Inhibition of nuclear factor-kappaB activation by IRFI 042, protects against endotoxin-induced shock

BACKGROUND: The aim of our study was to investigate the effect of IRFI 042, a novel dual vitamin E-like antioxidant, on nuclear factor-kappaB (NF-kappaB) activation, TNF-alpha gene priming and on the release of the mature protein during endotoxin shock. METHODS: Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg(-1) of Salmonella enteritidis lipopolysaccharide (LPS). Survival rate, mean arterial blood pressure, serum TNF-alpha and plasma malondialdehyde (MAL) levels were investigated. We then evaluated in the liver TNF-alpha mRNA levels, NF-kappaB binding activity and the inhibitory protein IkappaBalpha. Moreover we studied in LPS stimulated (50 microg ml(-1)) peritoneal macrophages (Mphi), NF-kappaB activation, cytoplasmic IkappaB-alpha degradation, the message for TNF-alpha, and TNF-alpha and MAL levels. RESULTS: LPS administration reduced survival rate (0%, 72 h after LPS administration), decreased mean arterial blood pressure, augmented serum TNF-alpha (60+/-11 ng ml(-1)) and enhanced plasma malondialdehyde (MAL) levels (55+/-7.1 nmol l(-1)). LPS shocked rats also had increased TNF-alpha mRNA levels, augmented liver NF-kappaB binding activity in the nucleus and decreased levels of the inhibitory protein IkappaBalpha. In addition, in vitro LPS stimulation (50 microg ml(-1)) significantly induced NF-kappaB activation and cytoplasmic IkappaBalpha degradation in Mphi, enhanced TNF-alpha mRNA levels and increased Mphi TNF-alpha and MAL. Treatment with IRFI 042 (20 mg kg(-1), i.v., 5 min after endotoxin challenge) protected against LPS-induced lethality (90% survival rate 24 h and 80% survival rate 72 h after LPS injection, respectively), reduced hypotension, blunted plasma MAL (9.0+/-0.9 nmol l(-1)) and decreased serum TNF-alpha (15+/-3 ng ml(-1)). The antioxidant also inhibited the loss of IkappaBalpha protein from the hepatic cytoplasm, blunted the increased NF-kappaB binding activity in the liver and decreased hepatic liver mRNA for TNF-alpha. Furthermore 'in vitro' IRFI 042 (50 microM) significantly inhibited activation of NF-kappaB through inhibition of IkappaBalpha degradation, reduced the amount of TNF-alpha mRNA, decreased LPS-induced TNF-alpha release and blunted lipid peroxidation (MAL) in LPS stimulated Mphi. CONCLUSIONS: These data suggest that IRFI 042 blocks the activation of NF-kappaB, reduces TNF-alpha mRNA levels, and finally reverses endotoxic shock.     

Persistent activation of nuclear factor kappa-B signaling pathway in patients with unstable angina and elevated levels of C-reactive protein evidence for a direct proinflammatory effect of azide and lipopolysaccharide-free C-reactive protein on human monocytes via nuclear factor kappa-B activation.

OBJECTIVES: Our study investigated: 1) the contribution of nuclear factor kappa-B (NF-kappaB) signaling pathway to the enhanced inflammatory response observed in unstable angina (UA) patients with elevated levels of C-reactive protein (CRP); and 2) whether CRP may have direct proinflammatory effects via NF-kappaB activation. BACKGROUND: Unstable angina patients with elevated CRP have enhanced inflammatory response and increased risk of persistent instability, myocardial infarction, and death. METHODS: We studied 28 patients with history of UA and persistently elevated CRP (>3 mg/l) followed for 24 months and free of symptoms for at least 6 months (group 1), 14 patients with history of UA and low CRP (group 2), and 24 patients with chronic stable angina and low CRP (group 3). Peripheral blood monocytes were analyzed for spontaneous NF-kappaB activation and interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha production. To assess the direct proinflammatory effects of CRP, monocytes from 8 healthy subjects were stimulated in vitro with increasing doses of CRP (5 to 10 to 25 microg/ml), lipopolysaccharide (LPS) (1 to 10 ng/ml), or both. RESULTS: Spontaneous NF-kappaB activation in vivo was demonstrated in 82% of group 1 versus 14% of group 2 and 21% of group 3 patients (p < 0.001). Interleukin-6 and TNF-alpha production was significantly correlated with the NF-kappaB activation status (r = 0.55, p < 0.001 and r = 0.53, p = 0.006, respectively). Patients with NF-kappaB activation had recurrence of acute coronary events (60% vs. 28%; p = 0.017). C-reactive protein induced a significant but modest in vitro NF-kappaB activation in human monocytes (p = 0.002). Coincubation with LPS produced a greater-than-additive response (p < 0.01 vs. CRP and LPS alone). CONCLUSIONS: Nuclear factor kappa-B activation might represent a mechanism by which CRP amplifies and perpetuates the inflammatory component of acute coronary syndromes and influences the clinical outcome.     

