Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

Evolocumab and lipoprotein apheresis combination therapy may have synergic effects to reduce low-density lipoprotein cholesterol levels in heterozygous familial hypercholesterolemia: A case report.

A 49 years old woman (weight 68 kg, BMI 27.3 kg/m2 ) with heterozygous familial hypercholesterolemia (HeFH) and multiple statin intolerance with muscle aches and creatine kinase elevation, presented at the Outpatient Lipid Clinic of Verona University Hospital in May 2015. Hypercholesterolemia was firstly diagnosed during adolescence, followed in adulthood by a diagnosis of Cogan's syndrome, a rheumatologic disorder characterized by corneal and inner ear inflammation. No xanthomas, corneal arcus, or vascular bruits were detectable at physical examination. Screening for macrovascular complications did not reveal relevant damages. Ongoing medical therapy included salicylic acid, methylprednisolone, methotrexate, and protonic-pump inhibitor. In the absence of specific lipid-lowering therapy, plasma lipid levels at first visit were: total-cholesterol = 522 mg/dL, LDL-cholesterol = 434 mg/dL, HDL-cholesterol = 84 mg/dL, triglycerides = 120 mg/dL, Lp(a) = 13 mg/dL. On December 2015, evolocumab 140 mg sc every 2 weeks was initiated. After a 24-week treatment, the LDL-cholesterol levels decreased by an average of 21.2% to 342 ± 22 mg/dL (mean ± SD). On May 2016, LDL-apheresis (H.E.L.P.system) was started as add-on therapy. Compared to the average levels obtained during the evolocumab monotherapy period, the LDL-cholesterol was reduced by 49.4%, thus reaching an inter-apheresis level (mean ± SD) of 173 ± 37 mg/dL. This report suggests that a combination therapy with evolocumab and lipoprotein-apheresis may have synergic effects on circulating lipid levels. Its relevance as a highly effective treatment option for hyperlipidemia in HeFH patients warrants further investigation in larger datasets.

The effects of treatment on lipoprotein subfractions evaluated by polyacrylamide gel electrophoresis in patients with autoimmune hypothyroidism and hyperthyroidism.

BACKGROUND: Atherogenic dyslipoproteinemia is one of the most important risk factor for atherosclerotic changes development. Hypothyroidism is one of the most common causes of secondary dyslipidemias which results from reduced LDL clearance and therefore raised levels of LDL and apoB. Association between small dense LDL (sdLDL) presentation and thyroid status has been examinated using polyacrylamide gel electrophoresis for lipoprotein subfractions evaluation. METHODS: 40 patients with diagnosed autoimmune hypothyroidism and 30 patients with autoimmune hyperthyroidism were treated with thyroxine replacement or thyreo-suppressive treatment. In both groups lipid profiles, LDL subractions, apolipoproteins (apoA1, apoB), apoA1/apoB ratio and atherogenic index of plazma (AIP) were examined before treatment and in state of euthyreosis. RESULTS: Thyroxine replacement therapy significantly reduced levels of total cholesterol (TC), LDL, triglycerides (TG) and also decreased levels of sdLDL (8,55±11,671 vs 0,83±1,693mg/dl; p<0,001), apoB and AIP. For estimation of atherogenic lipoprotein profile existence an AIP evaluation seems to be better than apoB measurement because of the more evident relationship with sdLDL (r=0,538; p<0,01). Thyreo-suppressive therapy significantly increased levels of TC, LDL, TG and apoB. The sdLDL was not found in hyperthyroid patients. CONCLUSIONS: Atherogenic lipoprotein profile was present in 52.5% of hypothyroid subjects, which is higher prevalence than in normal, age-related population. Substitution treatment leads to an improvement of the lipid levels, TG, apoB, AIP and LDL subclasses. It significantly changed the presentation of sdLDL - we noticed shift to large, less atherogenic LDL particles. Significantly positive correlation between sdLDL and TAG; sdLDL and VLDL alerts to hypertriglyceridemia as a major cardiovascular risk factor.

Genetic and biochemical analyses in dyslipidemic patients undergoing LDL apheresis.

