Impact of the B Cell Growth Factor APRIL on the Qualitative and Immunological Characteristics of Atherosclerotic Plaques

Studies on the role of B lymphocytes in atherosclerosis development, have yielded contradictory results. Whereas B lymphocyte-deficiency aggravates atherosclerosis in mice; depletion of mature B lymphocytes reduces atherosclerosis. These observations led to the notion that distinct B lymphocyte subsets have different roles. B1a lymphocytes exert an atheroprotective effect, which has been attributed to secretion of IgM, which can be deposited in atherosclerotic lesions thereby reducing necrotic core formation. Tumor necrosis factor (TNF)-family member ‘A Proliferation-Inducing Ligand’ (APRIL, also known as TNFSF13) was previously shown to increase serum IgM levels in a murine model. In this study, we investigated the effect of APRIL overexpression on advanced lesion formation and composition, IgM production and B cell phenotype. We crossed APRIL transgenic (APRIL-Tg) mice with ApoE knockout (ApoE^-/-) mice. After a 12-week Western Type Diet, ApoE^-/-APRIL-Tg mice and ApoE^-/- littermates showed similar increases in body weight and lipid levels. Histologic evaluation showed no differences in lesion size, stage or necrotic area. However, smooth muscle cell (α-actin stain) content was increased in ApoE^-/-APRIL-Tg mice, implying more stable lesions. In addition, increases in both plaque IgM deposition and plasma IgM levels were found in ApoE^-/-APRIL-Tg mice compared with ApoE^-/- mice. Flow cytometry revealed a concomitant increase in peritoneal B1a lymphocytes in ApoE^-/-APRIL-Tg mice. This study shows that ApoE^-/-APRIL-Tg mice have increased oxLDL-specific serum IgM levels, potentially mediated via an increase in B1a lymphocytes. Although no differences in lesion size were found, transgenic ApoE^-/-APRIL-Tg mice do show potential plaque stabilizing features in advanced atherosclerotic lesions.     

Effects of CRP infusion on endothelial function and coagulation in normocholesterolemic and hypercholesterolemic subjects

C-reactive protein (CRP) has been suggested to exert direct adverse effects on the vasculature in experimental setups, including endothelial dysfunction and proinflammatory changes. Here, we assessed the consequences of 1.25 mg/kg highly purified recombinant human CRP, administered as an intravenous bolus, in six patients with familial hypercholesterolemia (FH) and six normocholesterolemic subjects. Endothelium-dependent and -independent vasoreactivity to serotonin and nitroprusside, respectively, were assessed using venous occlusion plethysmography before and after CRP infusion. For biochemical analyses, blood was drawn at different time points. At baseline, FH patients showed blunted endothelium-dependent vasodilation (maximum, 89.2 +/- 30.0% vs. 117.7 +/- 13.1% in normolipidemic subjects; P = 0.037). Procoagulant activity was also higher in FH patients, illustrated by increased prothrombin fragment 1+2 (F(1+2)) levels (P = 0.030) and plasminogen activator inhibitor type-1 (PAI-1) activity (P = 0.016). Upon CRP challenge, endothelium-dependent vasodilator capacity further deteriorated in FH patients (P = 0.029), whereas no change in vascular reactivity was observed in normolipidemic subjects. Additionally, coagulation activation was augmented in FH patients compared with normolipidemic subjects (P = 0.009 for F(1+2) levels; P = 0.018 and P = 0.003 for PAI-1 antigen and activity, respectively). No difference in inflammatory responses was observed between groups. In hypercholesterolemic patients, CRP aggravates endothelial dysfunction and also evokes augmented procoagulant responses. These findings suggest that particularly in hypercholesterolemia, CRP-lowering strategies should be considered in addition to LDL reduction.     

Heterozygosity for a loss-of-function mutation in GALNT2 improves plasma triglyceride clearance in man

Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGalNAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.     

Pathophysiology of hypertriglyceridemia

The importance of triglycerides as risk factor for CVD is currently under debate. The international guidelines do not include TG into their risk calculator despite the recent observations that plasma TG is an independent risk factor for CVD. The understanding of the pathophysiology of triglycerides opens up avenues for development of new drug targets. Hypertriglyceridemia occurs through 1. Abnormalities in hepatic VLDL production, and intestinal chylomicron synthesis 2. Dysfunctional LPL-mediated lipolysis or 3. Impaired remnant clearance. The current review will discuss new aspects in lipolysis by discussing the role of GPIHBP1 and the involvement of apolipoproteins and in the process of hepatic remnant clearance with a focus upon the role of heparin sulfate proteoglycans. Finally we will shortly discuss future perspectives for novel therapies aiming at improving triglyceride homeostasis. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Partial recovery of the endothelial glycocalyx upon rosuvastatin therapy in patients with heterozygous familial hypercholesterolemia

