhibition abolished the effects of L-Arg on all measured parameters. Our results suggest that L-Arg abrogates chemokine protein and mRNA production in rat lung after LPS. This effect is dependent on NO and is mediated by stabilization of IkappaB-alpha levels and inhibition of NF-kappaB DNA binding.

Animals↗
PubMed2001

Living-unrelated renal transplantation at the University of Wisconsin.

Since 1984, we have performed 243 living-unrelated renal transplants at the University of Wisconsin. Rejection occurred in 47% of the patients. Graft loss occurred in 59 patients and 39 patients died. Graft survival in LURD transplants at 10 years is 54% and 43% at 15 years. Patient survival is 68% at 10 years and 54% at 15 years. These long-term results demonstrate that LURD is equivalent to haploidentical renal transplantation and superior to cadaveric transplantation. Husband-to-wife donation demonstrated improved graft survival when compared with wife-to-husband and nonspousal donation. Living-unrelated renal transplantation has been utilized successfully at the University of Wisconsin and may help to alleviate the donor shortage.

Academic Medical Centers↗
PubMed2000

Adenosine preconditioning reduces both pre and postischemic arrhythmias in human myocardium.

INTRODUCTION: Consistently, clinical series record supraventricular tachyarrhythmias in approximately 30% of patients following coronary artery bypass surgery (CABG). Ischemic preconditioning and adenosine preconditioning (Ado-PC) decrease postischemia/reperfusion (I/R) myocardial stunning, infarct size, and pharmacologically induced arrhythmias in all species including man. We hypothesized that adenosine preconditioning would decrease spontaneous pre- and postischemic atrial arrhythmias in human myocardium. The purposes of this study were to determine the effect of in vivo and in vitro Ado-PC on atrial arrhythmias. METHODS: Human atrial trabeculae were harvested from CABG patients, placed in organ baths, and paced (1 Hz). Developed force (DF) was recorded during simulated I/R (30/45 min). Prior to I/R, trabeculae were treated with Ado (125 microM) for 5 min (in vitro), or patients were treated with Ado (12 mg iv) 5 min (in vivo) prior to harvest of trabeculae. Contraction frequency >4 Hz (defined as atrial tachyarrhythmias) was recorded in all groups pre- and postischemia. RESULTS: Control trabeculae exhibited increased tachyarrhythmias pre- and postischemia. In vivo and in vitro Ado-PC suppressed both pre- and postischemic arrhythmias. CONCLUSIONS: Adenosine preconditioning suppresses the frequency of pre- and postischemic tachyarrhythmias against an ischemia/reperfusion insult in human myocardium. This antiarrhythmic effect occurs with both in vitro and in vivo administration of adenosine. Preconditioning with adenosine prior to elective ischemia/reperfusion is a promising strategy of reducing spontaneous atrial arrhythmias in patients undergoing myocardial revascularization.

Adenosine↗
PubMed2000

Chlamydia pneumoniae activates nuclear factor kappaB and activator protein 1 in human vascular smooth muscle and induces cellular proliferation.

BACKGROUND: Observational data strongly suggest an association between Chlamydia pneumoniae and atherosclerotic cardiovascular disease. However, few studies have mechanistically linked C. pneumoniae to vascular remodeling. The purpose of the present study was to examine the mechanistic relationship between C. pneumoniae and human vascular smooth muscle cell (VSMC) physiology. We sought to determine the influence of human VSMC infection by C. pneumoniae on (1) VSMC proliferation and (2) activation of the proinflammatory and proliferative transcription factors nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). MATERIALS AND METHODS: C. pneumoniae was grown and isolated from Hep 2 cells. Human aortic VSMCs were inoculated with C. pneumoniae in the presence and absence of the azalide antibiotic azithromycin. Cell proliferation was assayed by direct cell counting 48 h following infection. Two hours following infection, nuclear extracts were isolated, and activation of both NF-kappaB and AP-1 was assessed by electrophoretic mobility shift assay. RESULTS: Compared with control, C. pneumoniae infection stimulated VSMC proliferation (P < 0.05) and induced both NF-kappaB and AP-1 DNA binding activity. These effects were eliminated by concurrent treatment with azithromycin. CONCLUSIONS: VSMC infection with C. pneumoniae activates proliferative intracellular signals and stimulates cell growth. These data implicate C. pneumoniae as a pathogenic mediator and a potential therapeutic target in the prevention of atherosclerotic disease.

