Heterogeneous stock mice are susceptible to encephalomyelitis and antibody-initiated arthritis but not to collagen- and G6PI-induced arthritis.

The strategy of using heterogeneous stock (HS) mice has proven to be successful in fine mapping of quantitative trait loci in complex diseases. However, whether these mice can be used for arthritis, encephalomyelitis and autoimmune phenotypes has not been addressed. Here, we screened the Northport HS mice for arthritis phenotypes using three different models: collagen-induced arthritis (CIA), using rat, bovine or chicken collagen type II (CII); recombinant human glucose-6-phosphate isomerase (G6PI)-induced arthritis; and collagen antibody-induced arthritis (CAIA). Irrespective of the origin of collagen, we found HS mice to be fairly resistant to CIA and G6PI-induced arthritis, despite the development of antibodies against the respective antigens. On the other hand, HS mice were found to be susceptible for CAIA. Similarly, these mice developed encephalomyelitis (EAE) induced either with mouse or rat spinal cord homogenate (SCH), or with recombinant rat myelin oligodendrocyte glycoprotein, with elevated antibody levels against CNS proteins. Accordingly, we conclude that the use of HS mice for fine mapping and positional cloning of gene(s) involved in CAIA and EAE is possible, but not for collagen- and G6PI-induced arthritis.

3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition prevents endothelial NO synthase downregulation by atherogenic levels of native LDLs: balance between transcriptional and posttranscriptional regulation.

Atherogenic levels of native low density lipoproteins (nLDLs) decrease the bioavailability of endothelium-derived NO and downregulate endothelial NO synthase (eNOS) expression in cultured human endothelial cells. Here, we show that simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, within the therapeutic range (0.01 to 1 micromol/L) prevented the downregulation of eNOS mRNA and protein promoted by nLDL (180 mg cholesterol/dL, 48 hours) in human umbilical vein endothelial cells. This effect of simvastatin was completely reversed by mevalonate, the product of the reaction, and to a lesser extent by farnesol and geranyl geraniol. Simvastatin significantly stabilized eNOS mRNA in cells treated with nLDL during 48 hours (eNOS mRNA half-life approximately 11 hours in controls versus >24 hours in nLDL per 0.1 micromol/L simvastatin-treated cells). The downregulation of eNOS by nLDL was abrogated by cycloheximide, an inhibitor of protein synthesis, and by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor that reduces the catabolism of sterol regulatory element binding proteins. Sterol deprivation increased the downregulation produced by nLDL on eNOS and sterol regulatory element binding protein-2 expression levels. However, no differential modulation of the retardation bands corresponding to the putative sterol-responsive element present in the eNOS promoter was detected by electrophoretic mobility shift assay. Our results suggest that nLDL promote eNOS downregulation operating at a transcriptional level, whereas simvastatin prevents such an effect through a posttranscriptional mechanism.

Modulation of ERG25 expression by LDL in vascular cells.

BACKGROUND: Plasma low density lipoproteins (LDL) play a key role in the pathogenesis of atherosclerosis. LDL modify gene expression in vascular cells leading to disturbances in the functional state of the vessel wall. METHODS: Expression levels of C-4 sterol methyl oxidase gene (ERG25), sterol regulatory element binding protein (SREBP)-1 and -2 were evaluated in porcine aortic endothelial cells (PAEC), porcine and human smooth muscle cells (SMC) and in the vascular wall from normolipemic and hyperlipemic pigs by RT-PCR. SREBP-1 protein levels were assessed by Western blot and SREBP-SRE binding by EMSA. SREBP-2 was overexpressed by transient transfection with lipofectin. RESULTS: We have identified expression of the ERG25 in vascular cells and analyzed its regulation by LDL. ERG25, an enzyme involved in cholesterol biosynthesis, is expressed in vascular endothelial and SMC from porcine and human origin and is downregulated by LDL in a time- and dose-dependent manner. Downregulation of ERG25 by LDL was abolished by an inhibitor of neutral cysteine proteases (N-acetyl-leucyl-leucyl-norleucinal) that abrogates SREBP catabolism. LDL downregulated SREBP-2 mRNA levels but not SREBP-1 expression in these cells and both ERG25 and SREBP-2 gene expression was significantly decreased in the vascular wall of diet-induced hypercholesterolemic swine. Finally, in cell transfection experiments SREBP-2 overexpression blocks ERG25 downregulation caused by LDL. CONCLUSIONS: Our results indicate that LDL modulate ERG25 expression in the vascular wall and suggest the involvement of SREBP-2 in this mechanism.

Lysyl oxidase (LOX) down-regulation by TNFalpha: a new mechanism underlying TNFalpha-induced endothelial dysfunction.

OBJECTIVE: TNFalpha is a pro-inflammatory cytokine that induces endothelial dysfunction and promotes atherosclerosis progression. Down-regulation of lysyl oxidase (LOX), a key enzyme in extracellular matrix maturation, by pro-atherogenic risk factors such as LDL and homocysteine, is associated with an impairment of endothelial barrier function. Our hypothesis is that the inflammatory cytokine TNFalpha could also modulate LOX expression/function in endothelial cells. METHODS: The study was carried out in human umbilical vein endothelial cells (HUVEC), porcine aortic endothelial cells (PAEC) and bovine aortic endothelial cells (BAEC). LOX mRNA levels were analysed by real-time PCR and LOX activity was assessed by a high sensitive fluorescent assay. Promoter activity was determined by transient transfection using a luciferase reporter system. RESULTS: TNFalpha decreases LOX mRNA levels in endothelial cells in a dose- and time-dependent manner. The effect of TNFalpha was observed at low concentrations (0.1-1 ng/mL) and was maximal at 2.5 ng/mL (after 21 h). In transfection assays, TNFalpha reduced LOX transcriptional activity to a similar extent than LOX mRNA. Furthermore, TNFalpha decreases endothelial LOX enzymatic activity. By using both TNF receptor (TNFR) agonist and blocking antibodies we determined the involvement of TNFR2 on LOX down-regulation. Moreover, while TNFR-associated factor-2 (TRAF-2) did not mediate signalling events leading to LOX inhibition, PKC inhibitors counteracted the TNFalpha-induced decrease of LOX mRNA levels. Finally, TNFalpha administration significantly reduced vascular LOX expression in rat aorta. CONCLUSIONS: Endothelial dysfunction induced by TNFalpha is associated with a decrease of LOX expression/activity. Thus, LOX seems to be involved in the impairment of endothelial function triggered by different pathological conditions.