Differential effects of pyrrolidine dithiocarbamate on TNF-alpha-mediated liver injury in two different models of fulminant hepatitis

BACKGROUND/AIMS: Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of nuclear factor kappa B (NF-kappaB) activation. The present study aimed to investigate the effects of PDTC on lipopolysaccharide (LPS)-induced liver injury in two different models of fulminant hepatitis. METHODS: Mice infected with Bacillus Calmette Guerin (BCG) were challenged with LPS (0.2 mg/kg) to induce the model of inflammatory liver injury. Mice were injected with D-galactosamine (GalN, 600 mg/kg) and LPS (20 microg/kg) to induce the model of apoptotic liver injury. In the treatment groups, mice were pre-treated with PDTC (100 mg/kg), initiated 24 h prior to LPS. RESULTS: PDTC pretreatment reduced the infiltration of inflammatory cells, inhibited NF-kappaB activation and the expression of tumor necrosis factor alpha (TNF-alpha), attenuated nitric oxide production, and alleviated hepatic glutathione depletion. Correspondingly, PDTC reduced serum alanine aminotransferase, improved hepatic necrosis, and prolonged the survival in the BCG/LPS model. Conversely, PDTC accelerated death and aggravated liver apoptosis in the GalN/LPS model, although it reduced nitric oxide production, attenuated glutathione depletion, and inhibited the expression of TNF-alpha in liver. CONCLUSIONS: PDTC protects mice against BCG/LPS-induced inflammatory liver injury through the repression of NF-kappaB-mediated TNF-alpha release, while it seems to be detrimental in GalN/LPS-induced apoptotic liver damage.     

Low-molecular weight and unfractionated heparins induce a downregulation of inflammation: decreased levels of proinflammatory cytokines and nuclear factor-kappaB in LPS-stimulated human monocytes

Unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) are well defined anticoagulant agents. Recent data suggest that both LMWH and UFH may also have potent anti-inflammatory properties; however, their mechanism of action responsible for the anti-inflammatory effect is not yet fully elucidated. This study was designed to assess the effect of LMWH and UFH on human monocytes production of inflammatory markers and nuclear translocation of nuclear factor (NF)-kappaB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 microg and 1 microg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6 and IL-1beta release were subsequently measured by enzyme-linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF-kappaB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS-induced production of TNF-alpha, IL-8, IL-6 and IL-1beta as well as NF-kappaB translocation. These results indicate equivalent and significant heparin anti-inflammatory properties at low doses on monocyte-mediated immune response. The inhibition of NF-kappaB activation certainly represents one of the mechanisms by which heparin exerts its anti-inflammatory effect. LMWH and UFH therefore appear as potential therapeutic inhibitors of inflammation.     

Moderate alcohol intake in humans attenuates monocyte inflammatory responses: inhibition of nuclear regulatory factor kappa B and induction of interleukin 10

