Clinical practice recommendations on lipoprotein apheresis for children with homozygous familial hypercholesterolaemia: An expert consensus statement from ERKNet and ESPN.

Homozygous familial hypercholesterolaemia is a life-threatening genetic condition, which causes extremely elevated LDL-C levels and atherosclerotic cardiovascular disease very early in life. It is vital to start effective lipid-lowering treatment from diagnosis onwards. Even with dietary and current multimodal pharmaceutical lipid-lowering therapies, LDL-C treatment goals cannot be achieved in many children. Lipoprotein apheresis is an extracorporeal lipid-lowering treatment, which is used for decades, lowering serum LDL-C levels by more than 70% directly after the treatment. Data on the use of lipoprotein apheresis in children with homozygous familial hypercholesterolaemia mainly consists of case-reports and case-series, precluding strong evidence-based guidelines. We present a consensus statement on lipoprotein apheresis in children based on the current available evidence and opinions from experts in lipoprotein apheresis from over the world. It comprises practical statements regarding the indication, methods, treatment goals and follow-up of lipoprotein apheresis in children with homozygous familial hypercholesterolaemia and on the role of lipoprotein(a) and liver transplantation.

Current Diagnosis and Management of Primary Chylomicronemia.

Primary chylomicronemia (PCM) is a rare and intractable disease characterized by marked accumulation of chylomicrons in plasma. The levels of plasma triglycerides (TGs) typically range from 1,000 - 15,000 mg/dL or higher.PCM is caused by defects in the lipoprotein lipase (LPL) pathway due to genetic mutations, autoantibodies, or unidentified causes. The monogenic type is typically inherited as an autosomal recessive trait with loss-of-function mutations in LPL pathway genes (LPL, LMF1, GPIHBP1, APOC2, and APOA5). Secondary/environmental factors (diabetes, alcohol intake, pregnancy, etc.) often exacerbate hypertriglyceridemia (HTG). The signs, symptoms, and complications of chylomicronemia include eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, and acute pancreatitis with onset as early as in infancy. Acute pancreatitis can be fatal and recurrent episodes of abdominal pain may lead to dietary fat intolerance and failure to thrive.The main goal of treatment is to prevent acute pancreatitis by reducing plasma TG levels to at least less than 500-1,000 mg/dL. However, current TG-lowering medications are generally ineffective for PCM. The only other treatment options are modulation of secondary/environmental factors. Most patients need strict dietary fat restriction, which is often difficult to maintain and likely affects their quality of life.Timely diagnosis is critical for the best prognosis with currently available management, but PCM is often misdiagnosed and undertreated. The aim of this review is firstly to summarize the pathogenesis, signs, symptoms, diagnosis, and management of PCM, and secondly to propose simple diagnostic criteria that can be readily translated into general clinical practice to improve the diagnostic rate of PCM. In fact, these criteria are currently used to define eligibility to receive social support from the Japanese government for PCM as a rare and intractable disease.Nevertheless, further research to unravel the molecular pathogenesis and develop effective therapeutic modalities is warranted. Nationwide registry research on PCM is currently ongoing in Japan with the aim of better understanding the disease burden as well as the unmet needs of this life-threatening disease with poor therapeutic options.

Lipoprotein Lipase Deficiency Impairs Bone Marrow Myelopoiesis and Reduces Circulating Monocyte Levels.

OBJECTIVE: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. APPROACH AND RESULTS: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. CONCLUSIONS: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.

Severe dyslipidemia in pregnancy: The role of therapeutic apheresis.

During pregnancy physiological changes occur in the lipid metabolism due to changing hormonal conditions: the LDL cholesterol (LDL-C), triglycerides (TG) and lipoprotein(a) [Lp(a)] increase throughout pregnancy. Common lipoprotein disorders are associated in pregnancy with two major clinical disorders: severe hypertriglyceridemia (SHTG) is a potent risk factor for development of acute pancreatitis and elevated cholesterol due to greater concentrations of LDL and remnant lipoproteins and reduced levels of HDL promote atherosclerosis. The combination of homozygous Familial Hypercholesterolemia (HoFH) and pregnancy can be a fatal condition. Therapeutic plasma exchange (TPE) may be used for an urgent need of a fast and effective lowering of TG levels in order to prevent a severe pancreatitis episode or hypertriglyceridemia-induced complications during pregnancy. LDL apheresis can decrease LDL-C and prevent complications and can be considered in the treatment of pregnancies complicated by high LDL-C. These conditions are configured in patients with HeFH who were taking statins before pregnancy (selected cases), patients already receiving apheresis before pregnancy suffering from HoFH, patients suffering from hypertriglyceridemia due to familial hyperlipoproteinemia types I and V, and cases of hypertriglyceridemia secondary to diabetes.