OBJECTIVE: Familial hypercholesterolemia (FH) can be due to mutations in LDLR, PCSK9, and APOB. In phenotypically defined patients, a subset remains unresponsive to lipid-lowering therapies and requires low density-lipoprotein (LDL) apheresis treatment. In this pilot study, we examined the genotype/phenotype relationship in patients with dyslipidemia undergoing routine LDL apheresis. DESIGN: LDLR, APOB, and PCKS9 were analyzed for disease-causing mutations in seven patients undergoing routine LDL apheresis. Plasma and serum specimens were collected pre- and post-apheresis and analyzed for lipid concentrations, Lp(a) cholesterol, and lipoprotein particle concentrations (via NMR). RESULTS: We found that four patients harbored LDLR mutations and of these, three presented with xanthomas. While similar reductions in LDL-cholesterol (LDL-C), apolipoprotein B, and LDL particles (LDL-P) were observed following apheresis in all patients, lipid profile analysis revealed the LDLR mutation-positive cohort had a more pro-atherogenic profile (higher LDL-C, apolipoprotein B, LDL-P, and small LDL-P) pre-apheresis. CONCLUSION: Our data show that not all clinically diagnosed FH patients who require routine apheresis have genetically defined disease. In our small cohort, those with LDLR mutations had a more proatherogenic phenotype than those without identifiable mutations. This pilot cohort suggests that patients receiving the maximum lipid lowering therapy could be further stratified, based on genetic make-up, to optimize treatment.

Plaque regression and progenitor cell mobilization with intensive lipid elimination regimen (PREMIER) trial design.

Progression of lipid rich necrotic core elements of atherosclerotic vulnerable plaque (VP) or its rupture leads to a majority of cardiovascular events. Endothelial progenitor cells (EPC) contribute to vascular healing and play a crucial role in repair following ischemic injury primarily by endothelialization of VP and neovascularization of ischemic myocardium. We present the rationale and design of the Plaque Regression and Progenitor Cell Mobilization with Intensive Lipid Elimination Regimen or the PREMIER Trial, which is designed to address the question for the very first time whether a highly intensive low-density lipoprotein (LDL)-lowering therapy with LDL-apheresis could lead to a more rapid and detectable reduction in coronary atheroma volume, along with a robust mobilization of EPC compared to standard statin therapy, in patients selected for percutaneous coronary intervention for an acute coronary syndrome.

An international survey of pediatric apheresis practice.

INTRODUCTION: Pediatric apheresis (PA) has distinct characteristics compared to adult apheresis, and requires specialized knowledge and experience to perform safely, particularly in low-weight patients. As evidence-based medicine advances the field of therapeutic apheresis, increased attention must be paid to pediatric patients with conditions for which apheresis is indicated. METHODS: An electronic survey of >5,000 potential participants throughout the world was conducted to ascertain the scope and the current state of practice. RESULTS: The survey elicited 159 responses from 12 countries; 107 of the responses provided sufficient information for analysis. Participants performed an average of 176 PA procedures/year (range: 1-2,000). The types of PA procedures were therapeutic plasma exchange (92% of centers), red cell exchange (86%), leukocyte depletion (87%) and peripheral blood hematopoietic progenitor cell collection (72%). More than 65% of the centers had treated children older than 5 years with PA. Many centers had also performed PA on younger children; 40% have treated patients <12 months of age; 61% had treated patients 1-5 years old. 36% of centers reported that they would perform apheresis regardless of patient weight; 18% used a 5 kg threshold, 11% used 5-10 kg, and 17% used 10 kg as their weight threshold. CONCLUSION: This report is the largest single survey of centers performing PA. The results provide information about the scope and diversity of PA and identify areas where considerable variability in practice exists. Further exploration of these differences could establish best practices in PA through international research and collaboration.

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia.

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-ß1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.

Use of ultra high performance liquid chromatography-tandem mass spectrometry to demonstrate decreased serum statin levels after extracorporeal LDL-cholesterol elimination.