BACKGROUND: In contrast to the deleterious effects of chronic excessive alcohol consumption on the liver and cardiovascular system, modest alcohol intake, such as 1 to 2 drinks per day, has benefits on cardiovascular mortality. Little is known about the length of time or the amounts of alcohol consumed that may cause alterations in inflammatory cells such as monocytes that are crucial to atherosclerotic vascular disease. Here, we determine in vivo effects of acute alcohol consumption on inflammatory cytokine production and nuclear regulatory factor kappaB (NF-kappaB) binding in human monocytes. METHODS: Human blood monocytes were isolated by plastic adherence before and after acute alcohol consumption (2 ml vodka/kg body weight). Lipopolysaccharide (LPS)- and superantigen-induced tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1beta, and IL-10 production were then determined in monocytes by ELISA. Nuclear regulatory factor-kappaB activity of monocytes before and after alcohol consumption was estimated by electromobility shift assay and promoter-driven reporter activity. IkappaBalpha was determined by Western blotting in the cytoplasmic extracts. RESULTS: Eighteen hours after moderate alcohol consumption, we found a significant reduction in monocyte production of inflammatory mediators, TNF-alpha and IL-1beta, in response to LPS or staphylococcal enterotoxin B stimulation. Acute alcohol consumption inhibited LPS-induced DNA binding of the p65/p50 NF-kappaB in monocytes that regulates the expression of both the TNF-alpha and the IL-1beta genes. Consistent with this, acute alcohol treatment (25 mM) significantly reduced LPS-induced activation of an NF-kappaB-driven reporter gene suggesting inhibition of this proinflammatory signaling pathway. Further, LPS-induced IkappaBalpha degradation was not affected by acute alcohol consumption indicating an IkappaBalpha-independent mechanism, as observed earlier in the in vitro acute alcohol studies. In contrast, monocyte production of the anti-inflammatory cytokine, IL-10, was augmented by acute alcohol intake. CONCLUSIONS: Our findings suggest that acute alcohol consumption has dual anti-inflammatory effects that involve augmentation of IL-10 and attenuation of monocyte inflammatory responses involving inhibition of NF-kappaB. These mechanisms may contribute to the beneficial effects of moderate alcohol use on atherosclerosis.     

Mulberry leaf aqueous fractions inhibit TNF-alpha-induced nuclear factor kappaB (NF-kappaB) activation and lectin-like oxidized LDL receptor-1 (LOX-1) expression in vascular endothelial cells

Mulberry (Morus Alba L., family Moraceae) leaf extracts have various biological effects including inhibition of oxidative modification of low-density lipoprotein (LDL), which is the major cause of atherosclerosis. Endothelial dysfunction elicited by oxidized LDL (Ox-LDL) has been implicated in atherogenesis. Lectin-like Ox-LDL receptor-1 (LOX-1), a cell-surface receptor for atherogenic Ox-LDL, appears to mediate Ox-LDL-induced inflammation, which may be crucial in atherogenesis. Previous studies revealed that expression of LOX-1 is highly inducible by proinflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and transforming growth factor-beta (TGF-beta). Therefore, we examined whether mulberry leaf aqueous fractions inhibit LOX-1 expression induced by proinflammatory stimuli. Pretreatment of cultured bovine aortic endothelial cells (BAECs) with mulberry leaf aqueous fractions inhibited TNF-alpha- and LPS-induced expression of LOX-1 at both protein and mRNA levels in a time- and concentration-dependent manner. In contrast, mulberry leaf aqueous fractions did not affect TGF-beta-induced LOX-1 expression. Furthermore, mulberry leaf aqueous fractions inhibited TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) and phosphorylation of inhibitory factor of NF-kappaB-alpha (IkappaB-alpha) in a time- and concentration-dependent fashion. Thus, mulberry leaf aqueous fractions suppress TNF-alpha- and LPS-induced LOX-1 gene expression, by inhibiting NF-kappaB activation.     

IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-alpha production in macrophages

Interleukin-10 (IL-10) plays an important role in prevention of chronic inflammation in vivo. However, the molecular mechanism by which IL-10 exerts its anti-inflammatory response is poorly understood. Here, we performed a microarray analysis and identified Bcl-3 as an IL-10-inducible gene in macrophages. Lentiviral vector-mediated expression of Bcl-3 inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNF-alpha), but not IL-6, in macrophages. In Bcl-3-transduced and IL-10-pretreated macrophages, LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) p65 was not impaired. However, DNA binding by NF-kappaB p50/p65 was profoundly inhibited. Nuclear localization of Bcl-3 was associated with inhibition of LPS-induced TNF-alpha production. Overexpression of Bcl-3 suppressed activation of the TNF-alpha promoter, but not the IL-6 promoter. Bcl-3 interacted with NF-kappaB p50 and was recruited to the TNF-alpha promoter, but not the IL-6 promoter, indicating that Bcl-3 facilitates p50-mediated inhibition of TNF-alpha expression. Furthermore, Bcl-3-deficient macrophages showed defective IL-10-mediated suppression of LPS induction of TNF-alpha, but not IL-6. These findings suggest that IL-10-induced Bcl-3 is required for suppression of TNF-alpha production in macrophages.     