Arteriosclerosis↗
PubMed2000

Thirty years of clinical trials in acute respiratory distress syndrome.

OBJECTIVE: To systematically review clinical trials in acute respiratory distress syndrome (ARDS). DATA SOURCES: Computerized bibliographic search of published research and citation review of relevant articles. STUDY SELECTION: All clinical trials of therapies for ARDS were reviewed. Therapies that have been compared in prospective, randomized trials were the focus of this analysis. DATA EXTRACTION: Data on population, interventions, and outcomes were obtained by review. Studies were graded for quality of scientific evidence. MAIN RESULTS: Lung protective ventilator strategy is supported by improved outcome in a single large, prospective trial and a second smaller trial. Other therapies for ARDS, including noninvasive positive pressure ventilation, inverse ratio ventilation, fluid restriction, inhaled nitric oxide, almitrine, prostacyclin, liquid ventilation, surfactant, and immune-modulating therapies, cannot be recommended at this time. Results of small trials using corticosteroids in late ARDS support the need for confirmatory large clinical trials. CONCLUSIONS: Lung protective ventilator strategy is the first therapy found to improve outcome in ARDS. Trials of prone ventilation and fluid restriction in ARDS and corticosteroids in late ARDS support the need for large, prospective, randomized trials.

Clinical Trials as Topic↗
PubMed2000

Chemokines as mediators of diseases related to surgical conditions.

Chemokines are important mediators of inflammation. Animal studies suggest that inhibition of chemokine action results in a decrease in inflammation. Novel anti-inflammatory agents directed against chemokines are now available. Surgeons are uniquely positioned to treat multiple chemokine-mediated diseases. In this article, we review the biology and nomenclature of chemokines as well as their role in neutrophil migration. Further, the potential role of chemokines in various diseases related to surgical conditions, including adult respiratory distress syndrome, atherosclerosis, inflammatory bowel disease, and solid organ rejection, is reviewed. Finally, the idea that chemokines could be targets for novel therapeutic agents is discussed.

Animals↗
PubMed2000

Interleukin-1beta deficiency results in reduced NF-kappaB levels in pregnant mice.

Interleukin (IL)-1beta-deficient (IL-1beta(-/-)) mice were assessed for cytokine production during pregnancy. A significant reduction in nuclear factor (NF)-kappaB p65 protein content was observed in the uteri and spleens of pregnant IL-1beta(-/-) mice, as demonstrated by immunohistochemistry and Western immunoblot analysis. In addition, electromobility gel shift assay revealed less DNA binding activity of NF-kappaB p65-containing complex in pregnant IL-1beta(-/-) mice. To investigate differences in cytokine production regulated by NF-kappaB, the levels of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and interferon-gamma were measured in the uterine wall, spleen homogenates, and spleen cell cultures obtained from pregnant mice. Endocervical administration of lipopolysaccharide (LPS) increased cytokine levels in both wild-type (IL-1beta(+/+)) and IL-1beta(-/-) animals, but in IL-1beta(-/-) mice this response was 50-75% lower. Splenocytes from nonpregnant mice exhibited decreased LPS-induced cytokine production when primed in vitro with progesterone. This suppression was 25% greater in IL-1beta(-/-) than in IL-1beta(+/+) mice. These data suggest that constitutive NF-kappaB p65 protein synthesis is regulated by IL-1beta, particularly during pregnancy.