BACKGROUND: Using our statin analysis method, it was possible to uncover a significant drop in statin levels (atorvastatin, simvastatin, and metabolites) after extracorporeal LDL-cholesterol elimination (EE) in severe familial hypercholesterolemia (FH). The purpose of this work was to identify the mechanism underlying this drop and its clinical significance as well as to propose measures to optimize a pharmacotherapeutical regimen that can prevent the loss of statins. METHODS: Ultra High Performance Liquid Chromatography (UHPLC) connected to the triple quadrupole MS/MS system was used. Patients. A group of long-term treated patients (3-12 years of treatment) with severe FH (12 patients) and treated regularly by LDL-apheresis (immunoadsorption) or haemorheopheresis (cascade filtration) were included in this study. RESULTS: After EE, the level of statins and their metabolites decreased (atorvastatin before/after LDL-apheresis: 8.83/3.46 nmol/l; before/after haemorheopheresis: 37.02/18.94 nmol/l). A specific loss was found (concentration of atorvastatin for LDL-apheresis/haemorheopheresis: 0.28/3.04 nmol/l in washing fluids; 11.07 nmol/l in filters). To prevent substantial loss of statin concentrations, a pharmacotherapeutic regimen with a longer time interval between the dose of statins and EE is recommended (15 hours). CONCLUSIONS: A specific loss of statins was found in adsorbent columns and filters. The decrease can be prevented by the suggested dosage scheme.

Pancytopenia with severe thrombocytopenia in a patient treated with twice-weekly LDL-apheresis by polyacrylate adsorption from whole blood.

Pancytopenia with severe thrombocytopenia occurred in a patient treated with low-density lipoprotein (LDL)-apheresis by polyacrylate adsorption from whole blood, after treatment frequency had been increased from once to twice a week. Cell counts recovered with discontinuation of LDL-apheresis, but thrombocytopenia recurred after resumption of twice-weekly treatments. Thrombocyte counts remained stable following the replacement of polyacrylate adsorption from whole blood by double-filtration plasmapheresis. The complications' close coincidence with twice-weekly polyacrylate adsorption from whole blood suggests a causal relationship, although by a still unknown mechanism. Monitoring of thrombocytes should be advised in patients treated with LDL-apheresis by polyacrylate adsorption from whole blood.

Plasminogen activator inhibitor-1 removal using dextran sulphate columns. Evidence of PAI-1 homeostasis.

Patients with high plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels are prone to develop thrombosis. Lowering PAI-1 levels may offer a therapeutic option and help to better understand PAI-1 metabolism. We examined the effect on plasma PAI-1 levels of LDL-apheresis using dextran sulphate (DS) columns in 12 patients (9 male, 3 female, 49 +/- 10 years) with heterozygous familial hypercholesterolaemia and coronary artery disease. One plasma volume equivalent (2.3-4.0 l) was treated during each procedure (at flow rates of 23 +/- 2 ml/min). Lipids and PAI-1 antigen levels were measured in plasma before and immediately after 19 aphereses (once in 7 patients, twice in 3 patients and three times in 2 patients) and also at 3 and 7 days post apheresis in five of these patients and in the column eluates from 8 of these patients. DS-apheresis reduced plasma cholesterol (50 +/- 8%), triglyceride (45 +/- 27%), apolipoprotein B (59 +/- 10%) and PAI-1 antigen levels from 10.2 +/- 5.2 to 6.0 +/- 3.1 ng/ml (P = 0.005). The PAI-I changes were independent of circadian variation. PAI-I bound to the DS-columns (3.51 +/- 1.03 ng/ml filtered plasma) and the percent of filtered PAI-1 that was bound correlated inversely (r = -0.81, P < 0.02) with basal PAI-1 levels indicating a high affinity saturable binding process. In four patients, plasma PAI-1 levels post-apheresis were higher than expected based on the amount of PAI-removed by the DS columns. The difference between the expected and actual PAI-1 level post apheresis, reflecting PAI-1 secretion or extracellular redistribution, correlated inversely with basal PAI-1 levels (r = -0.83, P = 0.01). PAI-1 levels returned to baseline pre-apheresis values 7 days post apheresis. PAI-1 antigen may be removed from plasma without adverse effect, resulting temporarily in its extracellular redistribution and restoration to baseline levels over one week. PAI-1 redistribution particularly when baseline pre-apheresis values were low may reflect a homeostatic mechanism to maintain sufficient PAI-1 levels. Procedures that could selectively remove PAI-1 from plasma may offer a treatment option for those with very high plasma PAI-1 levels and thrombosis.

