Suppression of nuclear factor-κB activity in macrophages by chylomicron remnants: modulation by the fatty acid composition of the particles

Current evidence indicates that chylomicron remnants (CMR) induce macrophage foam cell formation, an early event in atherosclerosis. Inflammation also plays a part in atherogenesis and the transcription factor nuclear factor-κB (NF-κB) has been implicated. In this study, the influence of CMR on the activity of NF-κB in macrophages and its modulation by the fatty acid composition of the particles were investigated using macrophages derived from the human monocyte cell line THP-1 and CMR-like particles (CRLPs). Incubation of THP-1 macrophages with CRLPs caused decreased NF-κB activation and downregulated the expression of phospho-p65–NF-κB and phospho-IκBα (pIκBα). Secretion of the inflammatory cytokines tumour necrosis factor α, interleukin-6 and monocyte chemoattractant protein-1, which are under NF-κB transcriptional control, was inhibited and mRNA expression for cyclooxygenase-2, an NF-κB target gene, was reduced. CRLPs enriched in polyunsaturated fatty acids compared with saturated or monounsaturated fatty acids had a markedly greater inhibitory effect on NF-κB binding to DNA and the expression of phospho-p65–NF-κB and pIκB. Lipid loading of macrophages with CRLPs enriched in polyunsaturated fatty acids compared with monounsaturated fatty acids or saturated fatty acids also increased the subsequent rate of cholesterol efflux, an effect which may be linked to the inhibition of NF-κB activity. These findings demonstrate that CMR suppress NF-κB activity in macrophages, and that this effect is modulated by their fatty acid composition. This downregulation of inflammatory processes in macrophages may represent a protective effect of CMR which is enhanced by dietary polyunsaturated fatty acids

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype

The Crandell-Rees Feline Kidney Cell (CRFK) is an immortalised cell line derived from the feline kidney that is utilised for the growth of certain vaccinal viruses. Confusion exists as to whether CRFK are epithelial or mesenchymal in phenotype. The aim of this study was to characterise CRFK cells via immunofluorescence, enzyme cytochemistry, western blotting, RT-qPCR for S100A4 and comparison to primary feline proximal tubular epithelial cells (FPTEC) and feline cortical fibroblasts (FCF). CRFK cells were of fusiform morphology and appeared similar to FCF. CRFK expressed the mesenchymal intermediate filament (IF) protein vimentin together with two cell adhesion molecules associated with feline fibroblasts (CD29 and CD44), and lacked expression of the epithelial IF cytokeratin, myogenic IF desmin and endothelial marker von Willebrand factor (vWF). In addition, CRFK did not demonstrate brush border enzyme activity typical of FPTEC. S100A4 gene expression, implicated in both neoplastic transformation and epithelial to mesenchymal transition, was highly upregulated in CRFK in comparison to the primary feline renal cells. CRFK appear phenotypically similar to fibroblasts, rather than tubular epithelial cells, and may have undergone neoplastic transformation or epithelial-to-mesenchymal transition after extensive passaging. This finding may have potential implications for future research utilising this cell line