Molecular-genetic aspects of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is the world's most abundant and the most common heritable disorder of lipid metabolism. The prevalence of the disease in general population is 1:500. Therefore the approximate number of FH patients all over the world is 14 million. From the genetic point of view the disease originates as a result of mutations in genes affecting the processing of LDL particles from circulation, resulting in an increase in LDL cholesterol and hence total cholesterol. These are mutations in genes encoding LDL receptor, apolipoprotein B, proprotein convertase subtilisin/kexin 9 and LDL receptor adaptor protein 1. Cholesterol depositing in tissues and blood vessels of individuals creates tendon xanthoma, xanthelesma and arcus lipoides cornae. Due to the increased deposition of cholesterol in blood vessels, atherosclerosis process is accelerated, what leads to a significantly higher risk of premature cardiovascular diseases. Therefore, early clinical diagnosis confirmed by the DNA analysis, and effective treatment are crucial to reduce the mortality and high risk of premature atherosclerotic complications.

A novel apolipoprotein C-II mimetic peptide that activates lipoprotein lipase and decreases serum triglycerides in apolipoprotein E-knockout mice.

Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E-knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia.

Lipoprotein lipase deficiency: clinical, biochemical and molecular characteristics in three patients with novel mutations in the LPL gene.

Lipoprotein lipase (LPL) deficiency, caused by mutations in the LPL gene, is a rare autosomal recessive disorder manifesting in early childhood with recurrent abdominal pain, hepatosplenomegaly, acute pancreatitis, lipaemia retinalis and eruptive xanthomas. Typical laboratory findings are lactescent serum, extreme hypertriglyceridaemia and hypercholesterolaemia. The diagnostics is based on postheparin serum LPL assay and DNA analyses of the LPL gene. We report clinical, biochemical and molecular data of three children with LPL deficiency. One child manifested since the first week of life with recurrent abdominal pain (Patient 1), the second with abdominal distension and hepatosplenomegaly since the second month of life (Patient 3) and patient 2, asymptomatic younger brother of patient 1, was diagnosed in the first week of life. Lipaemia retinalis and splenomegaly were present in two symptomatic children, hepatomegaly in patient 3 and acute pancreatitis in patient 1. All children had lactescent serum, profound hypertriglyceridaemia (124 ± 25 mmol/l; controls < 2.2), hypercholesterolaemia (22.8 ± 7.3 mmol/l, controls < 4.2) and their LPL immunoreactive mass in serum did not increase after heparin injection. Molecular analyses revealed that both siblings are homozygous for novel mutation c.476C > G in the LPL gene changing the conserved amino acid of the catalytic centre. The third patient is a compound heterozygote for mutations c.604G>A and c.698A>G in the LPL gene, both affecting highly conserved amino acids. We conclude that LPL deficiency must be considered in neonates and young infants with abdominal pain and hypertriglyceridaemia because early treatment might prevent development of life-threatening acute pancreatitis.

Effect of alipogene tiparvovec (AAV1-LPL(S447X)) on postprandial chylomicron metabolism in lipoprotein lipase-deficient patients.