In vitro characterization of a monoclonal IgG(kappa) from a patient with planar xanthomatosis.

OBJECTIVE: To characterize a monoclonal IgG(kappa) (MAb) from a patient with planar xanthoma that precipitated with serum lipids at reduced temperature. METHODS: The molecular weight (Mr), sensitivity to proteases, and glycosylation of the purified MAb were analyzed. The specificity of the MAb was tested by measuring cryoprecipitation with pure high- (HDL) and low-density (LDL) lipoproteins. The effect of choline, phosphocholine, and glycerol 3-phosphate on the precipitation temperature of LDL by the MAb was studied. RESULTS: The MAb was larger than normal IgG due to hyperglycosylation of the MAb light chain. It formed cryoprecipitates with pure HDL and LDL as well as the lipids extracted from these lipoproteins. Fab fragments of the MAb lowered the temperature of its precipitation with LDL. Choline, phosphocholine, and glycerol 3-phosphate also lower the precipitation temperature. CONCLUSION: This is the first human IgG reported with apparent specificity for phosphocholine antigens. Its precipitation with lipids at reduced temperature suggests that it recognizes conformations of phospholipid head groups that develop below core body temperature.

Macrophage paraoxonase 2 (PON2) expression is upregulated by unesterified cholesterol through activation of the phosphatidylinositol 3-kinase (PI3K) pathway.

Advanced atherosclerotic lesions are characterized by a progressive increase in the unesterified cholesterol (UC) content and a decrease in its cholesteryl ester (CE) content. In the present study, we examined mechanisms involved in the effect of UC and CE on the expression of paraoxonase 2 (PON2) in macrophages. J774A.1 macrophages were enriched with CE or UC by incubation for 14-48 h with 50 microg acetylated low-density lipoprotein in the absence or presence of the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor 58035 (50 microg/ml), respectively. Macrophage PON2 mRNA expression, protein abundance and activity were increased only in the UC-enriched cells. In UC-enriched cells, inhibition of phosphatidylinositol 3-kinase (PI(3)K; using wortmannin or LY294002) attenuated the increase in PON2 mRNA expression by 50%, compared to untreated cells. In addition, we evidenced an increased phosphorylation of Akt in UC-enriched cells. Thus, we conclude from our data that macrophage PON2 expression is upregulated in UC-enriched macrophages through activation of the PI(3)K signal pathway.

Proteomic and lipid characterization of apolipoprotein B-free luminal lipid droplets from mouse liver microsomes: implications for very low density lipoprotein assembly.

The assembly of very low density lipoproteins involves the formation of a primordial, poorly lipidated apoB-containing particle in the endoplasmic reticulum, followed by the addition of neutral lipid from luminal lipid droplets (LLD). However, the lipid and protein compositions of LLD have not been determined. We have isolated LLD from mouse liver microsomes and analyzed their lipid and protein compositions. LLD are variably sized particles relatively poor in triacylglycerol (TG) content when compared with the lipid composition of cytosolic lipid droplets (CLD). They are devoid of apoB, adipophilin, and albumin but contain numerous proteins different from those found on CLD, including TG hydrolase (TGH), carboxylesterase 1 (Ces1), microsomal triglyceride transfer protein (MTP), and apoE. Ectopic expression of TGH in McArdle RH7777 hepatoma cells resulted in decreased cellular TG levels, demonstrating a role for TGH in the mobilization of hepatic neutral lipid stores. The isolation and characterization of LLD provide new supporting evidence for the two-step assembly of very low density lipoproteins.

[Effects of LDL-apheresis--more than reduction of cholesterol?]. / Effekte der LDL-Apherese--Mehr als nur Cholesterinsenkung?