Urinary active transforming growth factor ß in feline chronic kidney disease

The cytokine transforming growth factor beta 1 (TGF-β1) has been widely implicated in the development and progression of renal fibrosis in chronic kidney disease (CKD) in humans and in experimental models. The aims of this study were to assess the association between urinary active TGF-β1 and (a) development of CKD in a cross-sectional study, (b) deterioration of renal function over 1 year in a longitudinal study, and (c) renal histopathological parameters in cats. A human active TGF-β1 ELISA was validated for use in feline urine. Cross-sectional analysis revealed no significant difference in urinary active TGF-β1:creatinine ratio (aTGF-β1:UCr) between groups with differing renal function. Longitudinally, non-azotaemic cats that developed CKD demonstrated a significant (P = 0.028) increase in aTGF-β1:UCr approximately 6 months before the development of azotaemia, which remained elevated (P = 0.046) at diagnosis (approximately 12 months prior, 8.4 pg/mg; approximately 6 months prior, 22.2 pg/mg; at CKD diagnosis, 24.6 pg/mg). In the histopathology study, aTGF-β1:UCr was significantly higher in cats with moderate (P = 0.02) and diffuse (P = 0.005) renal fibrosis than in cats without fibrosis. Cats with moderate renal inflammation had significantly higher urinary active aTGF-β1 concentrations than cats with mild (P = 0.035) or no inflammatory change (P = 0.004). The parameter aTGF-β1:UCr was independently associated with Log urine protein:creatinine ratio in a multivariable analysis of clinicopathological parameters and interstitial fibrosis score in a multivariable analysis of histopathological features. These results suggest that urinary aTGF-β1 reflects the severity of renal pathology. Increases in urinary aTGF-β1 followed longitudinally in individual cats may indicate the development of CKD

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory drugs with nitroxy moiety

Copyright British Pharmacological Society and Nature Publishing GroupEffects of novel nitric oxide-releasing nonsteroidal anti-inflammatory compounds (NONSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a cultured murine macrophage cell line J774.Peer reviewe

Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells

'The final version of record is available at: http://www.biochemj.org/bj/tocprev/toc1999.htm ' --Copyright Biochemical SocietyThe signalling mechanisms involved in the induction of nitric oxide synthase (iNOS) and Larginine transport were investigated in bacterial lypopolysaccharide (LPS) and interferon-γ (IFN-γ)- stimulated rat cultured aortic smooth muscle cells (RASMC). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-γ were also examined. Control RASMC expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and L-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen activated kinases (MAPK), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 μM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors, genistein and herbimycin A. SB203580 differentially regulated iNOS expression and NO production, potentiating both processes at low μM concentrations and inhibiting at concentrations of ≥ 1μM. In conclusion, our data suggest that RASMC constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-γ. Moreover, stimulation of Larginine transport and induction of NO synthesis by LPS and IFN-γ appears to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced L-arginine transport and induced NO synthesis.Peer reviewe

Fatty acids composition of Chylomicron remnants like particles influences their uptake and induction of lipid accumulation in macrophages.

The influence of the fatty acid composition of chylomicron remnant-like particles (CRLPs) on their uptake and induction of lipid accumulation in macrophages was studied. CRLPs containing triacylglycerol enriched in saturated, monounsaturated, n−6 or n−3 polyunsaturated fatty acids derived from palm, olive, corn or fish oil, respectively, and macrophages derived from the human monocyte cell line THP-1 were used. Lipid accumulation (triacylglycerol and cholesterol) in the cells was measured after incubation with CRLPs for 5, 24 and 48 h, and uptake over 24 h was determined using CRLPs radiolabelled with [3H]triolein. Total lipid accumulation in the macrophages was significantly greater with palm CRLPs than with the other three types of particle. This was mainly due to increased triacylglycerol concentrations, whereas changes in cholesterol concentrations did not reach significance. There were no significant differences in lipid accumulation after incubation with olive, corn or fish CRLPs. Palm and olive CRLPs were taken up by the cells at a similar rate, which was considerably faster than that observed with corn and fish CRLPs. These findings demonstrate that CRLPs enriched in saturated or monounsaturated fatty acids are taken up more rapidly by macrophages than those enriched in n−6 or n−3 polunsaturated fatty acids, and that the faster uptake rate results in greater lipid accumulation in the case of saturated fatty acid-rich particles, but not monounsaturated fatty acid-rich particles. Thus, dietary saturated fatty acids carried in chylomicron remnants may enhance their propensity to induce macrophage foam cell formation

Differential regulation of L-arginine transport and nitric oxide synthase induction in cultured smooth muscle cells

"The Version of Record (VoR) is available at www.biochemj.org" [Full text of this article is not available in the UHRA]Peer reviewe