Hans Dieplinger

Elevated plasma concentrations of lipoprotein(a) in patients with end-stage renal disease are not related to the size polymorphism of apolipoprotein(a).

Patients with terminal renal insufficiency suffer from an increased incidence of atherosclerotic diseases. Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been established as a genetically controlled risk factor for these diseases. Variable alleles at the apo(a) gene locus determine to a large extent the Lp(a) concentration in the general population. In addition, other genetic and nongenetic factors also contribute to the plasma concentrations of Lp(a). We therefore investigated Apo(a) phenotypes and Lp(a) plasma concentrations in a large group of patients with end-stage renal disease (ESRD) and in a control group. Lp(a) concentrations were significantly elevated in ESRD patients (20.1 +/- 20.3 mg/dl) as compared with the controls (12.1 +/- 15.5 mg/dl, P < 0.001). However, no difference was found in apo(a) isoform frequency between the ESRD group and the controls. Interestingly, only patients with large size apo(a) isoforms exhibited two- to fourfold elevated levels of Lp(a), whereas the small-size isoforms had similar concentrations in ESRD patients and controls. Beside elevated Lp(a) concentrations, ESRD patients had lower levels of plasma cholesterol and apolipoprotein B. These results show that elevated Lp(a) plasma levels might significantly contribute to the risk for atherosclerotic diseases in ESRD. They further indicate that nongenetic factors related to renal insufficiency or other genes beside the apo(a) structural gene locus must be responsible for the high Lp(a) levels.

Afamin and Apolipoprotein A-IV: Novel Protein Markers for Ovarian Cancer

Comparative proteomics identified the vitamin E-binding plasma protein afamin as a potential novel tumor marker for ovarian cancer. In addition, we observed in a previous small study decreased plasma concentrations of apolipoprotein A-IV (apoA-IV) in preoperative patients with kidney cancer. The aim of this study was therefore to analyze afamin and apoA-IV in a large case-control study to evaluate the diagnostic utility of the two potential novel tumor markers in ovarian cancer patients. We measured plasma concentrations of afamin and apoA-IV by means of a specific sandwich-type ELISA using affinity-purified polyclonal and monoclonal antibodies in 181 ovarian cancer patients of various clinical stages, 399 patients with benign gynecologic diseases, including endometriosis, and 177 controls and compared results with those for the conventional ovarian cancer tumor marker cancer antigen 125 (CA125). Afamin concentrations decreased from a median of 70.7 mg/L (range, 34.6-116.1 mg/L) in healthy controls to 65.2 mg/L (range, 20.2-206.6 mg/L) in patients with benign gynecologic diseases to 56.0 mg/L (range, 4.7-96.0 mg/L) in ovarian cancer patients (P < 0.001 for all pairwise comparisons). Similar results were obtained with apoA-IV concentrations decreasing from 13.0 mg/dL (range, 5.5-34.0 mg/dL) in controls to 11.7 mg/dL (range, 2.0-32.3 mg/dL) in benign conditions to 9.4 mg/dL (range, 0.3-29.5 mg/dL) in ovarian cancer (all P < 0.001). Receiver operating characteristic analysis for differentiating ovarian cancer patients from healthy controls revealed for a specificity of 90% sensitivity values of 92.4%, 42.4%, and 40.8% for CA125, afamin, and apoA-IV, respectively. Afamin, but not apoA-IV, added independent diagnostic information to CA125 and age for differentiating ovarian cancer from benign and healthy samples; the odds ratio of ovarian cancer was reduced by 44% for each doubling of afamin (P = 0.032). The relatively low sensitivity, however, clearly indicates that afamin and apoA-IV alone are not sufficiently suitable as diagnostic markers for ovarian cancer. Afamin contributes, however, independent diagnostic information to CA125, thus establishing its potential as an adjunct marker to CA125. (Cancer Epidemiol Biomarkers Prev 2009;18(4):1127–33)

Familial hypercholesterolaemia: A global call to arms.