Animals↗
PubMed2000

Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin.

Inducible nitric oxide synthase (iNOS) is associated with vascular hypocontractility in systemic vessels after endotoxin lipopolysaccharide (LPS) administration. Although lung iNOS is increased after LPS, its role in the pulmonary circulation is unclear. We hypothesized that whereas iNOS upregulation is responsible for LPS-induced vascular dysfunction in systemic vessels, iNOS does not play a significant role in the pulmonary artery (PA). Using isolated aorta (AO) and PA rings, we examined the effect of nonselective NOS inhibition [N(G)-monomethyl-L-arginine (L-NMMA); 100 micromol/l] and selective iNOS inhibition (aminoguanidine, AG; 100 micromol/l) on alpha(1)-adrenergic-mediated vasoconstriction (phenylephrine; 10(-9) to 10(-3) M) after LPS (Salmonella typhimurium, 20 mg/kg ip). We also determined the presence of iNOS using Western blot and immunohistochemistry. LPS markedly impaired AO contractility (maximal control tension 1,076 +/- 33 mg vs. LPS 412 +/- 39 mg, P < 0.05), but PA contractility was unchanged (control 466 +/- 29 mg vs. LPS 455 +/- 27 mg, P > 0.05). Selective iNOS inhibition restored the AO's response to vasoconstriction (LPS + AG 1,135 +/- 54 mg, P > 0.05 vs. control and P < 0.05 vs. LPS), but had no effect on the PA (LPS + AG 422 +/- 38 mg, P > 0.05 vs. control and LPS). Western blot and immunohistochemistry revealed increased iNOS expression in the AO after LPS, but iNOS was not detected in the PA. Our results suggest that differential iNOS expression after LPS in systemic and pulmonary vessels contributes to the phenomenon of sepsis/endotoxemia-induced systemic hypotension and pulmonary hypertension.

Animals↗
PubMed1999

Inhibition of myocardial TNF-alpha production by heat shock. A potential mechanism of stress-induced cardioprotection against postischemic dysfunction.

Overproduction of tumor necrosis factor-alpha (TNF-alpha) contributes to cardiac dysfunction associated with systemic or myocardial stress, such as endotoxemia and myocardial ischemia/reperfusion (I/R). Heat shock has been demonstrated to enhance cardiac functional resistance to I/R. However, the protective mechanisms remain unclear. The purpose of this study was to determine: (1) whether cardiac macrophages express heat shock protein 72 (HSP72) after heat shock, (2) whether induced cardiac HSP72 suppresses myocardial TNF-alpha production during I/R, and (3) whether preservation of postischemic myocardial function by heat shock is correlated with attenuated TNF-alpha production during I/R. Rats were subjected to heat shock (42 degrees C for 15 min) and 24 h recovery. Immunoblotting confirmed the expression of cardiac HSP72. Immunofluorescent staining detected HSP72 in cardiac interstitial cells including resident macrophages rather than myocytes. Global I/R caused a significant increase in myocardial TNF-alpha. The increase in myocardial TNF-alpha was blunted by prior heat shock and the reduced myocardial TNF-alpha level was correlated with improved cardiac functional recovery. This study demonstrates for the first time that heat shock induces HSP72 in cardiac resident macrophages and inhibits myocardial TNF-alpha production during I/R. These observations suggest that inhibition of myocardial TNF-alpha production may be a mechanism by which HSP72 protects the heart against postischemic dysfunction.

Animals↗
PubMed1999

Cardiotrophin-1 attenuates endotoxin-induced acute lung injury.