BACKGROUND: Lipoprotein lipase-deficient (LPLD) individuals display marked chylomicronemia and hypertriglyceridemia associated with increased pancreatitis risk. The aim of this study was to determine the effect of i.m. administration of an adeno-associated viral vector (AAV1) for expression of LPL(S447X) in muscle (alipogene tiparvovec, AAV1-LPL(S447X)) on postprandial chylomicron metabolism and on nonesterified fatty acid (NEFA) and glycerol metabolism in LPLD individuals. METHODOLOGY: In an open-label clinical trial (CT-AMT-011-02), LPLD subjects were administered alipogene tiparvovec at a dose of 1 × 10(12) genome copies per kilogram. Two weeks before and 14 wk after administration, chylomicron metabolism and plasma palmitate and glycerol appearance rates were determined after ingestion of a low-fat meal containing (3)H-palmitate, combined with (continuous) iv infusion of [U-(13)C]palmitate and [1,1,2,3,3-(2)H]glycerol. PRINCIPAL FINDINGS: After administration of alipogene tiparvovec, the triglyceride (TG) content of the chylomicron fraction and the chylomicron-TG/total plasma TG ratio were reduced throughout the postprandial period. The postprandial peak chylomicron (3)H level and chylomicron (3)H area under the curve were greatly reduced (by 79 and 93%, 6 and 24 h after the test meal, respectively). There were no significant changes in plasma NEFA and glycerol appearance rates. Plasma glucose, insulin, and C-peptide also did not change. CONCLUSIONS/SIGNIFICANCE: Intramuscular administration of alipogene tiparvovec resulted in a significant improvement of postprandial chylomicron metabolism in LPLD patients, without inducing large postprandial NEFA spillover.

Relative hypoglycemia and hyperinsulinemia in mice with heterozygous lipoprotein lipase (LPL) deficiency. Islet LPL regulates insulin secretion.

Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL+/-). LPL+/- mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL+/+ littermates. Despite having lower glucose levels, LPL+/- mice had fasting insulin levels that were twice those of +/+ mice. Hyperinsulinemic clamp experiments showed no effect of genotype on basal or insulin-stimulated glucose utilization. LPL message was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, +/- islets expressed half the enzyme activity of +/+ islets. Islets isolated from +/+ mice secreted less insulin in vitro than +/- and -/- islets, suggesting that LPL suppresses insulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing human LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-beta-galactosidase-treated cells. INS-1 cells transfected with an antisense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They further suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.

Triglyceride-induced diabetes associated with familial lipoprotein lipase deficiency.

Raised plasma triglycerides (TGs) and nonesterified fatty acid (NEFA) concentrations are thought to play a role in the pathogenesis of insulin-resistant diabetes. We report on two sisters with extreme hypertriglyceridemia and overt diabetes, in whom surgical normalization of TGs cured the diabetes. In all of the family members (parents, two affected sisters, ages 18 and 15 years, and an 11-year-old unaffected sister), we measured oral glucose tolerance, insulin sensitivity (by the euglycemic-hyperinsulinemic clamp technique), substrate oxidation (indirect calorimetry), endogenous glucose production (by the [6,6-2H2]glucose technique), and postheparin plasma lipoprotein lipase (LPL) activity. In addition, GC-clamped polymerase chain reaction-amplified DNA from the promoter region and the 10 coding LPL gene exons were screened for nucleotide substitution. Two silent mutations were found in the father's exon 4 (Glu118 Glu) and in the mother's exon 8 (Thr361 Thr), while a nonsense mutation (Ser447 Ter) was detected in the mother's exon 9. Mutations in exons 4 and 8 were inherited by the two affected girls. At 1-2 years after the appearance of hyperchylomicronemia, both sisters developed hyperglycemia with severe insulin resistance. Because medical therapy (including high-dose insulin) failed to reduce plasma TGs or control glycemia, lipid malabsorption was surgically induced by a modified biliopancreatic diversion. Within 3 weeks of surgery, plasma TGs and NEFA and cholesterol levels were drastically lowered. Concurrently, fasting plasma glucose levels fell from 17 to 5 mmol/l (with no therapy), while insulin-stimulated glucose uptake, oxidation, and storage were all markedly improved. Throughout the observation period, plasma TG levels were closely correlated with both plasma glucose and insulin concentrations, as measured during the oral glucose tolerance test. These cases provide evidence that insulin-resistant diabetes can be caused by extremely high levels of TGs.

Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene.