Familial Hypercholesterolaemia (FH) is the commonest autosomalnco-dominantly inherited condition affecting man. It is causednby mutation in one of three genes, encoding the low-densitynlipoprotein (LDL) receptor, or the gene for apolipoprotein Bn(which is the major protein component of the LDL particle), ornin the gene coding for PCSK9 (which is involved in thendegradation of the LDL-receptor during its cellular recycling).nThese mutations result in impaired LDL metabolism, leading tonlife-long elevations in LDL-cholesterol (LDL-C) and developmentnof premature atherosclerotic cardiovascular disease (ASCVD)n[1], [2] and [3]. If left untreated, the relative risk ofnpremature coronary artery disease is significantly higher innheterozygous patients than unaffected individuals, with mostnuntreated homozygotes developing ASCVD before the age of 20 andngenerally not surviving past 30 years [2], [3], [4] and [5].nAlthough early detection and treatment with statins and othernLDL-C lowering therapies can improve survival, FH remainsnwidely underdiagnosed and undertreated [1], therebynrepresenting a major global public health challenge.

Lecithin cholesterol acyl transferase deficiency : molecular analysis of a mutated allele

SummaryThe enzyme, lecithin cholesterol acyltransferase (LCAT), is responsible for the esterification of plasma cholesterol mediating the transfer of an acyl group from lecithin to the 3-hydroxy group of cholesterol. Deficiency of the enzyme is a well-known syndrome with a widespread geographic occurrence. We have cloned an allele from a patient homozygous for the LCAT deficiency. The only change that we could detect is a C to T transition in the fourth exon of the gene; this causes a substitution of Arg for Trp at position 147 of the mature protein. The functional significance of such a substitution with respect to the enzyme defect was demonstrated by transfecting the mutated LCAT gene in the cell line COS-1.

In vivo stable-isotope kinetic study suggests intracellular assembly of lipoprotein(a)

OBJECTIVEnLipoprotein(a) [Lp(a)] consists of apolipoprotein B-100 (apoB-100) as part of an LDL-like particle and the covalently linked glycoprotein apolipoprotein(a) [apo(a)]. Detailed mechanisms of its biosynthesis, assembly, secretion and catabolism are still poorly understood. To address the Lp(a) assembly mechanism, we studied the in vivo kinetics of apo(a) and apoB-100 from Lp(a) and LDL apoB-100 in nine healthy probands using stable-isotope methodology.nnnMETHODSnThe level of isotope enrichment was used to calculate the fractional synthesis rate (FSR), production rate (PR) and retention time (RT) using SAAMII software and multicompartmental modeling.nnnRESULTSnWe observed a similar mean PR for apo(a) (1.15 nmol/kg/d) and apoB-100 (1.31 nmol/kg/d) from Lp(a), which differed significantly from the PR for apoB-100 from LDL (32.6 nmol/kg/d). Accordingly, mean FSR and RT values for Lp(a)-apo(a) were similar to those of Lp(a)-apoB and different from those for LDL-apoB.nnnCONCLUSIONnTwo different kinetic apoB pools within Lp(a) and LDL suggest intracellular Lp(a) assembly from apo(a) and newly synthesized LDL.

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood-brain barrier.

α‐Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH‐binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high‐density lipoprotein‐mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo.

Afamin secreted from nonresorbing osteoclasts acts as a chemokine for preosteoblasts via the Akt-signaling pathway

Although it is well known that osteoclastic bone resorption is followed by osteoblastic bone formation, questions remain as to when coupling factors are produced during bone resorption and which stages of bone formation are affected by these factors. To clarify these mechanisms, we established an in vitro system to investigate the coupling phenomenon. We obtained conditioned media (CM) from osteoclasts in the early and late stages of differentiation and from bone resorption stages. The collected CM was used to treat primary mouse calvarial osteoblasts and preosteoblastic MC3T3-E1 cells and to evaluate its influence on the migration, viability, proliferation, and differentiation of osteoblasts. We found that CM from osteoclasts in the early stage of differentiation predominantly stimulated the migration of osteoblastic lineages. By further performing fractional analyses of the CM with liquid chromatography-tandem mass spectrometry, we identified afamin, which has binding activity with vitamin E, as a possible coupling factor. The CM collected from afamin siRNA-transfected osteoclasts significantly suppressed preosteoblast migration. Afamin activated Akt in preosteoblasts, and pretreatment with Akt inhibitor significantly blocked afamin-stimulated preosteoblast migration. In conclusion, these results indicate that osteoclasts themselves play a central role in the coupling of bone resorption and formation by stimulating preosteoblast migration. In addition, we identified afamin as one of osteoclast-derived chemokines that affect preosteoblasts through the activation of the Akt-signaling pathway.

Afamin--A pleiotropic glycoprotein involved in various disease states.