Cardiotrophin-1 (CT-1) is a recently discovered member of the gp130 cytokine family, which includes IL-6, IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Recent evidence suggests that, like other members of this family, CT-1 may possess anti-inflammatory properties. We hypothesized that in vivo CT-1 administration would attenuate endotoxin (ETX)-induced acute lung injury. We studied the effects of CT-1 (100 microgram/kg ip, 10 min prior to ETX) in a rat model of ETX-induced acute lung injury (Salmonella typhimurium lipopolysaccharide, 20 mg/kg ip). Six hours after ETX, lungs were harvested for determination of neutrophil accumulation (myeloperoxidase, MPO, assay) and lung edema (wet-to-dry weight ratio). Mechanisms of pulmonary vasorelaxation were examined in isolated pulmonary artery rings at 6 h by interrogating endothelium-dependent (response to acetylcholine) and endothelium-independent (response to sodium nitroprusside) relaxation following alpha-adrenergic (phenylephrine)-stimulated preconstriction. CT-1 abrogated the endotoxin-induced lung neutrophil accumulation: 2.3 +/- 0.2 units MPO/g wet lung (gwl) vs 6. 3 +/- 0.3 units MPO/gwl in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Similarly, CT-1 prevented ETX-induced lung edema: wet-to-dry-weight ratio, 4.473 +/- 0.039 vs 4.747 +/- 0.039 in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Endotoxin caused significant impairment of both endothelium-dependent and -independent pulmonary vasorelaxation, and CT-1 attenuated this injury. Thus, cardiotrophin-1 possesses significant anti-inflammatory properties in a model of endotoxin-induced acute lung injury.

Acute Disease↗
PubMed1999

p38 MAPK inhibition decreases TNF-alpha production and enhances postischemic human myocardial function.

INTRODUCTION: TNF-alpha is a proinflammatory cytokine implicated in myocardial dysfunction following ischemia/reperfusion (I/R). I/R results in myocardial production of TNF-alpha and TNF-alpha suppresses myocardial contractility. p38 mitogen-activated protein kinase (MAPK) is a redox-sensitive protein kinase involved in intracellular signaling leading to TNF-alpha production. It remains unknown if the human heart produces TNF-alpha after I/R and, if so, whether p38 MAPK is involved. HYPOTHESIS: p38 MAPK inhibition enhances human myocardial post-I/R contractile function by inhibition of myocardial TNF-alpha production. METHODS: Human atrial trabeculae were suspended in organ baths, field simulated at 1 Hz, and force development was recorded. Following a 90-min equilibration, trabeculae were exposed to a p38 MAPK inhibitor (SB 203580, 1 microM) or vehicle (each n = 6) prior to simulated ischemia (45 min hypoxia, substrate-free, rapid pacing at 3 Hz) followed by 120 min reoxygenation. Myocardial TNF-alpha levels were measured by ELISA at end reoxygenation. RESULTS: I/R increased human myocardial TNF-alpha levels from 26.9 +/- 9.3 to 83.9 +/- 19.2 pg/g wet tissue (P < 0.05 perfusion vs I/R; ANOVA Bonferroni/Dunn), while p38 MAPK inhibition decreased post-I/R myocardial TNF-alpha levels to 32.3 +/- 8.0 pg/g wet tissue (P > 0.05 p38 MAPK inhibition vs I/R). p38 MAPK inhibition improved postischemic force development from 18.5 +/- 2.1 to 37.0 +/- 2.0% baseline developed force (%BDF; P < 0.05 I/R vs p38 MAPK inhibition). CONCLUSIONS: (1) The human heart produces TNF-alpha after I/R, (2) p38 MAPK mediates myocardial I/R-induced TNF-alpha production, (3) p38 MAPK inhibition limits functional impairment after I/R, and (4) inhibition of ischemia-induced TNF-alpha production may represent a potent therapeutic strategy for improving myocardial function after angioplasty, coronary bypass, or heart transplantation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.

Cells, Cultured↗
PubMed1999

Liposomal delivery of purified inhibitory-kappaBalpha inhibits tumor necrosis factor-alpha-induced human vascular smooth muscle proliferation.