GM-CSF priming of human monocytes is dependent on ERK1/2 activation

The ability to augment monocyte functions such as TNF-alpha-producing capacities confers a high immunostimulating potential to GM-CSF. In the present investigation, the mechanism of the GMCSF-mediated enhancement of monocyte cytokine production was analysed with regard to the involvement of intracellular signalling pathways. GM-CSF primes human monocytes dose- and time-dependently for enhanced LPS-stimulated TNF-alpha synthesis. Pre-incubation with 10 ng/ml GM-CSF for 6 h before LPS stimulation (10 ng/ml) caused a 3.4 +/- 1.9-fold increase in TNF-alpha release compared to unprimed controls. This was associated with increased phosphorylation of IkappaBalpha and elevated nuclear levels of the NF-kappaB components p50 and p65 and NF-kappaB binding to DNA. LPS-induced AP-1 binding to DNA was also enhanced in GM-CSF-pre-incubated cells. GMCSF treatment also caused a slight increase in TLR4 expression on monocytes while CD14 expression remained unchanged. GM-CSF-priming was unaffected by inhibitors of p38 MAPK (SB203580) and lipoxygenase (NDGA). In contrast, the broad-spectrum tyrosine kinase inhibitor genistein and the MEK-1 inhibitor (PD98059) abrogated GM-CSF priming of TNF-alpha release and activation of both NF-kappaB and AP-1. It is concluded that a tyrosine kinase of the GM-CSF-triggered ERK1/2 pathway augments the LPS-induced NF-kappaB and AP-1 activation.     

Implication of Galpha i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production

Previous studies have suggested that heterotrimeric G proteins and tyrosine kinases may be involved in lipopolysacchaide (LPS) signaling events. Signal transduction pathways activated by LPS we examined in human pomonocytic THP-l cells. We hypothesized that Gi proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa(NF-kappaB) activation. Post-receptor coupling to Ga, proteins were examined using pertussis toxin (PTx),which inhibits Galpha i receptor-coupling. The involvement of the Src family of tyrosine kinases was examined using the selective Src tyrosine kinase inhibitor pyrazolopyrimidine-2 (PP2). Pretreatment of THP-1 cells with PTx attenuated LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase, and production of tumor necrosis factor-alpha (TN-alpha) and thromboxane B2 (TXB2). Pretreatment with PP2 inhibited TNF-alpha and TxB2 production, but had no effect on p38 kinase or JNK signaling. Therefore, the Ga i-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF-alpha and TxB2 production, but differ in their effects on MAPK activation. Neither PTx nor PP2 inhibited LPS-induced activation of interleukin receptor activated kinase (IRAK) or inhibited translocation of NF-kappaB. However, PP2 inhibited LPS-induced NF-kappaB transactivation of a luciferase reporter gene construct in a concentration-dependent manner. Thus, LPS induction of Src tyrosine kinases may be essential in downstream NF-kappaB tansactivation of genes following DNA binding. PTx had no effect on NF-kaapaB activation of the reporter construct. These data suggest upstream divergence in signaling through Galpha i,pathways leading to MAPK activation and other signaling events leading to IkappaBalpha degradation and NF-kaapaB DNA binding.     

Genistein prevents nuclear factor-kappa B activation and acute lung injury induced by lipopolysaccharide.