BACKGROUND: Patients with lipoprotein lipase deficiency usually present with chylomicronemia in childhood. The syndrome has been considered nonatherogenic primarily because of the low levels of low-density lipoprotein (LDL) cholesterol. We prospectively evaluated patients with lipoprotein lipase deficiency for atherosclerosis. METHODS: Evidence of carotid, peripheral, and coronary atherosclerosis was sought in four patients (two men and two women) with the phenotype of familial chylomicronemia by clinical examination over a period of 14 to 30 years and by Doppler ultrasonography, B-mode ultrasonography [corrected], and exercise-tolerance testing after the age of 40. Angiography was performed when indicated. Lipoprotein lipase deficiency was assessed in vivo and in vitro by functional assays and DNA-sequence analysis. RESULTS: All four patients had a profound functional deficiency of lipoprotein lipase with a reduced enzymatic mass due to missense mutations on both alleles of the lipoprotein lipase gene. In all four patients, peripheral or coronary atherosclerosis (or both) was observed before the age of 55. Despite following a low-fat diet in which fat composed 10 to 15 percent of the daily caloric intake, the patients had hypertriglyceridemia (mean [+/- SD] triglyceride level, 2621 +/- 1112 mg per deciliter [29.59 +/- 12.55 mmol per liter]), low plasma levels of high-density lipoprotein cholesterol (17 +/- 7 mg per deciliter [0.43 +/- 0.18 mmol per liter]), and very low levels of LDL cholesterol (28 +/- 16 mg per deciliter [0.72 +/- 0.41 mmol per liter]). Three patients had one risk factor for atherosclerosis, whereas in one male patient, heavy smoking and diabetes were associated with an accelerated course of the disease. CONCLUSIONS: Premature atherosclerosis can occur in patients with familiar chylomicronemia as a result of mutations in the lipoprotein lipase gene. Defective lipolysis may increase susceptibility to atherosclerosis in humans.

Remnant-like lipoproteins stimulate whole blood platelet aggregation in vitro.

We have developed a simple, rapid assay method to measure remnant-like lipoproteins by using an immunoaffinity gel mixture of anti apo B-100 and apoA-1 antibodies to Sepharose 4B. Characterization of the unbound lipoproteins has shown that they represent chylomicron and VLDL remnant particles (RLP). Preincubation of whole blood with RLP resulted in the enhanced activation of aggregation with ADP and collagen. Such enhancement was not observed in the presence of lipoprotein deficient serum or albumin preparation. The extent of enhancement was 2.78 times by 7.5 microM of ADP and 44 times by 0.5 microgram/ml of collagen in the presence of RLP-preparation 1 (RLP-1), respectively. In the presence of RLP-2, the enhancement was 5.37 times by 7.5 microM of ADP and 102 times by 0.5 microgram/ml of collagen, respectively. On the other hand RLP slightly inhibited PRP aggregation by these agonists. Inhibitions were 19% by 7.5 microM of ADP and 18% by 1.0 microgram/of collagen in the presence of RLP-1, respectively. Incubation of whole blood with RLP did not result in the release of factors to stimulate platelets or ADP- or collagen-induced platelet aggregation in vitro. The extents of enhanced aggregation in whole blood or inhibition in PRP were not correlated with RLP-cholesterol nor RLP-protein concentrations of RLP preparations used. These results may indicate that RLP not only interact with platelets but with erythrocytes or leukocytes. Our findings support the hypothesis that the postprandial increase in remnant lipoproteins is an atherosclerotic risk factor and may be a part of the reasons of thrombotic complications by stimulating platelets in patients with remnant hyperlipoproteinemia.

[A case of the long-term observation of Bürger-Grütz disease]. / Sluchai dlitel'nogo nabliudeniia bolezni Biurgera--Griuttsa.

The authors observed Bürger-Grütz disease (primary hyperchylomicronemia, painful recurrent xanthomatosis, hepatosplenomegaly, hyperglycemia) for 20 years in a 54-year-old female patient. The disease ran a benign course. No marked vascular lesions, progressive atherosclerosis, diabetes mellitus arose. This is an original report of Bürger-Grütz disease long-term course and life-long heparin treatment in this country.

Accumulation of an apoE-poor subfraction of very low density lipoprotein in hypertriglyceridemic men.