Vessel injury results in the elaboration of various cytokines, including tumor necrosis factor-alpha (TNF-alpha), which may influence vascular smooth muscle cell (VSMC) function and contribute to atherogenesis. We tested the hypothesis that TNF-alpha-induced VSMC proliferation requires activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which could be prevented by delivery of the NF-kappaB inhibitory peptide, IkappaBalpha. TNF-alpha induced concentration-dependent human VSMC proliferation, and neutralizing antibody to interleukin-6 reduced TNF-alpha-induced VSMC proliferation by 65%. In TNF-alpha-stimulated VSMCs, there was a 3-fold increase in NF-kappaB-dependent luciferase reporter activity that was associated with degradation of IkappaBalpha. To determine an essential role for NF-kappaB in TNF-alpha-induced VSMC proliferation, recombinant IkappaBalpha was introduced into VSMCs via liposomal delivery. Under these conditions, TNF-alpha-induced NF-kappaB nuclear translocation and DNA binding were inhibited, NF-kappaB-dependent luciferase activity was reduced by 50%, there was no degradation of native IkappaBalpha detected, interleukin-6 production was reduced by 54%, and VSMC proliferation was decreased by 60%. In conclusion, the mitogenic effect of TNF-alpha on human arterial VSMCs is dependent on NF-kappaB activation and may be prevented by exogenously delivered IkappaBalpha. Furthermore, liposomal delivery of endogenous inhibitory proteins may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.

Cell Division↗
PubMed1999

Calcium preconditioning, but not ischemic preconditioning, bypasses the adenosine triphosphate-dependent potassium (KATP) channel.

BACKGROUND: Recent evidence has implicated the KATP channel as an important mediator of ischemic preconditioning (IPC). Indeed, patients taking oral sulfonylurea hypoglycemic agents (i.e., KATP channel inhibitors) for treatment of diabetes mellitus are resistant to the otherwise profoundly protective effects of IPC. Unfortunately, many cardiopulmonary bypass patients, who may benefit from IPC, are chronically exposed to these agents. Calcium preconditioning (CPC) is a potent form of similar myocardial protection which may or may not utilize the KATP channel in its mechanism of protection. The purpose of this study was to determine whether CPC may bypass the KATP channel in its mechanism of action. If so, CPC may offer an alternative to IPC in patients chronically exposed to these agents. METHODS: Isolated rat hearts (n = 6-8/group) were perfused (Langendorff) and received KATP channel inhibition (glibenclamide) or saline vehicle 10 min prior to either a CPC or IPC preconditioning stimulus or neither (ischemia and reperfusion, I/R). Hearts were subjected to global warm I/R (20 min/40 min). Postischemic myocardial functional recovery was determined by measuring developed pressure (DP), coronary flow (CF), and compliance (end diastolic pressure, EDP) with a MacLab pressure digitizer. RESULTS: Both CPC and IPC stimuli protected myocardium against postischemic dysfunction (P < 0.05 vs I/R; ANOVA with Bonferroni/Dunn): DP increased from 52 +/- 4 (I/R) to 79 +/- 2 and 83 +/- 4 mmHg; CF increased from 11 +/- 0.7 to 17 +/- 2 and 16 +/- 1 ml/min; and EDP decreased (compliance improved) from 50 +/- 7 to 27 +/- 5 and 31 +/- 7 mmHg. However, KATP channel inhibition abolished protection in hearts preconditioned with IPC (P < 0.05 vs IPC alone), but not in those preconditioned with CPC (P > 0.05 vs CPC alone). CONCLUSIONS: (1) Both IPC and CPC provide similar myocardial protection; (2) IPC and CPC operate via different mechanisms; i.e., IPC utilizes the KATP channel whereas CPC does not; and (3) CPC may offer a means of bypassing the deleterious effects of KATP channel inhibition in diabetic patients chronically exposed to oral sulfonylurea hypoglycemic agents.

Adenosine Triphosphate↗