Protein tyrosine kinase (PTK) inhibitors have been proposed to reduce lung injury and lethal toxicity. The mechanisms responsible for the effects of PTK inhibitors remain obscure. The purpose of the present study was to examine whether genistein, a specific inhibitor of PTK, inhibits nuclear factor-kappa B (NF-kappaB) activation during acute lung injury induced by lipopolysaccharide (LPS) and, if so, to enumerate the effects of inhibition of NF-kappaB activation on LPS-induced proinflammatory gene products, such as cytokine-inducible neutrophil chemoattractant (CINC) and matrix metalloproteinase-9 (MMP-9), as well as neutrophil influx into the lungs. Intratracheal treatment of rats with LPS (6 mg/kg) resulted in increases in total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid and activated DNA-binding activity of NF-kappaB in alveolar macrophages and lung tissue. A 2-h pretreatment with genistein (50 mg/kg, intraperitoneally) inhibited the LPS-induced changes in lung injury parameters and the induction of NF-kappaB activation. Furthermore, these inhibitory effects of genistein correlated with a depression of LPS-induced protein tyrosine phosphorylation (approximately molecular masses of 46, 48, and 54 kD) and phosphorylation of Jun N-terminal kinase (JNK) in lung tissue. Genistein also substantially reduced the LPS-induced CINC production and MMP-9 activity and suppressed neutrophil recruitment. These results suggest that genistein attenuates LPS-induced acute lung responses through inhibition of NF-kappaB activation. In addition, NF-kappaB activation appears to be an important mechanism mediating LPS-induced CINC production and MMP-9 activity and resulting neutrophil recruitment associated with acute lung inflammation and injury.     

Time-dependent effect of LPS on PGE2 and TNF-alpha production by rat glial brain culture: influence of COX and cytokine inhibitors

This study was undertaken to investigate the effect of lipopolysaccharide (LPS) stimulation on the time course of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-alpha) production by rat glial brain culture. A concentration of 10 microg/ml LPS from Escherichia coli was used as stimulation treatment. The effect of pentoxifylline (PXF), nimesulide (NIM), indomethacin (INDO) and dexamethasone (DEX) on the regulation of PGE2 and TNF-alpha production was tested. Stimulation of rat glial cells with LPS resulted in different time-dependent production patterns of PGE2 and TNF-alpha The time course of TNF-alpha elevation was short, reaching its peak at 6 h post LPS and decreasing to undetectable levels after 24 h. On the other hand, the time course of PGE2 elevation was longer, starting at 6 h post LPS treatment and increasing 100-fold compared with basal levels, 24 h post LPS exposure. The COX inhibitors (NIM and INDO) and DEX were found to inhibit the LPS-induced elevation in PGE2 production, while PXF lacked such an inhibitory effect. Furthermore, NIM, DEX and PXF were found to reduce the LPS-induced elevation in TNF-alpha levels, while INDO caused a greater elevation in TNF-alpha levels. These results may cast further light on the LPS-induced production of PGE2 and TNF-alpha by rat glial cell cultures and the relation between the two systems.     

The effect of a metalloproteinase inhibitor (GI5402) on tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha receptors during human endotoxemia.

Tumor necrosis factor-alpha (TNF-alpha) is released from the cell surface by cleavage of pro-TNF-alpha by metalloproteinases (MPs). In cell cultures, inhibition of MPs has been found not only to reduce the release of TNF-alpha, but also to enhance the surface expression of TNF-alpha and TNF-alpha receptors, which might lead to a proinflammatory effect. To determine the effect of MP inhibition during inflammation in humans, 7 healthy subjects were studied after intravenous injection of lipopolysaccharide (LPS; 4 ng/kg) preceded (-20 minutes) by an oral dose of the MP inhibitor GI5402 (100 mg) or matching placebo. GI5402 strongly reduced LPS-induced TNF-alpha release (P <.001), but did not influence the increase in monocyte-bound TNF-alpha. In addition, GI5402 attenuated the rise in plasma-soluble TNF-alpha receptors types I and II after LPS injection (both P <.001), but did not change the LPS-induced decreases in granulocyte and monocyte TNF-alpha receptor expression. These data suggest that MP inhibitors may be useful as a treatment modality in diseases in which excessive production of TNF-alpha is considered to play an important role. Furthermore, unlike in vitro, no evidence has been found in vivo with MP inhibition for a potential proinflammatory effect due to increases in membrane-bound TNF-alpha and TNF-alpha receptor number.     