The endothelial glycocalyx has been shown to serve as a protective barrier between the flowing blood and the vessel wall in experimental models. The aim of this study was to evaluate whether hypercholesterolemia is associated with glycocalyx perturbation in humans, and if so, whether statin treatment can restore this. We measured systemic glycocalyx volume (V(G)) in 13 patients with heterozygous familial hypercholesterolemia (FH) after cessation of lipid-lowering therapy for a minimum of 4 weeks and 8 weeks after initiating rosuvastatin therapy. Normocholesterolemic subjects were used as controls. V(G) was estimated by subtracting the intravascular distribution volume of a glycocalyx permeable tracer (dextran 40) from that of a glycocalyx impermeable tracer (labeled erythrocytes). V(G) in untreated FH patients [LDL 225 +/- 57 mg/dl (mean +/- SD)] was significantly reduced compared with controls (LDL 93 +/- 24 mg/dl) (V(G) 0.8 +/- 0.3 vs. 1.7 +/- 0.6, respectively, P < 0.001). After normalization of LDL levels (95 +/- 33 mg/dl) upon 8 weeks of statin treatment, V(G) recovered only partially (V(G) 1.1 +/- 0.4 L, P = 0.04). The endothelial glycocalyx is profoundly reduced in FH patients, which may contribute to increased atherogenic vulnerability. This perturbation is partially restored upon short-term statin therapy.     

Endothelial surface layer degradation by chronic hyaluronidase infusion induces proteinuria in apolipoprotein E-deficient mice

Objective

Functional studies show that disruption of endothelial surface layer (ESL) is accompanied by enhanced sensitivity of the vasculature towards atherogenic stimuli. However, relevance of ESL disruption as causal mechanism for vascular dysfunction remains to be demonstrated. We examined if loss of ESL through enzymatic degradation would affect vascular barrier properties in an atherogenic model.

Methods

Eight week old male apolipoprotein E deficient mice on Western-type diet for 10 weeks received continuous active or heat-inactivated hyaluronidase (10 U/hr, i.v.) through an osmotic minipump during 4 weeks. Blood chemistry and anatomic changes in both macrovasculature and kidneys were examined.

Results

Infusion with active hyaluronidase resulted in decreased ESL (0.32±0.22 mL) and plasma volume (1.03±0.18 mL) compared to inactivated hyaluronidase (0.52±0.29 mL and 1.28±0.08 mL, p<0.05 respectively).Active hyaluronidase increased proteinuria compared to inactive hyaluronidase (0.27±0.02 vs. 0.15±0.01 µg/µg protein/creatinin, p<0.05) without changes in glomerular morphology or development of tubulo-interstitial inflammation. Atherosclerotic lesions in the aortic branches showed increased matrix production (collagen, 32±5 vs. 18±3%; glycosaminoglycans, 11±5 vs. 0.1±0.01%, active vs. inactive hyaluronidase, p<0.05).

Conclusion

ESL degradation in apoE deficient mice contributes to reduced increased urinary protein excretion without significant changes in renal morphology. Second, the induction of compositional changes in atherogenic plaques by hyaluronidase point towards increased plaque vulnerability. These findings support further efforts to evaluate whether ESL restoration is a valuable target to prevent (micro) vascular disease progression.     

From evidence to practice: development of web-based Dutch lipid reference values

IntroductionIn the Netherlands, the total number of yearly measured lipid profiles exceeds 500,000. While lipid values are strongly affected by age and sex, until recently, no up-to-date age- and sex-specific lipid reference values were available. We describe the translation of big-cohort lipid data into accessible reference values, which can be easily incorporated in daily clinical practice.MethodsLipid values (total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides) from all healthy adults and children in the LifeLines cohort were used to generate age- and sex-specific percentiles. A combination of RStudio, Cascading Style Sheets and HyperText Markup Language was used to interactively display the percentiles in a responsive web layout.ResultsAfter exclusion of subjects reporting cardiovascular disease or lipid-lowering therapy at baseline, 141,611 subjects were included. On the website, input fields were created for age, sex and all main plasma lipids. Upon input of these values, corresponding percentiles are calculated, and output is displayed in a table and an interactive graph for each lipid. The website has been made available in both Dutch and English and can be accessed at www.lipidtools.com.ConclusionWe constructed the first searchable, national lipid reference value tool with graphical display in the Netherlands to use in screening for dyslipidaemias and to reduce the underuse of lipid-lowering therapy in Dutch primary prevention. This study illustrates that data collected in big-cohort studies can be made easily accessible with modern digital techniques and preludes the digital health revolution yet to come.     

Carriers of Loss-of-Function Mutations in EXT Display Impaired Pancreatic Beta-Cell Reserve Due to Smaller Pancreas Volume

Exotosin (EXT) proteins are involved in the chain elongation step of heparan sulfate (HS) biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF) in EXT1 and EXT2 result in hereditary exostoses (HME). Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME) affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT) followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS) in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI). OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46–1.16] vs. controls 1.53 [0.69–3.36] nmol·l⁻¹·min⁻¹, p<0.05). Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22–10.5] vs. controls 10.2 [7.91–12.70] nmol·l⁻¹·min⁻¹ p<0.05), indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm³ p = 0.04). In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.