The human glycoprotein afamin was discovered as the fourth member of the albumin gene family. Despite intense research over the last 20 years, our knowledge of afamins physiological or pathophysiological functions is still very limited. Circulating afamin is primarily of hepatic origin and abundant concentrations are found in plasma, cerebrospinal, ovarian follicular and seminal fluids. In vitro binding studies revealed specific binding properties for vitamin E. A previously performed analytical characterization and clinical evaluation study of an enzyme-linked immunosorbent assay for quantitative measurement of afamin in human plasma demonstrated that the afamin assay meets the quality specifications for laboratory medicine. Comparative proteomics has identified afamin as a potential biomarker for ovarian cancer and these findings were confirmed by quantitative immunoassay of afamin and validated in independent cohorts of patients with ovarian cancer. Afamin has also been investigated in other types of carcinoma. Most of these studies await further evaluation with validated quantitative afamin assays and require validation in larger patient cohorts. Transgenic mice overexpressing the human afamin gene revealed increased body weight and increased blood concentrations of lipids and glucose. These transgenic mouse data were in line with three large human population-based studies showing that afamin is strongly associated with the prevalence and development of the metabolic syndrome. This review summarizes and discusses the molecular, biochemical and analytical characterization of afamin as well as possible clinical applications of afamin measurement.

Genetic associations with lipoprotein subfractions provide information on their biological nature

Adverse levels of lipoproteins are highly heritable and constitute risk factors for cardiovascular outcomes. Hitherto, genome-wide association studies revealed 95 lipid-associated loci. However, due to the small effect sizes of these associations large sample numbers (>100 000 samples) were needed. Here we show that analyzing more refined lipid phenotypes, namely lipoprotein subfractions, can increase the number of significantly associated loci compared with bulk high-density lipoprotein and low-density lipoprotein analysis in a study with identical sample numbers. Moreover, lipoprotein subfractions provide novel insight into the human lipid metabolism. We measured 15 lipoprotein subfractions (L1-L15) in 1791 samples using (1)H-NMR (nuclear magnetic resonance) spectroscopy. Using cluster analyses, we quantified inter-relationships among lipoprotein subfractions. Additionally, we analyzed associations with subfractions at known lipid loci. We identified five distinct groups of subfractions: one (L1) was only marginally captured by serum lipids and therefore extends our knowledge of lipoprotein biochemistry. During a lipid-tolerance test, L1 lost its special position. In the association analysis, we found that eight loci (LIPC, CETP, PLTP, FADS1-2-3, SORT1, GCKR, APOB, APOA1) were associated with the subfractions, whereas only four loci (CETP, SORT1, GCKR, APOA1) were associated with serum lipids. For LIPC, we observed a 10-fold increase in the variance explained by our regression models. In conclusion, NMR-based fine mapping of lipoprotein subfractions provides novel information on their biological nature and strengthens the associations with genetic loci. Future clinical studies are now needed to investigate their biomedical relevance.

Pooling and expanding registries of familial hypercholesterolaemia to assess gaps in care and improve disease management and outcomes: Rationale and design of the global EAS Familial Hypercholesterolaemia Studies Collaboration

BACKGROUNDnThe potential for global collaborations to better inform public health policy regarding major non-communicable diseases has been successfully demonstrated by several large-scale international consortia. However, the true public health impact of familial hypercholesterolaemia (FH), a common genetic disorder associated with premature cardiovascular disease, is yet to be reliably ascertained using similar approaches. The European Atherosclerosis Society FH Studies Collaboration (EAS FHSC) is a new initiative of international stakeholders which will help establish a global FH registry to generate large-scale, robust data on the burden of FH worldwide.nnnMETHODSnThe EAS FHSC will maximise the potential exploitation of currently available and future FH data (retrospective and prospective) by bringing together regional/national/international data sources with access to individuals with a clinical and/or genetic diagnosis of heterozygous or homozygous FH. A novel bespoke electronic platform and FH Data Warehouse will be developed to allow secure data sharing, validation, cleaning, pooling, harmonisation and analysis irrespective of the source or format. Standard statistical procedures will allow us to investigate cross-sectional associations, patterns of real-world practice, trends over time, and analyse risk and outcomes (e.g. cardiovascular outcomes, all-cause death), accounting for potential confounders and subgroup effects.nnnCONCLUSIONSnThe EAS FHSC represents an excellent opportunity to integrate individual efforts across the world to tackle the global burden of FH. The information garnered from the registry will help reduce gaps in knowledge, inform best practices, assist in clinical trials design, support clinical guidelines and policies development, and ultimately improve the care of FH patients.