Studies were undertaken to investigate the mechanism of the marked accumulation of an apoE-poor very low density lipoprotein (VLDL) subfraction in untreated Type IV and IIb hypertriglyceridemic subjects. Heparin-Sepharose chromatography was used to separate large VLDL (Sf 60-400) from fasted subjects, into an apoE-poor, unbound fraction and an apoE-rich, bound fraction. As a percent of total VLDL protein, the apoE-poor fraction comprised 40 +/- 4% of total VLDL in hypertriglyceridemic subjects versus 25% in normal subjects. Compared to the apoE-rich, bound fraction, this apoE-poor material was found to have a 5-fold lower ratio of apoE to apoC (0.20 +/- 0.06 vs 0.91 +/- 0.18, P less than 0.005), but a 1.5-fold higher ratio of triglyceride to protein (11.41 +/- 0.85 vs 7.97 +/- 0.77, P less than 0.01). In addition, the apoE-poor fraction was found to be enriched 2-fold in apoB-48 (10.30 +/- 2.41% vs 5.73 +/- 1.59% of total apoB, P less than 0.005) compared to the apoE-rich fraction, suggesting that the apoE-poor fraction contains more chylomicron remnants. The amount of this apoE-poor VLDL was markedly reduced following a reduction in VLDL triglyceride levels (a decrease from 40 +/- 4% to 21 +/- 2% of VLDL protein following a 50% reduction in VLDL triglyceride levels). The large VLDL from Type I, III, and V hyperlipoproteinemic subjects subfractionated using heparin-Sepharose showed an equal distribution of apoE between the two fractions in contrast with the Type IV and IIb subjects. The separation of VLDL from Type I, III, and V subjects using heparin-Sepharose involves a mechanism other than apoE binding. Separation in the latter likely results from apoB-100 binding to heparin, as opposed to apoE binding of VLDL from Type IV and IIb subjects.

Binding of thyroid hormones to human plasma lipoproteins.

The binding of T4, T3, and rT3 to plasma lipoproteins was investigated in normal subjects and patients with abnormal lipoprotein metabolism. Gel filtration on Sepharose CL-6B demonstrated iodothyronine binding to all lipoprotein classes. In the total lipoprotein fraction (density less than 1.210 g/mL), high density lipoproteins (HDL) were the major binders, accounting for 92% of lipoprotein-bound T4, 99% of lipoprotein-bound T3, and 55% of lipoprotein-bound rT3. The estimated iodothyronine binding in normal plasma to HDL, low density lipoproteins (LDL), and very low density lipoproteins (VLDL) was 3%, 0.2%, and 0.03% for T4, 6%, 0.05%, and 0.02% for T3, and 0.1%, 0.1%, and 0.01% for rT3, respectively. These estimates may be low owing to possible dissociation during chromatography and the short incubation period used to avoid changes in lipoprotein structure. In VLDL and LDL deficiency (abetalipoproteinemia), HDL deficiency (Tangier disease), LDL excess (type IIa hyperlipoproteinemia), and VLDL excess (type III, IV, and V hyperlipoproteinemia), the distribution of iodothyronines reflected the lipoprotein abnormality. Variations resulting from altered distribution within HDL subclasses were also found. Binding was saturable, with approximate dissociation constants for VLDL, LDL, and HDL of 10(-5)-10(-6) mol/L. We conclude that thyroid hormones bind specifically to apolipoproteins, although additional binding by solubilization in the lipid components of the lipoproteins may also occur.

Peripheral neuropathy in cats with inherited primary hyperchylomicronaemia.

Primary hyperlipoproteinaemia (hyperchylomicronaemia) with a slight increase in very low density lipoprotein) is described in 20 cats. Fasting hyperlipaemia, lipaemia retinalis and peripheral neuropathies were the most frequently detected clinical signs. The disease is thought to be inherited as an autosomal recessive trait but the exact mode of inheritance has not been determined. Affected cats showed reduced lipoprotein lipase activity measured after heparin activation compared with the response in normal cats. Plasma triglyceride and cholesterol were increased in all the cats with the major proportion of triglyceride and cholesterol being present in chylomicrons. The peripheral nerve lesions were caused by compression of nerves by lipid granulomata. It is probable that the lipid granulomata result from trauma because the nerves most often affected were at sites like the spinal foraminae where they were susceptible to trauma.