The fact that an increased blood insulin level is observed in patients with coronary artery disease (CAD) confirms the hypothesis that insulin promotes the development of atherosclerosis. The low high-density lipoprotein (HDL) concentration observed in such patients may contribute to alteration in reverse cholesterol transport and promote the accumulation of sterols in vascular tissue. We examined the effect of insulin (20-1000 microU mL-1) on cholesterol efflux into HDL3 particles from human blood monocyte/macrophages and rat peritoneal macrophages preloaded with labelled cholesterol esters, and the influence of insulin on the accumulation of sterols by rat liver cells and HepG2 cell line in vitro models. Insulin at concentrations up to 250 microU mL-1 inhibited the efflux of cholesterol from rat macrophages and promoted high uptake of sterols by both types of hepatic cells. Pharmacological concentrations higher than 250 microU mL-1 exerted the opposite effect. In the case of human macrophages, an insulin concentration of 20 microU mL-1 increased cholesterol removal, whereas 100-200 microU mL-1 insulin inhibited cholesterol removal from cells, and very high concentrations (> 350 microU mL-1) again increased cholesterol removal. We have shown that insulin excess counteracts the beneficial effects of HDL in removing cellular cholesterol and, therefore, may promote development of atherogenesis.

Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10–30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology.

Atherosclerotic plaque formation is often associated with pathological angiogenesis. Modified phospholipids, including oxidized lipoproteins such as LDL, are found to induce adhesion of the monocytes to the endothelial cells and to stimulate their chemotaxis. Effects of oxidized 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) mimic actions of minimally modified LDL in vivo. Interleukin-8 (IL-8) and interleukin-15 (IL-15) are known to induce both inflammation and angiogenesis. The goal of our study was to analyze a potential synergism between ox-PAPC and IL-15 in the in vitro model of angiogenesis carried out in the human endothelial cells (HUVECs). Increasing IL-15 concentrations led to formation of the tube-like structures in the matrigel 3D-model of angiogenesis (P \ 0.05), in contrast to ox-PAPC that inhibited this process. HUVECs incubation with ox-PAPC led to reduced IL-15 gene basal expression (P = 0.033) along with parallel increase, however statistically insignificant, of basal gene expression of IL-8 (P = 0.086). Our findings point to the ox-PAPC opposite effects on the IL-8-and IL-15-mediated angiogenic responses that contribute to pathological angiogenesis induced by ox-LDL.

Background and objectives Mucopolysaccharidosis type VI (MPS VI) is a rare, autosomal recessive lysosomal storage disorder caused by deficient enzymatic activity of N -acetyl galactosamine-4-sulphatase, which is caused by mutations in the arylsulphatase B ( ARSB ) gene. To date, 163 different types of mutations in the ARSB have been reported. However, the full mutation spectrum in the MPS VI phenotype is still not known. The aim of this study was to perform molecular testing of the ARSB gene in the patient and his family members to confirm MPS VI. Methods Molecular characterisation of the ARSB gene was performed using Sanger sequencing. We studied a child suspected of having MPS VI and 16 other relatives. Results We identified a C-to-T transition resulting in an exchange of the Arg codon 160 for a premature stop codon (R160*, in exon 2). The transition was in CpG dinucleotides. Interpretation and conclusions The study provided some insights into the genotype-phenotype relationship in MPS VI and the importance of genetic testing when diagnosing MPS, which is not a mandatory test for the diagnosis and only very occasionally performed. Additionally, we present here the history of a family with confirmed MPS VI, which is extremely rare especially in south-eastern Poland. What is more, the position where the mutation is located is very interesting because it is the region of CpG, which is the site of the methylation process. Thus, this opens the possibility of a new approach indicating the involvement of an epigenetic mechanism that should be examined in the context of the pathomechanism of MPS.

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