LDL apheresis is a safe and very effective extracorporeal treatment of refractory hypercholesterolemia. LDL cholesterol levels can be reduced with this procedure by more than 60%. C-reactive protein (CRP) is a known marker of inflammation in atherosclerosis. Interestingly CRP can be effectively removed by a single LDL apheresis, but further studies are needed to substantiate the effect of extracorporeal reduction of CRP on the progression of atherosclerosis. However, adhesion molecules and activities of inflammatory cells were also found to be reduced after a single LDL apheresis. The biochemical composition of newly formed LDL particles after apheresis is altered: LDL particles isolated after LDL apheresis had an increased resistance to oxidative stress in vitro. In addition, antioxidants are not depleted by LDL apheresis. The extracorporal method itself does not have a negative impact on the oxidative/antioxidative balance. A recent investigation showed that LDL-cholesterol had a more pronounced effect on blood rheology than fibrinogen. This observation may explain why a single LDL apheresis leads to better myocardial perfusion, as demonstrated by PET in patients with hypercholesterolemia. These additional effects have so far only been known with statins. Further investigations are needed to substantiate the observed potentially beneficial effects of LDLapheresis beyond its effect of lowering LDL.

Changes in myocardial vasoreactivity after drastic reduction of plasma fibrinogen and cholesterol: a clinical study in long-term heart transplant survivors using positron emission tomography.

BACKGROUND: Given the central importance of the microvasculature in heart transplant recipients, we investigated the possibility of increasing cardiac perfusion after reduction of low-density lipoprotein (LDL)-cholesterol, lipoprotein (a), C-reactive protein (CRP) and fibrinogen plasma levels after apheresis treatment in transplanted patients. METHODS: Ten long-term heart transplant recipients were examined with positron emission tomography (PET) to measure myocardial perfusion before and after a single heparin-mediated extracorporeal LDL/fibrinogen precipitation (HELP)-apheresis treatment. PET studies were performed the mornings before and after the apheresis treatment. Myocardial blood flow at rest and during adenosine-induced hyperemia was measured using (13)N-ammonia. RESULTS: HELP-apheresis reduced the plasma levels of LDL-cholesterol, lipoprotein (a) and C-reactive protein by 48% (p < 0.001), fibrinogen by 42% (p = 0.02), plasma viscosity by 14% (p = 0.004) and erythrocyte aggregation by 28% (p < 0.02). Osmolality (<1%) and hematocrit (<2%) remained stable. A single apheresis treatment increased median corrected rest flow by 17.5% (p = 0.007) and median hyperemic flow by 27% (p = 0.02). Median coronary flow reserve increased by 8.1% (p = 0.09). Hyperemic flow after adenosine infusion increased plasma vascular endothelial growth factor levels only before HELP-apheresis (+60%), indicating better ischemic tolerance after apheresis (p = 0.01). CONCLUSIONS: Myocardial perfusion in transplanted hearts increases significantly after single HELP-apheresis treatment. The present study is only a proof of concept, providing complementary evidence to clinical long-term studies showing that cholesterol reduction either with statins and/or apheresis improves heart transplant outcome.

Selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity.

The role of adhesive selectin molecules in the process of atherogenesis is an open question. These molecules are known as markers of atherosclerosis activity, however, only some biological mechanisms are known up to now. In this study we examined the levels of soluble forms of E-, P-selectin and monocyte chemotactic protein (MCP-1) in the process of extracorporeal cholesterol elimination by LDL-apheresis. We measured the levels of sE-, sP-selectin and MCP-1 in the plasma before and after LDL-apheresis and in the washout solution from immunoabsorption columns Lipopak. Eighty measurements were performed repeatedly in 6 patients with severe familial hypercholesterolemia (FH) on long-term LDL-apheresis treatment. Before the procedure P-selectin levels were 204+/-179 ng/ml, E-selectin 32.1+/-33.7 ng/ml, MCP-1 323.8+/-121 pg/l, whereas after the procedure we found P-selectin levels 131.6+/-34 ng/ml, E-selectin 33.1+/-51 ng/ml, and MCP-1 200.4+/-15 pg/l. Levels of P-selectin were increased in the blood of patients with FH in spite of long-term intensive extracorporeal LDL-elimination, documenting thus the activity of atherosclerosis. The levels of P-selectin and MCP-1 decreased significantly after the hypolidemic procedure and could be used as another marker showing the effectivity of the extracorporeal LDL-cholesterol elimination (immediately after the procedure), and, after further verification, may serve as a marker for controlling the therapy efficacy.

