Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity.

BACKGROUND: Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs). METHODS: We developed a new cell-based high-content assay, the BioHybrid assay, to measure in vitro calcification. The BioHybrid assay was compared with the o-Cresolphthalein assay and the T50 assay. Serum and plasma were derived from different cohort studies including chronic kidney disease (CKD) stages III, IV, V and VD (on dialysis), pseudoxanthoma elasticum (PXE) and other cardiovascular diseases including serum from participants with mild and extensive coronary artery calcification (CAC). hVSMCs were exposed to serum and plasma samples, and in vitro calcification was measured using AlexaFluor®-546 tagged fetuin-A as calcification sensor. RESULTS: The BioHybrid assay measured the kinetics of calcification in contrast to the endpoint o-Cresolphthalein assay. The BioHybrid assay was more sensitive to pick up differences in calcification propensity than the T50 assay as determined by measuring control as well as pre- and post-dialysis serum samples of CKD patients. The BioHybrid response increased with CKD severity. Further, the BioHybrid assay discriminated between calcification propensity of individuals with a high CAC index and individuals with a low CAC index. Patients with PXE had an increased calcification response in the BioHybrid assay as compared to both spouse and control plasma samples. Finally, vitamin K1 supplementation showed lower in vitro calcification, reflecting changes in delta Agatston scores. Lower progression within the BioHybrid and on Agatston scores was accompanied by lower dephosphorylated-uncarboxylated matrix Gla protein levels. CONCLUSION: The BioHybrid assay is a novel approach to determine the vascular calcification propensity of an individual and thus may add to personalised risk assessment for CVD.

Targeting Coagulation Factor Xa Promotes Regression of Advanced Atherosclerosis in Apolipoprotein-E Deficient Mice.

Atherosclerosis is a progressive inflammatory vascular disorder, complicated by plaque rupture and subsequently atherothrombosis. In vitro studies indicate that key clotting proteases, such as factor Xa (FXa), can promote atherosclerosis, presumably mediated through protease activated receptors (PARs). Although experimental studies showed reduced onset of atherosclerosis upon FXa inhibition, the effect on pre-existing plaques has never been studied. Therefore, we investigated effects of FXa inhibition by rivaroxaban on both newly-formed and pre-existing atherosclerotic plaques in apolipoprotein-e deficient (ApoE-/-) mice. Female ApoE-/- mice (age: 8-9 weeks, n = 10/group) received western type diet (WTD) or WTD supplemented with rivaroxaban (1.2 mg/g) for 14 weeks. In a second arm, mice received a WTD for 14 weeks, followed by continuation with either WTD or WTD supplemented with rivaroxaban (1.2 mg/g) for 6 weeks (total 20 weeks). Atherosclerotic burden in aortic arch was assessed by haematoxilin & eosin immunohistochemistry (IHC); plaque vulnerability was examined by IHC against macrophages, collagen, vascular smooth muscle cells (VSMC) and matrix metalloproteinases (MMPs). In addition, PAR1 and -2 expressions and their main activators thrombin and FXa in the plaque were determined in the plaque. Administration of rivaroxaban at human therapeutic concentrations reduced the onset of atherosclerosis (-46%, p < 0.05), and promoted a regression of pre-existing plaques in the carotids (-24%, p < 0.001). In addition, the vulnerability of pre-existing plaques was reduced by FXa inhibition as reflected by reduced macrophages (-39.03%, p < 0.05), enhanced collagen deposition (+38.47%, p < 0.05) and diminished necrotic core (-31.39%, p < 0.05). These findings were accompanied with elevated vascular smooth muscle cells and reduced MMPs. Furthermore, expression of PARs and their activators, thrombin and FXa was diminished after rivaroxaban treatment. Pharmacological inhibition of FXa promotes regression of advanced atherosclerotic plaques and enhances plaque stability. These data suggest that inhibition of FXa may be beneficial in prevention and regression of atherosclerosis, possibly mediated through reduced activation of PARs.

ω6/ω3 Polyunsaturated fatty acid supplementations in renal cell model lead to a particular regulation through lipidome for preserved ω6/ω3 ratios.

Polyunsaturated fatty acids (PUFA) supplementations modify cell lipid composition leading to a change in cell function. However, the effect of PUFA supplementations in renal model cell on the kidney epithelial cells membrane fatty acid profile is not known. Therefore, the purpose of this study was to determine the effects of PUFAs with different ω6/ω3 ratios supplementations in the kidney epithelial cells and the type of supplementation that can be used as cellular protection during kidney transplantation. For that, we used as model the LLCPK1 cell and determined their membrane fatty acid (FA) composition after supplementation with three different commercial food supplements. These supplements consist of S1: docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) with ω6/ω3 ratio = 0.1, S2: DHA, EPA, linoleic acid (LA) and γ-linoleic acid (GLA) with ω6/ω3 ratio = 2.5, or S3: α-linolenic acid (ALA) and LA with ω6/ω3 ratio near 1. Cells were incubated for 24 hr with 30 µM of ω3 fatty acids from each supplement. Fatty acid composition of control and experimental groups was analysed by gas chromatography after extraction of lipids and fatty acids methylation. The efficiency of cell PUFA supplementation was achieved by showing 2 to 4 fold increases in cell PUFA incorporation. Whatever the supplementation used, the cell saturated fatty acids (SFA) were decreased by 50% following the three supplementations used (p<0.001) as compared to control group. These decreases in SFA were compensated in part by increasing monounsaturated fatty acid levels. All these changes were observed with constant of cell ω6/ω3 ratio whatever the supplementations used. These data suggest that the supplements, with long chain polyunsaturated fatty acids or their precursors, lead to important regulation in the lipidome (desaturases and elongases) associated to preserved ω6/ω3 ratios. The fatty acids remodeling may represent an interesting new mechanism by which renal FA homoestasis could occurred.