Effect of cholecystokinin on cytokines during endotoxic shock in rats

AIM To study the effect of cholecystokinin-octapeptide(CCK-8)on systemic hypotension and cytokine productionin lipopolysaccharide(LPS)-induced endotoxic shock(ES)rats.METHODS The changes of blood pressure were observedusing physiological record instrument in four groups ofrats:LPS(8mg·kg~(-1),iv)induced ES;CCK-8(40μg·kg~1,iv)pretreatment 10min before LPS(8mg.kg~(-1));CCK-8(40μg·kg~(-1),iv)or normal saline(control)groups.Differences in tissue and circulating specificity of theproinflammatory cytokines(TNF-α,IL-1β and IL-6)wereassayed with ELISA kits.RESULTS CCK-8 reversed LPS-induced decrease of meanartery blood pressure(MABP)in rats.Compared withcontrol,LPS elevated the serum level of IL-6 significantly(3567±687ng·L~(-1) vs 128±22ng·L~1,P<0.01),whilecontents of TNF-α and IL-1β elevated significantly(277±86ng.L~1 vs not detectable and 43±9ng·L~1 vs notdetectable,P<0.01)but less extent than IL-6.CCK-8significantly inhibited the LPS-induced increase in serumTNF-α,IL-1β and IL-6.LPS elevated spleen and lungcontent of IL-1β significantly(5184±85ng·L~1 vs 1047±21ng·L~1 and 4050±614ng·L~1 vs not detectable.P<0.01),while levels of TNF-α and IL-6 also rosesignificantly but in less extent than IL-1β.CCK-8 inhibitedthe LPS-induced increase of the cytokines in spleen andlung.In the heart,CCK-8 significantly inhibited LPS-induced increase of TNF-α(864±123ng·L~1 in CCK-8 LPS group vs 1599±227ng·L~1 in LPS group,P<0.01),and IL-1β(282±93ng·L~1 in CCK-8 LPS group vs 621±145ng·L~1 in LPS group,P<0.01).CONCLUSION CCK-8 reverses ES,which may be relatedto its inhibitory effect on the overproduction of cytokines.     

Countervailing effects of rapamycin (sirolimus) on nuclear factor-kappa B activities in neointimal and medial smooth muscle cells

OBJECTIVE: Local application of rapamycin (sirolimus) by drug-eluting stents prevents lumen obliteration after angioplasty by inhibition of neointimal hyperplasia. The effects of rapamycin on neointimal smooth muscle cells (niSMC) which are responsible for the occurrence of restenosis have not been investigated so far. METHODS AND RESULTS: Rat niSMC and medial SMC (mSMC) were obtained from balloon catheter-injured arteries. The niSMC exhibited higher basal NF-kappaB activity and TNF-alpha mRNA levels. Nuclear protein binding to NF-kappaB-DNA was attenuated in niSMC by incubation with rapamycin (0.1 and 1 microg/ml) for 24 and 48 h. In contrast in mSMC, 0.1 microg/ml rapamycin had no effect and at 1 microg/ml even increased nuclear protein binding to NF-kappaB-DNA. After 12 h incubation, rapamycin (0.001-10 microg/ml) induced IkappaB-alpha protein in niSMC, whereas in mSMC it stimulated IkappaB-alpha at much lower levels. Prolonged rapamycin treatment (1 microg/ml for 72 h) had no effect on TNF-alpha mRNA level and NF-kappaB activity in niSMC, whereas it led to their increase in mSMC. Vascular endothelial growth factor (VEGF) secretion was higher in mSMC than in niSMC; rapamycin decreased VEGF levels in both cell types. Ultrastructural analysis suggested that rapamycin caused early signs of degeneration in niSMC, but enhanced protein synthesis in mSMC. CONCLUSIONS: This study shows that rapamycin influences the inflammatory phenotypes of SMC in opposite directions: it reduces the high basal NF-kappaB activity in niSMC and enhances NF-kappaB activity and TNF-alpha expression in mSMC. In addition, rapamycin inhibits VEGF production regardless of the phenotype of SMC. These findings shed light on molecular mechanisms and structural changes underlying therapeutic applications of rapamycin in prevention of restenosis, inhibition of chronic transplant arteriosclerosis and reduction of secondary malignoma formation due to immunosuppression.