Effects of plant stanol and sterol esters on serum phytosterols in a family with familial hypercholesterolemia including a homozygous subject.

We studied the concentrations and ratios to cholesterol of noncholesterol sterols reflecting absorption (eg, campesterol) or synthesis (eg, lathosterol) of cholesterol off and on plant sterol and stanol ester spreads in serum and in different lipoproteins of a family with familial hypercholesterolemia, including heterozygous parents receiving no treatment and their homozygous offspring undergoing long-term treatment with statins and apheresis. Serum cholesterol levels were similar in the homozygous and heterozygous individuals, but the concentrations of sterols reflecting cholesterol absorption were as much as 10 times greater in the homozygous child than in the heterozygous parents, whereas the respective markers of cholesterol synthesis only tended to be higher. About 70% of squalene in the homozygous individual (60% in the heterozygous family members) and 85% to 90% of noncholesterol sterols (60%-80% in the heterozygous subjects) were transported by low-density lipoprotein. The ratios of absorption sterols to cholesterol were higher in high-density lipoprotein (HDL) than in very low-density lipoprotein (VLDL), whereas those of synthesis markers and plant stanols were highest in VLDL. The ratios of absorption sterols in serum were mostly lower than those in HDL but higher than in VLDL, whereas the ratios of synthesis sterols in serum were lower than they were in VLDL. Both spreads reduced serum total cholesterol by about 14% in the heterozygous family members and 9% in the homozygous individual. The sterol ester spread increased serum plant sterol concentrations (eg, campesterol in the homozygous family member increased from 5 to 9 mg/dL) and the ratios to cholesterol, but the stanol ester spread decreased them. Plant sterol esters seemed to similarly decrease serum cholesterol in this family with familial hypercholesterolemia, but the clinical role of increased plant sterol concentrations, almost doubled in the LDL of homozygous individuals, is not known.

[LDL-apheresis in the treatment of coronary heart disease. Rationale for a specific adjuvant therapy]. / Stellenwert der LDL-Apherese in der Behandlung der koronaren Herzerkrankung. Rationale für eine gezielte adjuvante Therapie.

Coronary artery disease (CHD) still remains the leading cause of death in all industrialized countries. In Germany, it claims the lives of an estimated 220,000 men and women and causes 200,000 non-lethal AMI's each year. Inspite of great efforts in the last 30 years, 25% of men and 40% of women still die within 12 months after they preliminary survived their first myocardial infarction. The total direct plus indirect expenses for CHD in the year 2000 sum up to approximately 57 billion Euro in Germany and to 100 billion US $ in the US. To date, management of coronary artery disease still consists mainly of a therapy designed to improve blood flow and oxygen supply to the heart or to reduce myocardial oxygen consumption. On this line angioplasty, bypass surgery, and more recently stenting of coronary arteries, have become leading techniques, but only a minority (<50%) of patients at risk for CHD received therapy to change the atherosclerotic process itself. However, only by means of a causal intervention with the pathmaker mechanism of atherosclerosis can we expect to decrease the financial burden of CHD. No question, one of the leading causal factors for early atherosclerosis and coronary heart disease is the abundance of LDL cholesterol in the blood, exceeding limits of 100 mg/dl. Thus, recommendations for therapy focus on LDL levels less than 100 mg/dl. With the introduction of the statins-a family of very potent lipid lowering agents-such target levels can be achieved in most patients, resulting in a drastic decrease of LDL concentrations, as well as in a reduction of cardiac and total mortality. There is, however, a remaining small group of patients, who are more or less resistant to an adequate combination of dietary and drug therapy. For these patients, various techniques of apheresis have been available for over 15 years. Some of them, e. g., H.E.L.P. System, KANEKA System, have been approved by the FDA in the US and comparable regulatory offices in Europe. The most extensive experimental and clinical experience was gathered with the H.E.L.P. System by B. Braun Melsungen, which differs from other apheresis techniques by its efficiency to eliminate LDL, Lp(a), fibrinogen and CRP simultaneously. The clinical results obtained to date with the H.E.L.P.-LDL-apheresis clearly demonstrate a significant reduction of risk factors and clinical events, as well as an excellent long-term tolerance. A comprehensive literature survey on H.E.L.P. is part of this communication.