Evolution of music by public choice.

Music evolves as composers, performers, and consumers favor some musical variants over others. To investigate the role of consumer selection, we constructed a Darwinian music engine consisting of a population of short audio loops that sexually reproduce and mutate. This population evolved for 2,513 generations under the selective influence of 6,931 consumers who rated the loops' aesthetic qualities. We found that the loops quickly evolved into music attributable, in part, to the evolution of aesthetically pleasing chords and rhythms. Later, however, evolution slowed. Applying the Price equation, a general description of evolutionary processes, we found that this stasis was mostly attributable to a decrease in the fidelity of transmission. Our experiment shows how cultural dynamics can be explained in terms of competing evolutionary forces.

Emulsification and lipolysis of triacylglycerols are altered by viscous soluble dietary fibres in acidic gastric medium in vitro.

This in vitro study was designed to test the hypothesis that soluble dietary fibres can alter the process of intragastric lipid emulsification and possibly subsequent triacylglycerol lipolysis. Three guar gums, two pectins and gum arabic were dissolved in acidic gastric medium in the concentration range 0.3-2.0% (w/v). Viscosities of fibre solutions were measured and apparent viscosities varied over a wide range (0.7-77 mPa/s). Emulsification of a lipid mixture (triolein/phosphatidylcholine/cholesterol) was performed under mild conditions in the presence of increasing concentrations of soluble fibres. The amount of emulsified lipid was not affected whereas the size of the emulsified droplets was increased by raising the concentration of viscous fibres only. The droplet size (r=0.75, P=0.006) and overall droplet surface area (r=-0.69, P=0.009) were strongly correlated with the medium viscosity in the range 0-20 mPa/s. The addition of solutions of viscous fibres to a preformed standard emulsion did not change the initial velocity of human gastric lipase reaction. Conversely, when emulsions prepared in the presence of fibres (i.e. with different droplet sizes) were incubated with excess gastric enzyme for 2 h, the high-viscosity guar gum significantly reduced the extent of triacylglycerol lipolysis, as compared with control and low- or medium-viscosity fibres. In conclusion, the data obtained show that reducing emulsification of dietary lipids in the mildly acid medium found in the stomach is a mechanism by which soluble viscous fibres can alter lipid assimilation.

Chronic oat bran intake alters postprandial lipemia and lipoproteins in healthy adults.

This study evaluates the possible interaction between chronic oat bran intake and the postmeal metabolic response. Six normolipidemic men consumed three different diets for 14 d, at the end of which they consumed a test meal. The diets were C (control), basal low-fiber diet (15.6 g fiber/d) and a low-fiber (2.8 g fiber) test meal; OB (oat bran), basal low-fiber diet and a 40-g oat bran-enriched test meal (12.8 g fiber); and OB-A (oat bran-adaptation), 14-d oat bran (40 g/d) supplemented diet (23.8 g fiber/d) and an oat bran test meal (12.8 g fiber). The diets were fed in a random order. Fasting and postmeal blood samples were obtained for 7 h and lipoproteins were isolated. Adding oat bran to the test meals markedly reduced the postmeal insulin rise (P < 0.05). Compared with the low-fiber control diet, the effects elicited postprandially by adding oat bran to a single meal were enhanced after 14 d of oat bran feeding, ie, increased plasma triglycerides, phospholipids, and free cholesterol; decreased plasma esterified cholesterol; increased chylomicron and small-sized triglyceride-rich lipoprotein triglycerides; increased LDL and HDL free cholesterol; and decreased HDL esterified cholesterol. Thus, chronic oat bran feeding alters the postmeal response in human subjects.

Effects of pea and soybean fibre on postprandial lipaemia and lipoproteins in healthy adults.

To evaluate some possible mechanisms whereby total dietary fibre (TDF) may affect lipid metabolism in humans, six normolipidaemic males ingested on separate days a low-fibre test meal (2.8 g TDF) containing 70 g fat and 756 mg cholesterol, enriched with 10 g TDF in the form of either pea fibre or soybean fibre. Fasting and post-meal blood samples were obtained for 7 h and chylomicrons (CM) were isolated. Lipoproteins (VLDL+CM remnants, LDL, HDL) were isolated from the baseline samples and the samples of the 2-3 h triglyceride peaks. As compared to the postprandial response given by the control low-fibre test meal, adding fibre induced no change in serum glucose, insulin or Apo A1 and Apo B variations. The serum triglyceride response was not altered by adding fibres but the 2-3 h chylomicron triglyceride rise was increased (P < or = 0.05) by soybean fibre. VLDL+CM remnants, LDL and HDL triglyceride variations were unchanged with fibres. Cholesterolaemia decreased postprandially for 6 h, and was further lowered in the presence of pea fibre. This resulted from a marked decrease in serum esterified cholesterol. The chylomicron cholesterol and phospholipid rise was lowered in the presence of either fibre. The postprandial changes in the free cholesterol concentrations of the various lipoprotein classes were not altered by fibre whereas changes from baseline in esterified cholesterol concentrations were reduced by soybean fibre in LDL and amplified by soybean and pea fibres in HDL. The results obtained show that dietary fibre present in legumes may alter postprandial lipaemia and lipoproteins in humans to a variable extent. These effects could be related to some long-term metabolic effects.